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Michael Philips

Michael Philips

Homologous Recombination & Site-Specific Recombination of DNA

Slide Duration:

Table of Contents

I. The Beginnings of Molecular Biology
Biochemistry Review: Importance of Chemical Bonds

53m 29s

Intro
0:00
Lesson Overview
0:14
Chemical Bonds
0:41
Attractive Forces That Hold Atoms Together
0:44
Types of Bonds
0:56
Covalent Bonds
1:34
Valence Number
1:58
H O N C P S Example
2:50
Polar Bonds
7:23
Non-Polar Bond
8:46
Non-Covalent Bonds
9:46
Ionic Bonds
10:25
Hydrogen Bonds
10:52
Hydrophobic Interactions
11:34
Van Der Waals Forces
11:58
Example 1
12:51
Properties of Water
18:27
Polar Molecule
13:34
H-bonding Between Water H20 Molecules
19:29
Hydrophobic Interactions
20:30
Chemical Reactions and Free Energy
22:52
Transition State
23:00
What Affect the Rate
23:27
Forward and Reserve Reactions Occur Simultaneously But at Different Rate
23:51
Equilibrium State
24:29
Equilibrium Constant
25:18
Example 2
26:16
Chemical Reactions and Free Energy
27:49
Activation Energy
28:00
Energy Barrier
28:22
Enzymes Accelerate Reactions by Decreasing the Activation Energy
29:04
Enzymes Do Not Affect the Reaction Equilibrium or the Change in Free Energy
29:22
Gibbs Free Energy Change
30:50
Spontaneity
31:18
Gibbs Free Energy Change Determines Final Concentrations of Reactants
34:36
Endodermic vs. Exothermic Graph
35:00
Example 3
38:46
Properties of DNA
39:37
Antiparallel Orientation
40:29
Purine Bases Always Pairs Pyrimidine Bases
41:15
Structure Images
42:36
A, B, Z Forms
43:33
Major and Minor Grooves
44:09
Hydrogen Bonding and Hydrophobic Interactions Hold the Two Strands Together
44:39
Denaturation and Renaturation of DNA
44:56
Ways to Denature dsDNA
45:28
Renature When Environment is Brought Back to Normal
46:05
Hyperchromiicity
46:36
Absorbs UV Light
47:01
Spectrophotometer
48:01
Graph Example?
49:05
Example 4
51:02
Mendelian Genetics & Foundational Experiments

1h 9m 27s

Intro
0:00
Lesson Overview
0:22
Gregor Johann Mendel
1:01
Was a Biologist and Botanist
1:14
Published Seminal Paper on Hybridization and Inheritance in the Pea Plant
1:20
Results Criticized
1:28
Father of Modern Genetics
1:59
Mendel’s Laws
2:19
1st Law: Principle of Independent Segregation of Alleles
2:27
2nd Law: Principle of Independent Assortment of Genes
2:34
Principle of Independent Segregation (of Alleles)
2:41
True Breeding Lines / Homozygous
2:42
Individuals Phenotypes Determined by Genes
3:15
Alleles
3:37
Alleles Can Be Dominant or Recessive
3:50
Genotypes Can be Experimentally Determined by Mating and Analyzing the Progeny
5:36
Individual Alleles Segregate Independently Into Gametes
5:55
Example 1
6:18
Principle of Independent Segregation (of Alleles)
16:11
Individual Genes Sort Independently Into Gametes
16:22
Each Gamete Receives One Allele of Each Gene: 50/50 Chance
16:46
Genes Act Independently to Determine Unrelated Phenotypes
16:57
Example: Punnett Square
17:15
Example 2
21:36
The Chromosomal Theory of Inheritance
30:41
Walter S Sutton Linked Cytological Studies with Mendels Work
31:02
Diploid Cells Have Two Morphologically Similar Sets of Chromosomes and Each Haploid Gamete Receives One Set
31:17
Genes Are on Chromosome
31:33
Gene for Seed Color’s on a Different Chromosome Than Gene for Seed Texture
31:44
Gene Linkage
31:55
Mendel’s 2nd Law
31:57
Genes Said to Be Linked To Each Other
32:09
Linkage Between Genes
32:29
Linkage is Never 100% Complete
32:41
Genes are Found on Chromosomes
33:00
Thomas Hunt Morgan and Drosophila Melanogaster
33:01
Mutation Linked to X Chromosome
33:15
Linkage of White Gene
33:23
Eye Color of Progeny Depended on Sex of Parent
33:34
Y Chromosome Does Not Carry Copy of White Gene
33:44
X Linked Genes, Allele is Expressed in Males
33:56
Example
34:11
Example 3
35:52
Discovery of the Genetic Material of the Cell
41:52
Transforming Principle
42:44
Experiment with Streptococcus Pneumoniae
42:55
Beadle and Tatum Proposed Genes Direct the Synthesis of Enzymes
45:15
One Gene One Enzyme Hypothesis
45:46
One Gene One Polypeptide Theory
45:52
Showing the Transforming Material was DNA
46:14
Did This by Fractionating Heat-Killed “S” Strains into DNA, RNA, and Protein
46:32
Result: Only the DNA Fraction Could Transform
47:15
Leven: Tetranucleotide Hypothesis
48:00
Chargaff Showed This Was Not the Case
48:48
Chargaff: DNA of Different Species Have Different Nucleotide Composition
49:02
Hershey and Chase: DNA is the Genetic Material
50:02
Incorporate Sulfur into Protein and Phosphorous into DNA
51:12
Results: Phosphorase Entered Bacteria and Progeny Phage, But no Sulfur
53:11
Rosalind Franklin’s “Photo 51” Showing the Diffraction Pattern of DNA
53:50
Watson and Crick: Double Helical Structure of DNA
54:57
Example 4
56:56
Discovery of the Genetic Material of the Cell
58:09
Kornberg: DNA Polymerase I
58:10
Three Postulated Methods of DNA Replication
59:22
Meselson and Stahl: DNA Replication is Semi-Conservative
1:00:21
How DNA Was Made Denser
1:00:52
Discovery of RNA
1:03:32
Ribosomal RNA
1:03:48
Transfer RNA
1:04:00
Messenger RNA
1:04:30
The Central Dogma of Molecular Biology
1:04:49
DNA and Replication
1:05:08
DNA and Transcription = RNA
1:05:26
RNA and Translation = Protein
1:05:41
Reverse Transcription
1:06:08
Cracking the Genetic Code
1:06:58
What is the Genetic Code?
1:07:04
Nirenberg Discovered the First DNA Triplet That Would Make an Amino Acid
1:07:16
Code Finished in 1966 and There Are 64 Possibilities or Triplet Repeats/ Codons
1:07:54
Degeneracy of the Code
1:08:53
II. Structure of Macromolecules
Structure of Proteins

49m 44s

Intro
0:00
Lesson Overview
0:10
Amino Acids
0:47
Structure
0:55
Acid Association Constant
1:55
Amino Acids Make Up Proteins
2:15
Table of 21 Amino Acid Found in Proteins
3:34
Ionization
5:55
Cation
6:08
Zwitterion
7:51
Anion
9:15
Example 1
10:53
Amino Acids
13:11
L Alpha Amino Acids
13:19
Only L Amino Acids Become Incorporated into Proteins
13:28
Example 2
13:46
Amino Acids
18:20
Non-Polar
18:41
Polar
18:58
Hydroxyl
19:52
Sulfhydryl
20:21
Glycoproteins
20:41
Pyrrolidine
21:30
Peptide (Amide) Bonds
22:18
Levels of Organization
23:35
Primary Structure
23:54
Secondary Structure
24:22
Tertiary Structure
24:58
Quaternary Structure
25:27
Primary Structure: Specific Amino Acid Sequence
25:54
Example 3
27:30
Levels of Organization
29:31
Secondary Structure: Local 3D
29:32
Example 4
30:37
Levels of Organization
32:59
Tertiary Structure: Total 3D Structure of Protein
33:00
Quaternary Structure: More Than One Subunit
34:14
Example 5
34:52
Protein Folding
37:04
Post-Translational Modifications
38:21
Can Alter a Protein After It Leaves the Ribosome
38:33
Regulate Activity, Localization and Interaction with Other Molecules
38:52
Common Types of PTM
39:08
Protein Classification
40:22
Ligand Binding, Enzyme, DNA or RNA Binding
40:36
All Other Functions
40:53
Some Functions: Contraction, Transport, Hormones, Storage
41:34
Enzymes as Biological Catalysts
41:58
Most Metabolic Processes Require Catalysts
42:00
Most Biological Catalysts Are Proteins
43:13
Enzymes Have Specificity of Reactants
43:33
Enzymes Have an Optimum pH and Temperature
44:31
Example 6
45:08
Structure of Nucleic Acids

1h 2m 10s

Intro
0:00
Lesson Overview
0:06
Nucleic Acids
0:26
Biopolymers Essential for All Known Forms of Life That Are Composed of Nucleotides
0:27
Nucleotides Are Composed of These
1:17
Nucleic Acids Are Bound Inside Cells
2:10
Nitrogen Bases
2:49
Purines
3:01
Adenine
3:10
Guanine
3:20
Pyrimidines
3:54
Cytosine
4:25
Thymine
4:33
Uracil
4:42
Pentoses
6:23
Ribose
6:45
2' Deoxyribose
6:59
Nucleotides
8:43
Nucleoside
8:56
Nucleotide
9:16
Example 1
10:23
Polynucleotide Chains
12:18
What RNA and DNA Are Composed of
12:37
Hydrogen Bonding in DNA Structure
13:55
Ribose and 2! Deoxyribose
14:14
DNA Grooves
14:28
Major Groove
14:46
Minor Groove
15:00
Example 2
15:20
Properties of DNA
24:15
Antiparallel Orientation
24:25
Phosphodiester Linkage
24:50
Phosphate and Hydroxyl Group
25:05
Purine Bases Always Pairs Pyramidine Bases
25:30
A, B, Z Forms
25:55
Major and Minor Grooves
26:24
Hydrogen Bonding and Hydrophobic Interactions Hold Strands Together
26:34
DNA Topology - Linking Number
27:14
Linking Number
27:31
Twist
27:57
Writhe
28:31
DNA Topology - Supercoiling
31:50
Example 3
33:16
III. Maintenance of the Genome
Genome Organization: Chromatin & Nucleosomes

57m 2s

Intro
0:00
Lesson Overview
0:09
Quick Glossary
0:24
DNA
0:29
Gene
0:34
Nucleosome
0:47
Chromatin
1:07
Chromosome
1:19
Genome
1:30
Genome Organization
1:38
Physically Cellular Differences
3:09
Eukaryotes
3:18
Prokaryotes, Viruses, Proteins, Small Molecules, Atoms
4:06
Genome Variance
4:27
Humans
4:52
Junk DNA
5:10
Genes Compose Less Than 40% of DNA
6:03
Chart
6:26
Example 1
8:32
Chromosome Variance - Size, Number, and Density
10:27
Chromosome
10:47
Graph of Human Chromosomes
10:58
Eukaryotic Cell Cycle
12:07
Requirements for Proper Chromosome Duplication and Segregation
13:07
Centromeres and Telomeres
13:28
Origins of Replication
13:38
Illustration: Chromosome
13:44
Chromosome Condensation
15:52
Naked DNA to Start
16:00
Beads on a String
16:13
Mitosis
16:52
Start with Two Different Chromosomes
17:18
Split Into Two Diploid Cells
17:26
Prophase
17:42
Prometaphase
17:52
Metaphase
19:10
Anaphase
19:27
Telophase
20:11
Cytokinesis
20:31
Cohesin and Condensis
21:06
Illustration: Cohesin and Condensis
21:19
Cohesin
21:38
Condensin
21:43
Illustration of What Happens
21:50
Cohesins
27:23
Loaded During Replication and Cleaved During Mitosis
27:30
Separase
27:36
Nucleosomes
27:59
Histone Core
28:50
Eight Histone Proteins
28:57
Octamer of Core Histones Picture
29:14
Chromosome Condensation via H1
30:59
Allows Transition to Compact DNA
31:09
When Not in Mitosis
31:37
Histones Decrease Available Binding Sites
32:38
Histone Tails
33:21
Histone Code
35:32
Epigenetic Code
35:56
Phosphorylation
36:45
Acetylation
36:57
Methylation
37:01
Ubiquitnation
37:04
Example 2
38:48
Nucleosome Assembly
41:22
Duplication of DNA Requires Duplication of Histones
41:50
Old Histones Are Recycled
42:00
Parental H3-H4 Tetramers Facilitate the Inheritance of Chromatin States
44:04
Example 3
46:00
Chromatin Remodeling
48:12
Example 4
53:28
DNA Replication

1h 9m 55s

Intro
0:00
Lesson Overview
0:06
Eukaryotic Cell Cycle
0:50
G1 Growth Phase
0:57
S Phase: DNA & Replication
1:09
G2 Growth Phase
1:28
Mitosis
1:36
Normal Human Cell Divides About Every 24 Hours
1:40
Eukaryotic DNA Replication
2:04
Watson and Crick
2:05
Specific Base Pairing
2:37
DNA Looked Like Tetrinucleotide
2:55
What DNA Looks Like Now
3:18
Eukaryotic DNA Replication - Initiation
3:44
Initiation of Replication
3:53
Primer Template Junction
4:25
Origin Recognition Complex
7:00
Complex of Proteins That Recognize the Proper DNA Sequence for Initiation of Replication
7:35
Prokaryotic Replication
7:56
Illustration
8:54
DNA Helicases (MCM 2-7)
11:53
Eukaryotic DNA Replication
14:36
Single-Stranded DNA Binding Proteins
14:59
Supercoils
16:30
Topoisomerases
17:35
Illustration with Helicase
19:05
Synthesis of the RNA Primer by DNA Polymerase Alpha
20:21
Subunit: Primase RNA Polymerase That Synthesizes the RNA Primer De Navo
20:38
Polymerase Alpha-DNA Polymerase
21:01
Illustration of Primase Function Catalyzed by DnaG in Prokaryotes
21:22
Recap
24:02
Eukaryotic DNA Replication - Leading Strand
25:02
Synthesized by DNA Polymerase Epsilon
25:08
Proof Reading
25:26
Processivity Increased by Association with PCNA
25:47
What is Processivity?
26:19
Illustration: Write It Out
27:03
The Lagging Strand/ Discontinuing Strand
30:52
Example 1
31:57
Eukaryotic DNA Replication - Lagging Strand
32:46
Discontinuous
32:55
DNA Polymerase Delta
33:15
Okazaki Fragments
33:36
Illustration
33:55
Eukaryotic DNA Replication - Okazaki Fragment Processing
38:26
Illustration
38:44
When Does Okazaki Fragments Happen
40:32
Okazaki Fragments Processing
40:41
Illustration with Okazaki Fragments Process Happening
41:13
Example 2
47:42
Example 3
49:20
Telomeres
56:01
Region of Repetitive Nucleotide Sequences
56:26
Telomeres Act as Chromosome Caps by Binding Proteins
57:42
Telomeres and the End Replication Problem
59:56
Need to Use a Primer
59:57
DNA Mutations & Repairs

1h 13m 8s

Intro
0:00
Lesson Overview
0:06
Damage vs. Mutation
0:40
DNA Damage-Alteration of the Chemical Structure of DNA
0:45
DNA Mutation-Permanent Change of the Nucleotide Sequence
1:01
Insertions or Deletions (INDELS)
1:22
Classes of DNA Mutations
1:50
Spontaneous Mutations
2:00
Induced Mutations
2:33
Spontaneous Mutations
3:21
Tautomerism
3:28
Depurination
4:09
Deamination
4:30
Slippage
5:44
Induced Mutations - Causes
6:17
Chemicals
6:24
Radiation
7:46
Example 1
8:30
DNA Mutations - Tobacco Smoke
9:59
Covalent Adduct Between DNA and Benzopyrene
10:02
Benzopyrene
10:20
DNA Mutations - UV Damage
12:16
Oxidative Damage from UVA
12:30
Thymidine Dimer
12:34
Example 2
13:33
DNA Mutations - Diseases
17:25
DNA Repair
18:28
Mismatch Repair
19:15
How to Recognize Which is the Error: Recognize Parental Strand
22:23
Example 3
26:54
DNA Repair
32:45
Damage Reversal
32:46
Base-Excision Repair (BER)
34:31
Example 4
36:09
DNA Repair
45:43
Nucleotide Excision Repair (NER)
45:48
Nucleotide Excision Repair (NER) - E.coli
47:51
Nucleotide Excision Repair (NER) - Eukaryotes
50:29
Global Genome NER
50:47
Transcription Coupled NER
51:01
Comparing MMR and NER
51:58
Translesion Synthesis (TLS)
54:40
Not Really a DNA Repair Process, More of a Damage Tolerance Mechanism
54:50
Allows Replication Past DNA Lesions by Polymerase Switching
55:20
Uses Low Fidelity Polymerases
56:27
Steps of TLS
57:47
DNA Repair
1:00:37
Recombinational Repair
1:00:54
Caused By Ionizing Radiation
1:00:59
Repaired By Three Mechanisms
1:01:16
Form Rarely But Catastrophic If Not Repaired
1:01:42
Non-homologous End Joining Does Not Require Homology To Repair the DSB
1:03:42
Alternative End Joining
1:05:07
Homologous Recombination
1:07:41
Example 5
1:09:37
Homologous Recombination & Site-Specific Recombination of DNA

1h 14m 27s

Intro
0:00
Lesson Overview
0:16
Homologous Recombination
0:49
Genetic Recombination in Which Nucleotide Sequences Are Exchanged Between Two Similar or Identical Molecules of DNA
0:57
Produces New Combinations of DNA Sequences During Meiosis
1:13
Used in Horizontal Gene Transfer
1:19
Non-Crossover Products
1:48
Repairs Double Strand Breaks During S/Gs
2:08
MRN Complex Binds to DNA
3:17
Prime Resection
3:30
Other Proteins Bind
3:40
Homology Searching and subsequent Strand Invasion by the Filament into DNA Duplex
3:59
Holliday Junction
4:47
DSBR and SDSA
5:44
Double-Strand Break Repair Pathway- Double Holliday Junction Model
6:02
DSBR Pathway is Unique
6:11
Converted Into Recombination Products by Endonucleases
6:24
Crossover
6:39
Example 1
7:01
Example 2
8:48
Double-Strand Break Repair Pathway- Synthesis Dependent Strand Annealing
32:02
Homologous Recombination via the SDSA Pathway
32:20
Results in Non-Crossover Products
32:26
Holliday Junction is Resolved via Branch Migration
32:43
Example 3
34:01
Homologous Recombination - Single Strand Annealing
42:36
SSA Pathway of HR Repairs Double-Strand Breaks Between Two Repeat Sequences
42:37
Does Not Require a Separate Similar or Identical Molecule of DNA
43:04
Only Requires a Single DNA Duplex
43:25
Considered Mutagenic Since It Results in Large Deletions of DNA
43:42
Coated with RPA Protein
43:58
Rad52 Binds Each of the Repeated Sequences
44:28
Leftover Non-Homologous Flaps Are Cut Away
44:37
New DNA Synthesis Fills in Any Gaps
44:46
DNA Between the Repeats is Always Lost
44:55
Example 4
45:07
Homologous Recombination - Break Induced Replication
51:25
BIR Pathway Repairs DSBs Encountered at Replication Forks
51:34
Exact Mechanisms of the BIR Pathway Remain Unclear
51:49
The BIR Pathway Can Also Help to Maintain the Length of Telomeres
52:09
Meiotic Recombination
52:24
Homologous Recombination is Required for Proper Chromosome Alignment and Segregation
52:25
Double HJs are Always Resolved as Crossovers
52:42
Illustration
52:51
Spo11 Makes a Targeted DSB at Recombination Hotspots
56:30
Resection by MRN Complex
57:01
Rad51 and Dmc1 Coat ssDNA and Promote Strand Invasion and Holliday Junction Formation
57:04
Holliday Junction Migration Can Result in Heteroduplex DNA Containing One or More Mismatches
57:22
Gene Conversion May Result in Non-Mendelian Segregation
57:36
Double-Strand Break Repair in Prokaryotes - RecBCD Pathway
58:04
RecBCD Binds to and Unwinds a Double Stranded DNA
58:32
Two Tail Results Anneal to Produce a Second ssDNA Loop
58:55
Chi Hotspot Sequence
59:40
Unwind Further to Produce Long 3 Prime with Chi Sequence
59:54
RecBCD Disassemble
1:00:23
RecA Promotes Strand Invasion - Homologous Duplex
1:00:36
Holliday Junction
1:00:50
Comparison of Prokaryotic and Eukaryotic Recombination
1:01:49
Site-Specific Recombination
1:02:41
Conservative Site-Specific Recombination
1:03:10
Transposition
1:03:46
Transposons
1:04:12
Transposases Cleave Both Ends of the Transposon in Original Site and Catalyze Integration Into a Random Target Site
1:04:21
Cut and Paste
1:04:37
Copy and Paste
1:05:36
More Than 40% of Entire Human Genome is Composed of Repeated Sequences
1:06:15
Example 5
1:07:14
IV. Gene Expression
Transcription

1h 19m 28s

Intro
0:00
Lesson Overview
0:07
Eukaryotic Transcription
0:27
Process of Making RNA from DNA
0:33
First Step of Gene Expression
0:50
Three Step Process
1:06
Illustration of Transcription Bubble
1:17
Transcription Starting Site is +1
5:15
Transcription Unit Extends From the Promoter to the Termination Region
5:40
Example 1
6:03
Eukaryotic Transcription: Initiation
14:27
RNA Polymerase II Binds to TATA Box to Initiate RNA Synthesis
14:34
TATA Binding Protein Binds the TATA Box
14:50
TBP Associated Factors Bind
15:01
General Transcription Factors
15:22
Initiation Complex
15:30
Example 2
15:44
Eukaryotic Transcription
17:59
Elongation
18:07
FACT (Protein Dimer)
18:24
Eukaryotic Transcription: Termination
19:36
Polyadenylation is Linked to Termination
19:42
Poly-A Signals Near the End of the pre-mRNA Recruit to Bind and Cleave mRNA
20:00
Mature mRNA
20:27
Dissociate from Template DNA Strand
21:13
Example 3
21:53
Eukaryotic Transcription
25:49
RNA Polymerase I Transcribes a Single Gene That Encodes a Long rRNA Precursor
26:14
RNA Polymerase III Synthesizes tRNA, 5S rRNA, and Other Small ncRNA
29:11
Prokaryotic Transcription
32:04
Only One Multi-Subunit RNA Polymerase
32:38
Transcription and Translation Occurs Simultaneously
33:41
Prokaryotic Transcription - Initiation
38:18
Initial Binding Site
38:33
Pribnox Box
38:42
Prokaryotic Transcription - Elongation
39:15
Unwind Helix and Expand Replication Bubble
39:19
Synthesizes DNA
39:35
Sigma 70 Subunit is Released
39:50
Elongation Continues Until a Termination Sequence is Reached
40:08
Termination - Prokaryotes
40:17
Example 4
40:30
Example 5
43:58
Post-Transcriptional Modifications
47:15
Can Post Transcribe your rRNA, tRNA, mRNA
47:28
One Thing In Common
47:38
RNA Processing
47:51
Ribosomal RNA
47:52
Transfer RNA
49:08
Messenger RNA
50:41
RNA Processing - Capping
52:09
When Does Capping Occur
52:20
First RNA Processing Event
52:30
RNA Processing - Splicing
53:00
Process of Removing Introns and Rejoining Exons
53:01
Form Small Nuclear Ribonucleoproteins
53:46
Example 6
57:48
Alternative Splicing
1:00:06
Regulatory Gene Expression Process
1:00:27
Example
1:00:42
Example 7
1:02:53
Example 8
1:09:36
RNA Editing
1:11:06
Guide RNAs
1:11:25
Deamination
1:11:52
Example 9
1:13:50
Translation

1h 15m 1s

Intro
0:00
Lesson Overview
0:06
Linking Transcription to Translation
0:39
Making RNA from DNA
0:40
Occurs in Nucleus
0:59
Process of Synthesizing a Polypeptide from an mRNA Transcript
1:09
Codon
1:43
Overview of Translation
4:54
Ribosome Binding to an mRNA Searching for a START Codon
5:02
Charged tRNAs will Base Pair to mRNA via the Anticodon and Codon
5:37
Amino Acids Transferred and Linked to Peptide Bond
6:08
Spent tRNAs are Released
6:31
Process Continues Until a STOP Codon is Reached
6:55
Ribosome and Ribosomal Subunits
7:55
What Are Ribosomes?
8:03
Prokaryotes
8:42
Eukaryotes
10:06
Aminoacyl Site, Peptidyl tRNA Site, Empty Site
10:51
Major Steps of Translation
11:35
Charing of tRNA
11:37
Initiation
12:48
Elongation
13:09
Termination
13:47
“Charging” of tRNA
14:35
Aminoacyl-tRNA Synthetase
14:36
Class I
16:40
Class II
16:52
Important About This Reaction: It Is Highly Specific
17:10
ATP Energy is Required
18:42
Translation Initiation - Prokaryotes
18:56
Initiation Factor 3 Binds at the E-Site
19:09
Initiation Factor 1 Binds at the A-Site
20:15
Initiation Factor 2 and GTP Binds IF1
20:50
30S Subunit Associates with mRNA
21:05
N-Formyl-met-tRNA
22:34
Complete 30S Initiation Complex
23:49
IF3 Released and 50S Subunit Binds
24:07
IF1 and IF2 Released Yielding a Complete 70S Initiation Complex
24:24
Deformylase Removes Formyl Group
24:45
Example 1
25:11
Translation Initiation - Eukaryotes
29:35
Small Subunit is Already Associated with the Initiation tRNA
29:47
Formation of 43S Pre-Initiation Complex
30:02
Circularization of mRNA by eIF4
31:05
48S Pre-Initiation Complex
35:47
Example 2
38:57
Translation - Elongation
44:00
Charging, Initiation, Elongation, Termination All Happens Once
44:14
Incoming Charged tRNA Binds the Complementary Codon
44:31
Peptide Bond Formation
45:06
Translocation Occurs
46:05
tRNA Released
46:51
Example 3
47:11
Translation - Termination
55:26
Release Factors Terminate Translation When Ribosomes Come to a Stop Codon
55:38
Release Factors Are Proteins, Not tRNAs, and Do Not Carry an Amino Acid
55:50
Class I Release Factors
55:16
Class II Release Factors
57:03
Example 4
57:40
Review of Translation
1:01:15
Consequences of Altering the Genetic Code
1:02:40
Silent Mutations
1:03:37
Missense Mutations
1:04:24
Nonsense Mutations
1:05:28
Genetic Code
1:06:40
Consequences of Altering the Genetic Code
1:07:43
Frameshift Mutations
1:07:55
Sequence Example
1:08:07
V. Gene Regulation
Gene Regulation in Prokaryotes

45m 40s

Intro
0:00
Lesson Overview
0:08
Gene Regulation
0:50
Transcriptional Regulation
1:01
Regulatory Proteins Control Gene Expression
1:18
Bacterial Operons-Lac
1:58
Operon
2:02
Lactose Operon in E. Coli
2:31
Example 1
3:33
Lac Operon Genes
7:19
LacZ
7:25
LacY
7:40
LacA
7:55
LacI
8:10
Example 2
8:58
Bacterial Operons-Trp
17:47
Purpose is to Produce Trptophan
17:58
Regulated at Initiation Step of Transcription
18:04
Five Genes
18:07
Derepressible
18:11
Example 3
18:32
Bacteriophage Lambda
28:11
Virus That Infects E. Coli
28:24
Temperate Lifecycle
28:33
Example 4
30:34
Regulation of Translation
39:42
Binding of RNA by Proteins Near the Ribosome- Binding Site of the RNA
39:53
Intramolecular Base Pairing of mRNA to Hide Ribosome Binding Site
40:14
Post-transcriptional Regulation of rRNA
40:35
Example 5
40:08
Gene Regulation in Eukaryotes

1h 6m 6s

Intro
0:00
Lesson Overview
0:06
Eukaryotic Transcriptional Regulations
0:18
Transcription Factors
0:25
Insulator Protein
0:55
Example 1
1:44
Locus Control Regions
4:00
Illustration
4:06
Long Range Regulatory Elements That Enhance Expressions of Linked Genes
5:40
Allows Order Transcription of Downstream Genes
6:07
(Ligand) Signal Transduction
8:12
Occurs When an Extracellular Signaling Molecule Activates a Specific Receptor Located on the Cell
8:19
Examples
9:10
N F Kappa B
10:01
Dimeric Protein That Controls Transcription
10:02
Ligands
10:29
Example 2
11:04
JAK/ STAT Pathway
13:19
Turned on by a Cytokine
13:23
What is JAK
13:34
What is STAT
13:58
Illustration
14:38
Example 3
17:00
Seven-Spanner Receptors
20:49
Illustration: What Is It
21:01
Ligand Binding That Is Activating a Process
21:46
How This Happens
22:17
Example 4
24:23
Nuclear Receptor Proteins (NRPs)
28:45
Sense Steroid and Thyroid Hormones
28:56
Steroid Hormones Bind Cytoplasmic NRP Homodimer
29:10
Hormone Binds NRP Heterodimers Already Present in the Nucleus
30:11
Unbound Heterodimeric NRPs Can Cause Deacetylation of Lysines of Histone Tails
30:54
RNA Interference
32:01
RNA Induced Silencing Complex (RISC)
32:39
RNAi
33:54
RISC Pathway
34:34
Activated RISC Complex
34:41
Process
34:55
Example
39:27
Translational Regulation
41:17
Global Regulation
41:37
Competitive Binding of 5 Prime CAP of mRNA
42:34
Translation-Dependent Regulation
44:56
Nonsense Mediated mRNA Decay
45:23
Nonstop Mediated mRNA Decay
46:17
Epigenetics
48:53
Inherited Patterns of Gene Expression Resulting from Chromatin Alteration
49:15
Three Ways to Happen
50:17
DNA Sequence Does Not Act Alone in Passing Genetic Information to Future Generations
50:30
DNA Methylation
50:57
Occurs at CpG Sites Via DNA Methyltransferase Enzymes
50:58
CpG Islands Are Regions with a High Frequency of CpG Sites
52:49
Methylation of Multiple CpG Sites Silence Nearby Gene Transcription
53:32
DNA Methylation
53:46
Pattern Can Be Passed to Daughter Cells
53:47
Prevents SP1 Transcription Factors From Binding to CpG Island
54:02
MECP2
54:10
Example 5
55:27
Nucleosomes
56:48
Histone Core
57:00
Histone Protein
57:03
Chromosome Condensation Via J1
57:32
Linker Histone H1
57:33
Compact DNA
57:37
Histone Code
57:54
Post-translational Modifications of N-Terminal Histone Tails is Part of the Epigenetic Code
57:55
Phosphorylation, Acetylation, Methylation, Ubiquitination
58:09
Example 6
58:52
Nucleosome Assembly
59:13
Duplication of DNA Requires Duplication of Histones by New Protein Synthesis
59:14
Old Histones are Recycled
59:24
Parental H3-H4 Tetramers
58:57
Example 7
1:00:05
Chromatin Remodeling
1:01:48
Example 8
1:02:36
Transcriptionally Repressed State
1:02:45
Acetylation of Histones
1:02:54
Polycomb Repressors
1:03:19
PRC2 Protein Complex
1:03:38
PRC1 Protein Complex
1:04:02
MLL Protein Complex
1:04:09
VI. Biotechnology and Applications to Medicine
Basic Molecular Biology Research Techniques

1h 8m 41s

Intro
0:00
Lesson Overview
0:10
Gel Electraophoresis
0:31
What is Gel Electraophoresis
0:33
Nucleic Acids
0:50
Gel Matrix
1:41
Topology
2:18
Example 1
2:50
Restriction Endonucleases
8:07
Produced by Bacteria
8:08
Sequence Specific DNA Binding Proteins
8:36
Blunt or Overhanging Sticky Ends
9:04
Length Determines Approximate Cleavage Frequency
10:30
Cloning
11:18
What is Cloning
11:29
How It Works
12:12
Ampicillin Example
12:55
Example 2
13:19
Creating a Genomic DNA Library
19:33
Library Prep
19:35
DNA is Cut to Appropriate Sizes and Ligated Into Vector
20:04
Cloning
20:11
Transform Bacteria
20:19
Total Collection Represents the Whole Genome
20:29
Polymerase Chain Reaction
20:54
Molecular Biology Technique to Amplify a Small Number of DNA Molecules to Millions of Copies
21:04
Automated Process Now
21:22
Taq Polymerase and Thermocycler
21:38
Molecular Requirements
22:32
Steps of PCR
23:40
Example 3
24:42
Example 4
34:45
Southern Blot
35:25
Detect DNA
35:44
How It Works
35:50
Western Blot
37:13
Detects Proteins of Interest
37:14
How It Works
37:20
Northern Blot
39:08
Detects an RNA Sequence of Interest
39:09
How It Works
39:21
Illustration Sample
40:12
Complementary DNA (cDNA) Synthesis
41:18
Complementary Synthesis
41:19
Isolate mRNA from Total RNA
41:59
Quantitative PCR (qPCR)
44:14
Technique for Quantifying the Amount of cDNA and mRNA Transcriptions
44:29
Measure of Gene Expression
44:56
Illustration of Read Out of qPCR Machine
45:23
Analysis of the Transcriptome-Micrarrays
46:15
Collection of All Transcripts in the Cell
46:16
Microarrays
46:35
Each Spot Represents a Gene
47:20
RNA Sequencing
49:25
DNA Sequencing
50:08
Sanger Sequencing
50:21
Dideoxynucleotides
50:31
Primer Annealed to a DNA Region of Interest
51:50
Additional Presence of a Small Proportion of a ddNTPs
52:18
Example
52:49
DNA Sequencing Gel
53:13
Four Different Reactions are Performed
53:26
Each Reaction is Run in a Lane of a Denaturing Polyacrylamide Gel
53:34
Example 5
53:54
High Throughput DNA Sequencing
57:51
Dideoxy Sequencing Reactions Are Carried Out in Large Batches
57:52
Sequencing Reactions are Carried Out All Together in a Single Reaction
58:26
Molecules Separated Based on Size
59:19
DNA Molecules Cross a Laser Light
59:30
Assembling the Sequences
1:00:38
Genomes is Sequenced with 5-10x Coverage
1:00:39
Compare Genomes
1:01:47
Entered Into Database and the Rest is Computational
1:02:02
Overlapping Sequences are Ordered Into Contiguous Sequences
1:02:17
Example 6
1:03:25
Example 7
1:05:27
VII. Ethics of Modern Science
Genome Editing, Synthetic Biology, & the Ethics of Modern Science

45m 6s

Intro
0:00
Lesson Overview
0:47
Genome Editing
1:37
What is Genome Editing
1:43
How It Works
2:03
Four Families of Engineered Nucleases in Use
2:25
Example 1
3:03
Gene Therapy
9:37
Delivery of Nucleic Acids Into a Patient’s Cells a Treatment for Disease
9:38
Timeline of Events
10:30
Example 2
11:03
Gene Therapy
12:37
Ethical Questions
12:38
Genetic Engineering
12:42
Gene Doping
13:10
Synthetic Biology
13:44
Design and Manufacture of Biological Components That Do Not Exist in Nature
13:53
First Synthetic Cell Example
14:12
Ethical Questions
16:16
Stem Cell Biology
18:01
Use Stem Cells to Treat or Prevent Diseases
18:12
Treatment Uses
19:56
Ethical Questions
20:33
Selected Topic of Ethical Debate
21:30
Research Ethics
28:02
Application of Fundamental Ethical Principles
28:07
Examples
28:20
Declaration of Helsinki
28:33
Basic Principles of the Declaration of Helsinki
28:57
Utmost Importance: Respect for the Patient
29:04
Researcher’s Duty is Solely to the Patient or Volunteer
29:32
Incompetent Research Participant
30:09
Right Vs Wrong
30:29
Ethics
32:40
Dolly the Sheep
32:46
Ethical Questions
33:59
Rational Reasoning and Justification
35:08
Example 3
35:17
Example 4
38:00
Questions to Ponder
39:35
How to Answer
40:52
Do Your Own Research
41:00
Difficult for People Outside the Scientific Community
41:42
Many People Disagree Because They Do Not Understand
42:32
Media Cannot Be Expected to Understand Published Scientific Data on Their Own
42:43
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Lecture Comments (2)

1 answer

Last reply by: Professor Michael Philips
Wed Mar 23, 2016 11:23 AM

Post by Jinhai Zhang on March 8, 2016

Professor:
Is Homologous recombination is supposed to happen in prophase-I of meiosis?

Homologous Recombination & Site-Specific Recombination of DNA

    Long, 5 examples, 5 practice questions

  • Homologous recombination is a process that uses complementarity between homologous molecules to repair double strand DNA damage.
  • Homologous recombination that occurs during DNA repair tends to result in non-crossover products.
  • In eukaryotes, double strand-break repair can be resolved by the DSBR or SDSA pathways.
  • Homologous recombination via the SDSA pathway occurs in cells that divide through mitosis and meiosis and most often results in non-crossover products
  • In prokaryotes, double strand-break repair is often resolved via the RecBCD pathway.

Homologous Recombination & Site-Specific Recombination of DNA

True/False: Homologous recombination that occurs during DNA repair tends to result in non-crossover products
  • True
  • False
Which of the following pathways utilizes the double Holliday junction to resolve crossovers?
  • DSBR
  • SDSA
  • SSA
  • BIR
What protein complex is involved in double strand break repair in prokaryotes?
  • RecA
  • RecBCD
  • MRX
  • Rad51
More than 40% of the entire human genome is composed of repeated sequences. What is likely responsible for the presence of a large portion of those sequences?
  • Transposons
  • DNA replication
  • Meosis
  • Mitosis
HR repairs double strand breaks using long homologous sequences as templates for synthesis during what phase of the cell cycle?
  • G0
  • G1
  • S/G2
  • Mitosis

*These practice questions are only helpful when you work on them offline on a piece of paper and then use the solution steps function to check your answer.

Answer

Homologous Recombination & Site-Specific Recombination of DNA

Lecture Slides are screen-captured images of important points in the lecture. Students can download and print out these lecture slide images to do practice problems as well as take notes while watching the lecture.

  1. Intro
    • Lesson Overview
      • Homologous Recombination
      • Double-Strand Break Repair Pathway- Double Holliday Junction Model
      • Example 1
        • Example 2
          • Double-Strand Break Repair Pathway- Synthesis Dependent Strand Annealing
          • Example 3
            • Homologous Recombination - Single Strand Annealing
            • Example 4
              • Homologous Recombination - Break Induced Replication
              • Meiotic Recombination
              • Double-Strand Break Repair in Prokaryotes - RecBCD Pathway
              • Comparison of Prokaryotic and Eukaryotic Recombination
                • Site-Specific Recombination
                • Transposons
                • Example 5
                  • Intro 0:00
                  • Lesson Overview 0:16
                  • Homologous Recombination 0:49
                    • Genetic Recombination in Which Nucleotide Sequences Are Exchanged Between Two Similar or Identical Molecules of DNA
                    • Produces New Combinations of DNA Sequences During Meiosis
                    • Used in Horizontal Gene Transfer
                    • Non-Crossover Products
                    • Repairs Double Strand Breaks During S/Gs
                    • MRN Complex Binds to DNA
                    • Prime Resection
                    • Other Proteins Bind
                    • Homology Searching and subsequent Strand Invasion by the Filament into DNA Duplex
                    • Holliday Junction
                    • DSBR and SDSA
                  • Double-Strand Break Repair Pathway- Double Holliday Junction Model 6:02
                    • DSBR Pathway is Unique
                    • Converted Into Recombination Products by Endonucleases
                    • Crossover
                  • Example 1 7:01
                  • Example 2 8:48
                  • Double-Strand Break Repair Pathway- Synthesis Dependent Strand Annealing 32:02
                    • Homologous Recombination via the SDSA Pathway
                    • Results in Non-Crossover Products
                    • Holliday Junction is Resolved via Branch Migration
                  • Example 3 34:01
                  • Homologous Recombination - Single Strand Annealing 42:36
                    • SSA Pathway of HR Repairs Double-Strand Breaks Between Two Repeat Sequences
                    • Does Not Require a Separate Similar or Identical Molecule of DNA
                    • Only Requires a Single DNA Duplex
                    • Considered Mutagenic Since It Results in Large Deletions of DNA
                    • Coated with RPA Protein
                    • Rad52 Binds Each of the Repeated Sequences
                    • Leftover Non-Homologous Flaps Are Cut Away
                    • New DNA Synthesis Fills in Any Gaps
                    • DNA Between the Repeats is Always Lost
                  • Example 4 45:07
                  • Homologous Recombination - Break Induced Replication 51:25
                    • BIR Pathway Repairs DSBs Encountered at Replication Forks
                    • Exact Mechanisms of the BIR Pathway Remain Unclear
                    • The BIR Pathway Can Also Help to Maintain the Length of Telomeres
                  • Meiotic Recombination 52:24
                    • Homologous Recombination is Required for Proper Chromosome Alignment and Segregation
                    • Double HJs are Always Resolved as Crossovers
                    • Illustration
                    • Spo11 Makes a Targeted DSB at Recombination Hotspots
                    • Resection by MRN Complex
                    • Rad51 and Dmc1 Coat ssDNA and Promote Strand Invasion and Holliday Junction Formation
                    • Holliday Junction Migration Can Result in Heteroduplex DNA Containing One or More Mismatches
                    • Gene Conversion May Result in Non-Mendelian Segregation
                  • Double-Strand Break Repair in Prokaryotes - RecBCD Pathway 58:04
                    • RecBCD Binds to and Unwinds a Double Stranded DNA
                    • Two Tail Results Anneal to Produce a Second ssDNA Loop
                    • Chi Hotspot Sequence
                    • Unwind Further to Produce Long 3 Prime with Chi Sequence
                    • RecBCD Disassemble
                    • RecA Promotes Strand Invasion - Homologous Duplex
                    • Holliday Junction
                  • Comparison of Prokaryotic and Eukaryotic Recombination 1:01:49
                  • Site-Specific Recombination 1:02:41
                    • Conservative Site-Specific Recombination
                    • Transposition
                  • Transposons 1:04:12
                    • Transposases Cleave Both Ends of the Transposon in Original Site and Catalyze Integration Into a Random Target Site
                    • Cut and Paste
                    • Copy and Paste
                    • More Than 40% of Entire Human Genome is Composed of Repeated Sequences
                  • Example 5 1:07:14

                  Transcription: Homologous Recombination & Site-Specific Recombination of DNA

                  Hi, and welcome back to www.educator.com.0000

                  Today, we are going to talk about homologous recombination and site-specific recombination of DNA.0002

                  This is linking back on the previous unit where we introduced the concept of homologous recombination.0007

                  Now, we are really going to get into the details.0013

                  As an overview, we are going to talk about all things, homologous recombination.0017

                  Double Holliday junctions, resolution vs. the synthesis dependent strand annealing, which we introduced last time.0023

                  And then, we are also going to talk about single strand annealing, break induced replication, meiotic recombination,0032

                  as well as double strand break repair in prokaryotes.0038

                  Finally, we will have a short overview on site-specific recombination.0042

                  Homologous recombination, once again remember, we can call this HR, is genetic recombination which the nucleotide sequences are exchanged0051

                  between two similar which are called homologous or identical molecules of DNA.0061

                  Two similar molecules of DNA are called homologous DNA.0068

                  This will produce new combinations of DNA sequences during meiosis.0073

                  HR is often used in horizontal gene transfer to exchange genetic material between different strains and species of bacteria and viruses.0078

                  That is called horizontal gene transfer, it is the passing of DNA from one bacteria to the other bacteria.0088

                  As opposed to the passage through the new generations of proliferation.0098

                  Homologous recombination that occurs during DNA repair can result in what is called a non-crossover product.0107

                  Meaning, it is fully repaired back to normal as if nothing had happened.0115

                  We are also going to talk about forms of homologous recombination where we get what are called crossover products.0121

                  Homologous recombination, reminders from last unit.0130

                  It repairs double strand breaks during S phase or G2 phase using long homologous sequences, as templates for synthesis.0137

                  If we look here, the red and blue chromosomes are called homologs or homologous chromosomes.0145

                  They are not the exact same sequence but they are very similar.0157

                  The process of homologous recombination encounters several of these things that we are going to talk about.0164

                  Generally, we start with resection, we then go to strand invasion, followed by Holliday junction formation.0172

                  And then, either double strand break repair or synthesis depended strand annealing.0183

                  After a double strand break occurs, the MRN complex will bind to DNA on either side of that break.0199

                  We then have 5 prime to 3 prime resection which allows us to have a 3 prime overhang.0210

                  And then, we have proteins coming in, binding these 3 prime overhangs, specifically RPN RAD51, we are talking about eukaryotic right now.0218

                  This forms a nuclear protein filament.0228

                  What that means is it is a filament of nucleic acid, our DNA and proteins, our RPA, RAD51, and others.0231

                  We then go through what is called homology searching.0239

                  We are looking for a piece of DNA that has roughly the same sequent, and then,0242

                  we go through a process called strand invasion.0248

                  That nuclear protein filament invades the other homologous chromosome.0251

                  It goes into that DNA duplex.0258

                  If we are talking about being in mitosis, you would be invading a sister chromatid.0261

                  Those are identical chromosomes, that is the best time that you can do this.0268

                  Or if we are in meiosis, you are using homologous chromosome.0271

                  It is not the exact sequence but basically the same, it is very similar.0274

                  When you go to strand invasion, you form what is called a displacement loop or a D loop.0279

                  DNA polymerase, once we got through strand invasion,0289

                  DNA polymerase will extend the end of the invading 3 prime strand via normal DNA synthesis.0292

                  It is using the new DNA from that homologous chromosome as a template molecule.0300

                  This whole strand invasion and initiation of synthesis will form a cross shaped structure which we call a Holliday junction.0308

                  DNA synthesis will continue and then that is continuing on the invading strand,0323

                  the original piece of DNA not the homologous new piece, effectively actively restoring the strand on the homologous chromosome0330

                  that was just placed during the strand invasion.0338

                  The double strand break repair is then completed by one of the two pathways.0342

                  We either have the double strand break repair which we can also call the double Holliday junction model.0346

                  Or SDSA which is the synthesis dependent strand annealing model.0354

                  Let us talk about the first one being the double Holliday junction model.0364

                  The double strand break repair pathway is unique, and that the second 3 prime overhang0372

                  which is not involved in strand invasion, will also form a Holliday junction with the homologous chromosome.0377

                  Now, we have what is called the double Holliday junction0384

                  that will then get converted into recombination products by endonucleases.0386

                  We have cleavage events occurring here.0393

                  The double strand break pathway often results in crossovers.0396

                  This is the model of how a crossover of homologous recombination occurs during meiosis.0406

                  We will talk specifically about crossing over during meiosis in a few slides, after we have explained these models.0414

                  For example 1, back to this picture again.0423

                  What are the combinations in the Holliday junction pathway in that model, results in chromosomal crossover or not,0427

                  is determined by how the double Holliday junction gets resolved.0436

                  Chromosomal crossover will occur if one Holliday junction, let us call them the crossing strand and the other on the non crossing strand.0442

                  What that means is, if we look down here, this is the double strand break repair side.0449

                  If we cut at the purple here and the purple here, we will get a non-crossover product.0456

                  If we cut at the orange arrows here and the orange arrows here, we will also not get any type of crossover.0475

                  If we cut at the orange arrow at this first Holliday junction and the purple arrow at the second one, we will get a crossover, and vice versa.0488

                  Same thing, if we cut at the purple arrow here and the orange arrow here, we will get a crossover.0500

                  If we cut at both purples or both oranges, no crossover.0508

                  If we cut at one orange, one purple, we will have a cross over.0514

                  Having a crossover is more common than having a non-crossover product.0520

                  Let us draw this out, so we can see what is going on.0529

                  We will use different colors so we can see the different chromosomes.0536

                  First things first, we have a break in the chromosome.0546

                  What I’m going to do to keep track of what our DNA has is, I'm going to label 3 different low side, that is just a piece of region of DNA.0563

                  I’m going to say that this is region A, locus A, locus B, and locus D.0574

                  The homologous chromosome is going to be unbroken.0586

                  We are going to label these with a, so as to differentiate them, all lowercase.0598

                  First things first, after that break, we go through resection.0608

                  We are not going to mess with the homologous chromosome because there is nothing wrong with that one.0633

                  What we have had here is resection 5 prime the 3 prime, that leaves us with some overhangs, it is a 3 prime overhangs.0686

                  We can go through strand invasion.0702

                  Let us continue on down here.0724

                  What we have, we have our 3 prime, we have our 5 prime, we have both a that have been resected.0728

                  We have b and d, we have a.0743

                  What we are going to do, let us draw out this one.0755

                  What happens here is that, this strand starts to invade and pop up, it pops up this top 5 prime strand up here.0780

                  It moves this up.0804

                  This right here, this dotted line, that is all new synthesis that is going to be occurring.0814

                  This is the strand invasion process happening.0822

                  We are going to start synthesizing this way.0826

                  What we are going to be doing, since we are synthesizing using this bottom strand as the template,0834

                  remember the top strand up here, it was BB.0840

                  What we are doing this is, since we are using this is a template, this is actually being made as a b, that locus.0844

                  It is matching the homologous chromosome not the original chromosome.0852

                  For example, let us just say what this B vs. b could be.0858

                  Let us say that B that we are seeing, let us say that is an AT base pair and let us say that b is a GC base pair.0865

                  That might make it more easily understandable.0876

                  There is the start of our strand invasion.0882

                  What we can proceed with is the full double Holliday model, double strand break repair.0887

                  This is what we are going to have.0910

                  What is happening here is that, this is being synthesized using this piece of red as the template.1010

                  What we can do is if we call the crossing points, this is where we are going to have to resect in.1028

                  If we say that this, we are going to cut here and let us do it in different color so we do not get confused.1042

                  We can resolve it by cleaving there, in those places.1059

                  Here is what we can do.1081

                  What we are going to do is go through resolution.1095

                  If we resolve this, we have separated it.1104

                  If we resolve it at both green and green, or purple and purple, this is what we will get.1109

                  This is at both green or both purple arrows.1198

                  If we do this at one green, one purple.1219

                  Let us say the first one green, the second one purple, or vice versa.1232

                  The first one purple, the second one green, this is what we will get.1236

                  This right here are considered non-crossover.1288

                  These down here are considered crossover.1299

                  Why, first of all, what we are looking at is a, locus A with respect to D.1306

                  The original molecule, A is found with D, a found with d.1316

                  That is what we consider as normal or wild type.1325

                  After all of the finishing of this, the resolution, we see that A is still on the same chromosome as A with D and a with d.1329

                  We are not looking at the middle right now.1344

                  That is, we do not have a crossover.1348

                  Down here, we see that A is now with d.1350

                  A is with D, A with respect to D, there has been a crossover.1356

                  Before I mention something that you might have already looked at,1370

                  right here, we are going to keep this for one more second.1379

                  As you can see, we have B and b.1389

                  First, how do I get these, how do I get that from this.1392

                  Let us show you, 3 prime, A, B, D, 5 prime.1401

                  Let us show you how we get this.1470

                  The easiest way to look at is what if I cut it in both purples?1473

                  What that is basically saying is, what I’m going to do is, if I cut right here,1478

                  it is basically likely erasing, connecting these right here, connecting that right there.1484

                  You erase this, connect it right there, connect it right there.1492

                  What we are seeing is this being A, B, D, A, b, D.1496

                  That is what is happening over there.1511

                  What is happening down here, the bottom strand is not touched though, just like the top strand is not touched.1513

                  We have a, b, d.1519

                  What do we have when we follow it?1521

                  We can just follow it, a follow up, b follow up that, d.1524

                  That is the same thing we get over there.1533

                  What if we cut above the greens, what is that look like?1563

                  What we can think of is we are not touching the inner strands at all.1572

                  What we are doing is, when we make the cuts here, we are cutting both strands.1577

                  Cut there, cut there, cut there, cut there.1583

                  We are just making a big X.1588

                  This down to the bottom and up to the top, this one, up to the top, all the way over, down to the bottom.1590

                  All you have to do is follow along.1603

                  What we can see is we have A, A down to b up to D.1606

                  The other strand is A, b, no movement, D.1618

                  Over here, a, b, d, no change.1629

                  Up here, a, B, down to d.1635

                  A with respect to D, we still have no change.1643

                  You might get a little difference between the locus in the middle at B,1647

                  but there is no change A with respect to D, because A is still with D, a still with d.1650

                  When we cross, when we do one of each, we are going to see something different.1659

                  Let us say we cut here and here.1688

                  What is that going to look like, once again, it is just like you erased, you complete those.1703

                  One here, we have cut here, you just make the X.1711

                  What is it look like?1722

                  Top strand A, B, we will follow it in black.1724

                  A, B, down to d.1731

                  We can already see that A with respect to D is different.1737

                  This next one, we have A, b, d, so A with respect to d crossover.1742

                  Down here, a, b, up to D, so a with respect to D crossover.1753

                  Down here, a, b, all the way up to D.1764

                  Sorry, the previous strand, this is a, b, still D, but it is the bottom D.1772

                  On both strands, a with respect to d has had a crossover.1779

                  We are looking down here.1783

                  The last thing that I pointed out and I said let us take a look at this.1789

                  With each of these, we can have what is called a hetero duplex.1792

                  That is, when we have, let us say this strand is B, maybe it is a T up here.1798

                  This is a C, just for example.1807

                  That has to be repaired.1811

                  If the hetero duplex is repaired by mismatch repair, back to the original,1813

                  let us say in this case, it is supposed to be B, B, then everything is okay.1820

                  See down here, it is B, B, everything is okay.1829

                  B, B, up here, everything is okay.1834

                  But down here, what if I say that this is repaired the opposite way.1835

                  Let us say over here, this is repaired to b, b.1842

                  That is what we call gene conversion because it was supposed to be B, it should have been AT, but now it is a GC bond.1849

                  That is going to lead to what we call LOH or loss of heterozygocity.1862

                  Remember, right here, originally, it was an AT pair and this chromosome was a GC pair,1868

                  that you have heterozygocity at the B allele.1879

                  But if they are both repaired back to an AT or a GC, you lose the hetrozygocity,1883

                  you are homozygous at that allele and that could be problematic.1891

                  It can lead to disease, for example, this can occur when you have a loss of hetorozygocity at a certain locus,1896

                  that can lead to something called retinoblastoma which is a type of cancer that affects the eye, the retina.1906

                  This can be highly problematic.1916

                  We talked about the Holliday junction model.1924

                  Now, let us say the other type of double strand break repair.1926

                  We have synthesis dependent strand annealing which is SDSA.1932

                  Homologous recombination via the SDSA pathway occurs in the cells that divide through mitosis and meiosis.1939

                  This will always result in non-crossover products, repaired back to as if nothing ever happened.1946

                  Whereas, homologous recombination via the double Holliday junction model, more often results in crossover products.1953

                  Let us talk this through, we have the Holliday junction being formed,1964

                  just as if it were the other way as well but instead it is resolved via branch migration.1968

                  Now the invading 3 prime strand gets extended along the homologous, the recipient DNA duplex.1975

                  This is done via DNA polymerase.1984

                  It gets released as the Holliday junction slides.1988

                  We call them branch migration, it is sliding, it is moving.1992

                  The newly synthesized 3 prime end of the invading strand, remember, is that nuclear protein filament, nucleic acid proteins.1997

                  That can then anneal some base pair to the other 3 prime overhang in the damage chromosome, through complimentary base pairing.2007

                  After annealing, we can cleave off some small flaps of DNA.2019

                  However, this is not resolved, the Holliday junction is not resolved via cleavage, like the Holliday junction model is.2023

                  This one is resolved by moving and basically pulling back up the strand.2032

                  I will draw that out for you in the next slide.2036

                  Here is an example, let us show our SDSA.2042

                  Here is our 3 prime, we still our A, we have a break.2050

                  Here is our B and D, A, A, B, D.2057

                  By the way, on both of these, I have just said that this is a double strand break here.2079

                  Anything that is missing there, if it is a part of a gene, that is going to break that gene.2085

                  If you add any part of the missing gene, you are going to have problems down the line in transcription and translation.2090

                  As I have written in here, I’m saying that it is not an important piece of DNA.2095

                  Just like the Holliday junction model, after double strand break, there is still the resection.2103

                  Remember, this is SDSA, we have resection and we have 3 prime.2110

                  Remember, right here, nothing is happening on the homologous chromosome.2160

                  We once again have strand invasion.2180

                  We always want to get our polarity, right.2186

                  We know which way we are going to be synthesizing.2187

                  There is strand invasion.2255

                  Now, we get to the process of branch migration.2258

                  What is happening there is, what we are seeing, let us draw this out.2270

                  This is being synthesized right.2313

                  This is moving in that direction.2341

                  The Holliday junctions are just moving and then what we end up with is,2346

                  instead of cleavage at these spots, we have what is called dissolution.2355

                  No cleavage.2372

                  What we are basically doing is we are pulling this blue strand, it was pulling it up and pulling it up back to its own chromosome.2380

                  It is pulling this red one back down to its proper complimentary strand.2392

                  What we end up having is, if we follow it, we have our A, B, and our D.2398

                  We are just following all the way across this line.2419

                  This one, since we are pulling up, we are going to have A, b, D, because it is just going to pull that back up.2422

                  A with respect to D, no crossover.2439

                  Let us look at the homologous chromosome to check it.2443

                  We have the bottom one, it is the easy one.2448

                  a, b, d, it did not have to worry about anything.2451

                  This one, remember, we are just going to pull it down.2457

                  a, b, d, there is not going to be any cleavage.2460

                  These are a non-crossover.2471

                  However, let us still look, we do, the original damaged strand, we have a hetero duplex.2479

                  This hetero duplex still has to be repaired via MMR.2489

                  If it is repaired back to the BB, it is like nothing ever happened.2496

                  If it is repaired to bb, we have gene conversion and loss of heterozygocity.2502

                  Remember, instead of cleavage, SDSA goes through dissolution and that is where we have the 5 prime blue strand,2513

                  just pulling back up and becoming the template for the 3 prime blue strand.2520

                  We do the synthesis here right, then, it comes back up.2526

                  This synthesis, it is just coming back off of the blue strand, not on the red strand,2532

                  as seen in homologous recombination via double Holliday junction.2539

                  Once again, we can still have that hetero duplex and that can be affected,2544

                  whether we have gene conversion or not, based on how mismatch repair repairs that.2548

                  Those are the two big boys when we talk about homologous recombination.2559

                  There are other types of homologous recombination.2563

                  We are going to go a few of them, starting with single strand annealing.2568

                  The SSA pathway repairs double strand breaks between two repeat sequences and this is important.2571

                  The sequences have to be repeat.2580

                  This does not require any type of homology between two different homologous chromosomes or identical chromosomes.2584

                  This is using just a single double stranded helix.2595

                  This is unlike the Holliday junction or SDSA pathways.2601

                  This only requires that single DNA duplex and uses repeat sequences for the repair.2605

                  Those repeat sequences need to be at least 30 base pairs long.2613

                  This is considered extremely mutagenic because it results in large lesions of DNA as you are searching for the repeats.2620

                  I will show you an example of this in just a couple of slides, once I finish talking about SSA.2631

                  If we are going to talk about specifics, as DNA around the double stranded break gets resected,2642

                  the single stranded 3 prime overhangs are coated with RPA protein.2647

                  Remember, RPA is going to help protect this from endonucleases, as well as,2655

                  help form that nuclear protein filament that can go through strand invasion.2662

                  RAD52 is another protein that would bind each of these repeat sequences on either side of the break2668

                  and align them so that they can anneal.2674

                  After they anneal, their leftover non-homologous flaps of these 3 prime overhangs get cut away by endonucleases and then,2677

                  new DNA synthesis will fill in the gaps and DNA ligase will ligate those gaps making a new continuous strand.2687

                  The DNA between the repeat that bring off these flaps are always lost, as these one of the two repeat.2695

                  This is highly mutagenic because you lose a lot of sequent.2703

                  Let us see what that will look like.2707

                  We have our 5 prime to 3 prime resection, our homology search, our annealing in those overhangs,2712

                  the cleavage of the flaps, and then synthesis, and ligation, to finish it.2718

                  Let us see what this looks like.2722

                  Here is our strand, these are regions of homology.2732

                  We get a double strand break.2743

                  Those are the repeats.2765

                  First things first, we undergo resection to find that homology.2768

                  We are going to resect, we will continue on this line, 5 prime to 3 prime resection.2779

                  It is basically like your eraser.2792

                  Let us make sure we still know that we have the repeats right here.2809

                  The resection in the 5 prime to 3 prime manner, it is basically just doing this, it is an erase function.2816

                  I will draw this back.2827

                  The next step will be single strand annealing.2831

                  We find that we do the homology search, we are looking for these red spots, and then, we do the annealing of those overhangs.2839

                  What it looks like is this.2847

                  This is going to be single strand annealing.2853

                  What it looks like is this.2867

                  What you are doing is you are just moving this close in that direction.2895

                  You are pushing them together until these repeat sequences overlap.2901

                  This right here would be the two of them.2906

                  You are going to lose one of them so that you can make.2909

                  You will lose the bottom half of this piece and the top half of that piece to make one together.2914

                  There is the single strand annealing.2922

                  The next step, we are going to go through cleavage.2929

                  We have to cleave those flaps.2936

                  What we are doing, what we have done here is we have nicked it to cut it off.2940

                  And then finally, we go through the DNA synthesis and ligation to close the gaps.2985

                  Here is that one repeat, we have lost one of the repeats.3014

                  As well as, let us say for example, what we have ended up losing if we compare this to the original,3019

                  we have lost everything from here to here.3052

                  We have lost a lot of sequent, that all gets lost.3060

                  It is better to lose that and be somewhat mutagenic, than for you to have to completely lose this piece of chromosome,3065

                  however, not our preferred mechanism of repair.3077

                  Another form of homologous recombination is called break induce replication or BIR.3087

                  This pathway repairs double strand breaks encountered at replication forks.3094

                  This is because DNA helicase is trying to unwind the template strand and it leads to find the double strand break.3101

                  The exact mechanisms of BIR are not exactly clear.3110

                  There are several proposed mechanisms.3114

                  3 of them all have strand invasion as the first step, but then they differ in how the deal with migrates,3116

                  as well as some of the subsequent event.3124

                  But we do know that BIR pathway is actually very useful in maintaining the length of telomeres,3128

                  when telomerase, the enzyme, is either inactive or not present in certain cells.3135

                  When we talk about meiotic recombination, remember, homologous recombination is what is occurring.3148

                  In meiosis, homologous recombination is required of a proper chromosome alignment and segregation.3154

                  In meiosis, double Holliday junction always get resolve this crossover.3162

                  If we think about our homologous chromosomes during meiosis, we have homologous chromosome.3166

                  When they come together, they will form what is called a chiasma.3181

                  Right here, this is called a chiasma.3214

                  This helps a cell go through proper chromosomal segregation.3223

                  Without chiasma, you are actually increasing, by far, your occurrence of what is called non-disjunction.3229

                  That is when you have, let say both homologous chromosomes ending up in the same sperm or egg cell.3244

                  And then, one egg cell being without that complete set of genes.3252

                  Let us say for example, one sperm would get two chromosome 21, whereas, another cell will get 0 of them.3258

                  If this chromosome 21 sperm cell fertilizes the egg cell that was produced normally,3273

                  it has one copy of every gene or one copy of every chromosome,3282

                  but this sperm has one copy of every chromosome but two chromosome 21.3287

                  If these guys fuse, you are going to produce a fertilized egg with 3, it is a diploid cell,3292

                  but instead of two chromosome 21, it has 3 chromosome 21.3304

                  It is what we call trisomy 21 that may lead to Down syndrome.3309

                  Chiasma are very important.3321

                  In a chiasma, we are undergoing crossing over.3324

                  We are crossing over genetic material.3327

                  This black one, when we resolve this, what this looks like is that we have a little bit of the red.3329

                  This one has a little bit of the black.3352

                  Not only does it help us with non-disjunction events, but we actually have some gene transfer3354

                  which helps with the variability of the genetic sequence and can help with evolution.3360

                  This will always how we resolve this in meiotic recombination, this will always result in a crossover product.3372

                  In meiotic recombination, we can talk a little bit of a detail.3392

                  We have this protein called SPO11.3395

                  It is going to make a double strand break at what is called a recombination hot spots.3399

                  It is a place where it is likely to occur.3404

                  We then resect, we cut it by using our MRN complex.3409

                  Here is SPO11 occurring, here is the resection.3418

                  We have RAD51 and DMC1 as the proteins that coat the single stranded DNA and help with the strand invasion.3424

                  These are involved in making the nuclear protein filament and making the Holliday junction.3431

                  This is occurring in here, from the Holliday juncture migration can result in hetero duplex DNA containing mismatches.3432

                  We are coming down here.3452

                  The gene conversion can result in hetero duplex that we have talked about before.3456

                  We can have a loss of hetorozygocity.3461

                  Instead of BB on one strand, bb on one strand, maybe we have both of them being resolved to all b.3464

                  You will have all one way vs. none the second way.3472

                  We are resolving our crossovers.3480

                  We have already talked about having it resolved.3486

                  That was just a quick overview of just another few specific proteins involved in the meiotic recombination.3488

                  We have only talked about eukaryotes, so far.3496

                  We have to talk just a little bit about double strand break repair in prokaryotes.3499

                  There are pathways called the recBCD pathway.3503

                  There is basically just one pathway that they will go through.3508

                  What happens is that, we have this recBCD protein complex binding to and unwinding our double stranded DNA end.3512

                  That results, whether they are still binding here, it unwinds creating single stranded ends, as well as a single stranded loop.3522

                  The two tails will then anneal, re-anneal, to produce our second loop.3536

                  This is loop 1, this is loop 2.3546

                  Both loops can move and get bigger.3550

                  What we then have is recA is going to be binding.3553

                  It is going to be binding to the DNA, to protect the single strand.3559

                  We do not want it to get nicked by an endonuclease.3565

                  We have recBCD coming in, adding on.3571

                  It is already there, but now we are going to actually cause a nick.3577

                  We are going to nick this 3 prime strand at what is called the chi sequence.3582

                  This is just a hot spot for where we have nicks from the recBCD complex.3589

                  We then unwind further, and then we have what we see as this really long 3 prime end with the chi sequence that it is in.3594

                  This chi sequence right here, if we follow it around the back.3611

                  Here is the chi sequence, that is just a hotspot of where we are going to nick.3616

                  If we continue on to the next side, we have already shown that recBCD loads recA, that is down here.3620

                  Once recA is loaded, recBCD can disassemble.3630

                  It comes off of the DNA.3634

                  The recA promotes the strand invasion in the homologous DNA duplex,3636

                  producing our D loop, the displacement loop.3643

                  And then, the D loop gets cut and anneals with our gap in the DNA.3649

                  This is happening right here.3654

                  And then, we can resolve this DNA, this complex, this Holliday junction complex via the ruv AB complex, that is going to bind.3656

                  The ruv AB is going to see this Holliday junction and come and bind.3670

                  That is then going to recruit ruv C and we are going to resolve it.3676

                  This is all occurring here as well.3683

                  This is our final product.3685

                  That is how prokaryotes would do this, or e coli, specifically.3688

                  We did all the details in eukaryotes, then we zoomed over the prokaryotes a little bit.3692

                  This is the overview of how prokaryotic homologous recombination repair occurs.3700

                  To compare our prokaryotic and eukaryotic recombination.3710

                  The pairing of our homologous DNA, that is recA in E. Coli.3714

                  RAD51 and dcm1 in eukaryotes, dcm1 specifically in meiosis.3720

                  When we are generating that single stranded DNA for strand invasion, that is recBCD in E. Coli or MRN complex in eukaryotes.3726

                  MRX is used in human.3736

                  And then, Holliday junction recognition branch migration and resolution,3740

                  that is the ruv AB complex and ruv C for resolution for E. Coli.3745

                  In eukaryotes, we have a much more complex set of proteins handling that.3750

                  That is all of our homologous recombination.3763

                  There is a different type of recombination.3768

                  Instead of doing a whole long unit on that, I just added a few slides in this unit just to keep it all in recombination.3770

                  We have what is called site-specific recombination.3781

                  There are two types that we are going to talk about.3786

                  One is called conservative site-specific recombination.3788

                  That is involving protein enzymes called recombinases.3795

                  They act really similar to topoisomerases.3800

                  They cleave and rejoin DNA molecules to either invert DNA fragment.3805

                  They turn it upside down and flip the sequence of events or to insert them into a new site.3814

                  You either take it from one side and move it to the other.3822

                  We also have what is called transposition.3825

                  This uses a different type of enzyme called a transposase.3829

                  This transposase enzyme will cleave two ends, other transposable element.3834

                  It will randomly insert it into another place on DNA which we call the target site.3840

                  What are these going to look like?3853

                  Let us talk about our transposons first.3855

                  A transposases, we cleave both ends of the transposon in the original site and catalyzes integration in the random target site.3861

                  We saw them on previous slide.3869

                  We can do this in what I like to call a cut and paste mode or a copy and paste mode.3872

                  The difference between that is such.3878

                  Here is double stranded DNA and here is our transposon or a transposable element.3884

                  We have two options, we have the cut and paste option.3898

                  That is this, that is where we remove it from here.3905

                  It was here, we have cut it out of the DNA and now we have moved it to another spot on DNA which is right here.3913

                  That is what I call the cut and paste version.3925

                  We can also have the copy and paste which as you can probably already imagine.3935

                  We leave the original piece of DNA that sometimes we call these jumping genes transposons.3944

                  We leave the original one and we copy it, and add a second one in the new site.3955

                  We started with one, the cut and paste we still only have a one.3962

                  In the copy and paste, now we have two.3968

                  As we see down here, more than 40% of the entire human genome is composed of repeated sequences.3976

                  It is very likely that a transposon got into our ancient ancestors.3990

                  Throughout evolution, throughout the time, it has not only cut and pasted itself but more often,3999

                  more likely, copied and pasted itself making those repeat show up in many different parts of our genome.4007

                  Meaning that we have also lengthen our genome, as we have grown as well.4016

                  The transposons are very likely responsible for a large portion of these repeated sequence.4020

                  We have a lot of repeated sequence in our genome.4027

                  I want to give you an example of our site-specific recombinases.4036

                  Here is an example, what if we have a circular piece of DNA.4042

                  It wants to recombine with a linear piece of DNA.4054

                  This can sometimes happen when a bacterial plasmid, a vector, is going to join the into the main genome.4060

                  What is important here is that we have to see these recombinases work with a polarity.4073

                  This arrow that I’m showing is not just for no reason.4086

                  This means that they have the same polarity.4091

                  What you can do is to insert into this, A will cross like this.4093

                  We will cross like this.4112

                  What we end up with, we just follow the polarity, this is actually pretty easy.4114

                  We can start at X, we have a rightward arrow, and then we follow it out.4122

                  It goes to B, it comes all the way around.4136

                  DNA sequence to A, and then A crosses back down, rightward arrow, and then we get to Y.4142

                  This could be one like, nice long linear sequent from a circular sequence and a small linear sequence.4152

                  This is what we call insertion.4158

                  We have inserted this piece of DNA, the circular piece, into this linear piece of DNA.4168

                  There is also what is called inversion.4175

                  What we would have here is, if we start with X, rightward arrow to A, long piece of DNA B.4181

                  We have a different arrow, a leftward arrow to Y.4200

                  These polarities have to match up.4203

                  What has to happen to this, we actually have to curve this around.4205

                  Because look, the arrows pointing at B so it is always going to be pointing at B.4223

                  How we do this, we are going to once again make a nice X.4228

                  We can start with X.4237

                  We are going to go this way.4240

                  What we have, we start with X, we have a rightward arrow.4245

                  We come to B, follow it all the way around to A, this long piece of DNA to A.4254

                  What do we have, we have an arrow that is going to be pointing at A.4262

                  A leftward pointing arrow and then down to Y.4270

                  This is what we call inversion.4276

                  This one we can flip a DNA sequent, as we see here, look what has happened.4283

                  We still have X and Y on the outside but in this case, A to B went from left to right.4289

                  A to B goes right to left.4300

                  The last one that we are going to show is a deletion.4308

                  How can we take something out of the genome?4311

                  If we see here, we have X rightward arrow to B.4318

                  Nice stretch of DNA, A rightward arrow to Y.4325

                  What we can do is we can circle that up.4334

                  X to B, A to Y.4344

                  We can make our nice red cross again and we can draw it out.4358

                  Coming back down here, what do we have?4366

                  We are going to go X down to Y, that is the end of the DNA.4372

                  We are going to have a plus.4380

                  What about this one?4383

                  We can say this is A going to B and it is circular.4385

                  This is A going to B and it is circling around back to A.4397

                  This is almost back to up here right.4405

                  We have started with this again.4410

                  This is called deletion.4412

                  This is how you could invert insert something in.4420

                  You could insert a piece of DNA into a whole cell genome, think of viruses do this.4425

                  You can invert something, a piece of gene.4430

                  Or you can cut yourself out of the genome.4435

                  We think of our viruses that jump into the human gene and lay dormant for a while.4440

                  They have inserted in there.4447

                  When they want to be active again, they can cut themselves out, deletion, and become active again.4450

                  These are some examples of our site-specific recombination.4459

                  I hope you enjoyed this lesson and I hope to see you back.4462

                  Thank you very much for joining us at www.educator.com.4465

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