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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 at 10:25:41 AM

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 exchanged 0051

                  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 overhang 0372

                  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 junction 0384

                  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 transfer 3354

                  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