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MBIOS 503 Unit 2 > Unit 6: DNA recombination > Flashcards

Unit 6: DNA recombination Flashcards

1
Q

Fill in the blank: recombination is the result of crossing over that occurs at a ________ during _________ and involves _______ of the four chromatids

A

recombination is the result of crossing over that occurs at a CHIASMA during MEIOSIS and involves TWO of the four chromatids

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2
Q

What is a chiasma?

A

the point of exchange or crossing over in recombination. This occurs between 2 of the nonsister chromatids.

-occurs at prophase I or meiosis

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3
Q

What will be the result following recombination of two sister chromatids (AB) and two sisters chromatids (ab) ?

A

Two of the chromatids remain parental or non-recombinant (AB and ab), while the other two chromatids will recombinant and contain material from each parent and will now have new genetic combinations (Ab and aB)

OR two haploid gametes will be recombinant and two haploid gametes will be non-recombinant

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4
Q

What is homologous recombination essential for in meiosis and mitosis?

A
  • it is essential in meiosis for generating diversity and for chromosome segregation (we could not have evolution by natural selection w/o homologous recombination)
  • in mitosis it is essential to repair DNA damage and stalled replication forks
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5
Q

TRUE OR FALSE: the process of recombination allows organisms to have genes shuffled around and favorable changes get passed on while the harmful or deleterious ones are eliminated

A

TRUE: in meiosis this occurs via homologous recombination

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6
Q

What are the three types of recombination?

A

1) Homologous
2) Site-specific
3) Somatic

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7
Q

TRUE OR FALSE: recombination also changes the overall organization of DNA?

A

FALSE: Although crossing over can occur anywhere on the DNA duplex there is no change in the overall DNA or organization.

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8
Q

What is site-specific recombination? When was this first observed?

A

specific recombination involves specific DNA sequences, meaning the enzymes involve in this type of recombination target a specific DNA sequence. EXAMPLE: circular DNA AB gets incorporated into linear DNA XY becoming linear DNA X-A-B-Y.

this type of recombination was first observed in bacteria where the phage genome gets incorporated into the bacterial genome at specific sequences.

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9
Q

What is somatic recombination?

A

Recombination that occurs in nongerm cells or somatic cells (i.e., does not occur during meiosis); most commonly used to refer to recombination in the immune system.

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10
Q

What is chromosome pairing or synapsis?

A

chromosome pairing or synapses is when homologous chromosomes approach each other and pair up along the entire length. this process occurs after the first prophase (chromosomes have replicated and consist of two sister chromatids).

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11
Q

What is synaptonemal complex?

A

the synaptonemal complex is when all the chromosomes that are undergoing synapsis/chromosome pairing completely pair up and complete the process of synapse.

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12
Q

Fill in the blank: recombination requires a _____ _______ ______ in one chromatid for there to be a physical exchange of DNA

A

recombination requires a DOUBLE STRAND BREAK in one chromatid for there to be a physical exchange of DNA

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13
Q

Fill in the blank: Chromosomes must ___________ in order for chiasmata to form where crossing over occurs

A

Chromosomes must SYNAPSE (PAIR) in order for chiasmata to form where crossing over occurs

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14
Q

What is the 5’ end resection?

A

occurs after the double strand break repair model is initiated

-exonuclease activity generates 3’-single stranded ends that invade the other donor duplex

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15
Q

What is single strand invasion? what does this create?

A

when a single strand (free 3’ end) from one duplex displaces its counterpart in the other duplex

-this creates a branched structure called a D loop (displacement loop)

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16
Q

What is heteroduplex DNA and how does it form?

A

heteroduplex DNA consists of one strand from each parent and occurs after strand exchange

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17
Q

How is the double-strand break repair (DSBR) model of recombination initiated?

A

-it is initiated by making a double-strand break in one (recipient) DNA duplex

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18
Q

What are the steps in the double strand break repair model (DSBR) of homologous recombination?

A

1) Recombination is initiated by a double strand break.
2) Double strand break resection: Following nuclease degradation of the ends, single-strand tails w/3’-OH ends are formed.
3) Strand invasion by one end into homologous sequences forms a D-loop. Extension of the 3’-OH end by repair DNA synthesis enlarges/extends the D loop.
4) Once the displaced loop can pair with the other side of the break, the second double-strand break ends is captured. DNA synthesis to complete the break repair followed by ligation results in the formation of two Holliday junctions.
5) Resolution results in a non-crossover and a crossover product

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19
Q

During double strand break repair model, how is the D loop extended? What is used for this extension

A

The D loop is extended by repair DNA synthesis using the free 3’ end as a primer to make the double strand break.

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20
Q

What occurs once the DNA loop can pair up w/the other side of the break in DSBR?

A

the second double strand break is captured and DNA synthesis results in the formation of two Holliday junctions.

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21
Q

In the DSBR, what occurs after the formation of the two Holliday junctions?

A

The joint molecule is resolved into two separate duplex molecules by nicking two of the connecting strands

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22
Q

In DSBR, what determines whether recombinants are formed?

A

whether recombinants are formed depends on if the strands involved in the original exchange or the other pair of strands are nicked during resolution.

  • If the nicks are made in the strands that were not originally nicked, all four of the strands will now have been nicked and this will produce crossover recombinant DNA molecules.
  • if the second nick is in the original strands that were nicked before you will end up w/a duplex w/on strand intact while the second strand will have a sequence from the donor DNA. These are non-crossover products but are still considered recombinant

***Whether recombinants are formed depends on if the strands involved in the original exchange or the other pair of strands are nicked during resolution.

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23
Q

TRUE OR FALSE: heteroduplex DNA that is created by recombination can have mismatched sequences where the recombining alleles are not identical

A

TRUE: it is possible that the heteroduplex DNA following recombination can have mismatched sequences.

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24
Q

How are mismatched sequences of heteroduplex DNA repaired?

A

repair systems can remove these mismatches by changing one of the strands so its sequence is complementary to the other.

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25
Q

What can be generated by the mismatch (gap) repair of heteroduplex DNA ?

A

nonreciprocal recombinant products called gene conversions

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26
Q

Why is the synthesis-dependent strand-annealing model important?

A

b/c it produces gene conversions from double strand breaks but w/o crossovers

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27
Q

What are the steps in the synthesis-dependent strand annealing model?

A

1) the initial steps are the same as the double strand break repair model except that in this case there is no second end capture.
2) after strand invasion of the 3’ end and synthesis of the complementary strand (which is now identical to the sequence of the display strand) the D loop itself gets displaced and the single strand reanneals with the other end of the double strand break.
4) Synthesis and ligation repair the remaining break and no crossing over and no Holliday junctions are formed. The product is ALWAYS noncrossover

This model works well for mitotic gene conversions

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28
Q

When does the single strand annealing mechanism occur?

A

at double strand breaks between direct repeats

  • the ends are resected (or cut out) to give single strands. Resection proceeds until the 3’ end tails are homologous
  • Complementarity between the repeats allows for annealing of the single strand. In the end of the single strand annealing, the sequence between the repeats is deleted
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29
Q

What is BIR (break-induced replication)?

A

another type of repair mechanism that is induced by double strand breaks but at a different type of repeat sequences called fragile sites.

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30
Q

How does BIR (break-induced replication) repair fragile sites?

A

by using the homologous sequence from a repeat on a non-homologous chromosome (that has a repeated sequence) creating a nonreciprocal translocation.

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31
Q

During synapsis (in meiosis), each chromosome or sister chromatid pair condenses around what element?

A

a proteinaceous complex called axial element

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32
Q

How is the tripartite synaptonemal complex formed? What happens to the axial elements in this complex?

A

The axial elements of the homologous chromosomes line up to form the tripartite synaptonemal complex (in meiosis).

In this complex, the axial elements now become lateral elements and are separated from each other by central elements.

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33
Q

Central elements are formed by what proteins?

A

zip proteins.

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34
Q

What are recombination nodes?

A

dense objects present on the synaptonemal complex; they may represent protein complexes involved in crossing over.

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35
Q

TRUE OR FALSE: The synaptonemal complex is necessary for DNA exchange and must form first to initiate double strand breaks

A

FALSE: Double strand breaks that initiate recombination occur before the synaptonemal complex forms. Thus synaptonemal complex is more of a result of recombination

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36
Q

What are the hot spots for double strand breaks?

A

DSB tend to occur in the promoter regions and generally in areas where the chromatin is accessible.

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37
Q

What happens if chromosome pairing is prevented or abolished?

A

recombination does not occur and the synaptonemal complex fails to form

this is seen in yeast cells that have mutations and the chromosomes are not able to pair up. However, mutants that lack the synaptonemal complex can still generate recombination so this suggest that the synaptonemal complex is not necessary for recombination.

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38
Q

What are the two phases involved in meiotic recombination?

A

1) one that results in gene conversion w/o crossover

2) one that results in crossover products

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39
Q

What is the spo11 protein?

A

a protein that generates double strand breaks.

-Following the dissociation of spo11 from the DNA, the nucleases then play a role in expanding the break to a gap and generating the 3’-OH single strand ends and the next steps of the repair mechanism

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40
Q

TRUE OR FALSE: Chromosome pairing and synaptonemal complex formation depend on each other

A

FALSE: pairing and synaptonemal complex formation are independent

-mutations in either chromosome pairing or the formation of the synaptonemal complex do not appear to affect the other process making each of these processes mutually exclusive

41
Q

Recombination in bacteria can be initiated in different ways but requires the formation of what?

A

formation of free single strand 3’ ends/

42
Q

What is the chi sequence

A

the chi sequence stimulates recombination in bacteria.

43
Q

What is the function of RecBCD complex? Describe how it accomplishes its function

A

RecBCD acts on chi sites and has nuclease and helicase activity that generates a 3’-OH end and chi site which becomes a substrate for recombination

1) RecBCD complex binds downstream of the chi sequence; it unwinds the duplex and degrades one strand from the 3’ to 5’ as it moves towards the chi sequence
2) once the chi site is reached, the recD subunit dissociates, eliminating the nuclease activity
3) RecBC, with its helicase activity, is left to continue with the recB subunit driving the translocation.
4) this results in the generation of the 3’-OH and the chi site, which now becomes a substrate for recombination

44
Q

What are presynaptic filaments?

A

single stranded DNA bound in a helical nucleoprotein filament w/a strand transfer protein such as Rad51 or RecA

45
Q

Fill in the blanks: ______ and _______ belong to a family of proteins called the strand transfer proteins, which play a key role in ___________.

A

recA and rad 51 belong to a family of proteins called the strand transfer proteins, which play a key role in recombination

46
Q

What is the process of single strand assimilation (aka single strand invasion)?

A

When a single strand with a free 3’ end displaces its homolog in a duplex. RecA enables the displacement

47
Q

What is the recA protien?

A

the rec a could take the single strand of the three prime end and can use it to react with the homologous duplex creating a joint molecule. the rec a protein forms filament with the DNA and interaction between the DNA takes place within these filaments. Reca bound to single stranded DNA is a presynaptic filament. throughout the single stranded simulation process the rec a remains bound to the original single strand so that at the end of the process the RecA is now bound to the duplex. large amounts of ATP are used up during this reaction

48
Q

In single strand assimilation what results in strand exchange? What are the steps

A

if the reaction is between a partially duplex molecule and a fully duplex molecule it results in the exchange of strands.

1) the free strand initiates the exchange and starts to display its homolog in the duplex
2) when it reaches a region that is duplex in both molecules the invading strand will unpair from its partner and pair up w/the displaced strand

49
Q

In recombination, why is resolution of the holliday junctions important?

A

because resolution of the holliday junction is what results in either crossover recombinants or non-crossover recombinants.

50
Q

What genes are involved in the resolution if the holliday juntoin? How do they resolve this junction?

A
  • The products of the RUV genes (RuvA ,RuvB, and RuvC) are involved in stabilization and resolution of the holliday junction.
    1) RuvA recognizes the structur of the junction and binds to all four strands forming two tetramers that sandwich the DNA
    2) RuvB is a helicase that catalyzes branch migration. It is a ring shaped hexamer that encircles each duplex upstream of the crossover point.

**the RuvAB complex can cause branch migration at a rate of 10 to 20 base pairs per seconds

3) RuvC cleaves the holliday junction to generate recombination intermediates

**resolution in eukaryotes is less well understood, but a number of meiotic and mitotic proteins are implicated

51
Q

What is patch recombinant?

A

DNA that results from a Holliday junction being resolved by cutting the exchanged strands. The duplex is largely unchanged, except for a DNA sequence on one strand that came from the homologous chromosome

52
Q

What is splice recombinant?

A

DNA that results from a Holliday junction being resolved by cutting the non-exchanged strands

-both strands of DNA before the exchange point come from one chromosome; the DNA after the exchange point come from the homologous chromosome

53
Q

What is the difference between MRX and MRN complex? What is a similarity?

A

SIMILARITY: They both resect double strand breaks by binding to broken ends along with an endonuclease. The MRN or MRX complex along with the endonuclease prepares the ends by removing any proteins or other adducts that may interfere w/the resection

Difference:

MRX complex performs this task in yeast (Exo1, Sgs1/Dna2 are also part of this)

MRN complex performs this task in mammalian cells (BLM is also part of this)

54
Q

In the MXR and MRN complex what behaves as exonucleases and helicase?

A

MRX:
exonucleases: Exo1 and Dna2

helicase: Sgs1

MRN: BLM

55
Q

What is the role of single strand binding protein RPA?

A

once the 3’-OH single strand is generated by either MRN or MRX complex, RPA binds to the strand and removes any secondary structure. RPA is then removed/displaced by rad51 w/the help of mediator proteins

RPA has inhibitory effects

56
Q

TRUE OR FALSE: Rad51 is required for all homologous recombination processes except single strand annealing

A

TRUE

57
Q

What is BRCA2 required for?

A

homologous recombination

58
Q

Strand invasion requires what proteins in yeast? What about mammalian cells?

A

Yeast: Rad54 and Rdh54

Mammalian cells: Rad54 and Rad54B

59
Q

Mutation in Rad53, RDH54, aand Rad54B results in?

A

-increased sensitivity to radiation. In mouse and chicken cells the deletion of both rad54 and rad54B is not lethal like the deletion of other genes involved in homologous recombination.

60
Q

TRUE OR FALSE: In eukaryotes, Proteins involved in the step of heteroduplex extension and branch migration have not been identified

A

TRUE: Proteins involved in the step of heteroduplex extension and branch migration have not been identified

61
Q

What polymerases/proteins are thought to carry out the extension from the strand invasion intermediate terminus?

A

DNA polymerase delta is believed to be the polymerase that carries out the repair synthesis although recent study suggest it could be DNA polymerase eta or rad30 that carries out the extension from the strand invasion

62
Q

In eukaryotes, resolution of the holliday junction is thought to involve what proteins?

A
  • BLM, MUS81/EME1 in humans

- Sgs1, Mus81/Mms4 in yeast

63
Q

What is hemicatenane?

A

-this occurs in eukaryotes when the two Holliday junctions converge by branch migration to form the middle structure called the hemicatenane, which is basically the DNA strands are twisted around each other

64
Q

In eukaryotes, what resolves the hemicatenane structure?

A

-This structure is resolved by a DNA topoisomerase (TopIII), associated with Sgs1 in the case of yeast and hTOPO3alpha associated with BLM in mammalian cells.

65
Q

make a question about this: the meiotic resolvases that generate crossover product from the dissolution of the holliday junctions have not been identified although recently Gen 1 in humans and YenN1 in yeast have been shown to resolve holliday junctions in vitro but not in vivo

A

the meiotic resolvases that generate crossover product from the dissolution of the holliday junctions have not been identified although recently Gen 1 in humans and YenN1 in yeast have been shown to resolve holliday junctions in vitro but not in vivo

66
Q

What is recombinase?

A

an enzyme that catalyzes site-specific recombination

67
Q

What is specialized recombination? When does it occur

A

recombination that involves reaction between specific sites that are not necessarily homologous.

  • these sites are short (14 to 50 base pairs.
  • this type of reaction is used to either insert phage DNA into the bacterial chromosome or remove the integrated phage DNA from the chromosome.
68
Q

What are integrases?

A

enzymes involved in phage integration

69
Q

What is the difference between a lysogenic phage and lytic phage?

A

lysogenic phage is where the phage DNA is incorporated into the bacterial chromosome, while in the lytic form the phage DNA is independent of the bacterial chromosome

transition between these states involves site specific. recombination

70
Q

How does a phage DNA become lysogenic?

A

to become lysogenic, the phage DNA needs to be INTEGRATED into the bacterial DNA by the action of INTEGRASE and this is called prophage

71
Q

What does entry into the lytic phage form require?

A

it requires excision of the phage DNA from the host DNA. In the case of phage lambda, integration and excision of the phage DNA into or out of the bacterial chromosome are at specific sites called att sites

72
Q

What is a core sequence?

A

the segment of DNA that is common to the attachment sites on both the phage lambda and bacterial genomes

-it is the location of the recombination event that allows phage lambda to integrate. The phage is excised from the chromosome by recombination between the sites at the end of the linear prophage.

73
Q

Phage lambda int codes for what?

A

an integrase that catalyzes the integration reaction

74
Q

Fill in the blanks: Circular phage DNA is converted to an ________ __________ by a __________ recombination between attP and attB

A

Circular phage DNA is converted to an integrated prophage by a reciprocal recombination between attP and attB

75
Q

The cre-lox system is an example of what type of recombination?

A

site-specific recombination between phage and bacteria

76
Q

TRUE OR FALSE: The Cre-lox system is less efficient and more complex than the lambda phage system

A

FALSE: Cre-Lox system is much simpler and more efficient than the phage Lambda system and has been adapted for use in site specific recombination in eukaryotic cells. recombining sequences are identical unlike phage lambda

77
Q

Describe the cre-lox system

A

1) cre recombinase, encoded by the P1 phage, catalyzes recombination between two target sequences
2) the recombining sequence is a 34 base pair sequence called LoxP.

The recombining sequences are identical (unlike phage lambda) and no accessory proteins are involved.

78
Q

Explain recombination at att sites

A
  • Phage DNA is circular and has an attachment site labeled attP and a sequence component POP’.
  • Bacterial DNA is linear and has a BOB’ sequence and an attachment site at attB.
  • the O in BOB’ and POP’ is common and called the core sequence and this is where recombination occurs.
  • The flanking regions B and B’ and P and P’ are called the arms and are distinct in this sequence.
  • The circular lambda phage will integrate into host bacterial DNA as a linear sequence.
  • This results in the creation of two new att sites on either side of the inserted region, attL and attR
  • Now, for excision to occur, the reaction sequences are no longer attB/attP but rather attL/attR.

THUS, integration and excision do not involve the same pair of reacting sequences.

79
Q

TRUE OR FALSE: att recombination is reversible

A

TRUE: but the direction of the reaction is controlled by different conditions

80
Q

What conditions control the direction of site specific recombo (integration vs excision)?

A
  • it depends on the proteins around.
  • The integration reaction requires the presence of the integrase Int and integration host factor IHF. The integrase is a phage protein, while IHF is a bacterial protein.
  • The excision requires the phage integrase enzyme Int, bacterial host IHF, and an additional phage enzyme called Xis,, which inhibits integration
81
Q

P1 bacteriophage uses what kind of site specific recombination?

A

the cre-lox system, which uses a cre recombinase (phage enzyme that catalyzes recombination between two target sequences. Recombining sequences are identical (unlike phage lambda)

82
Q

In the Cre-Lox system what is the recombining sequences?

A

a 34bp sequences called LoxP. Recombining sequences are identical in the Cre-Lox system and no accessory proteins are involved.

83
Q

What is the difference between the attB and att P site?

A

-attP site is much bigger than the attB site, with 240 base pairs compared to 23 base pairs for att B.

84
Q

Is recombination between attP and attB a result of sequential exchange or is it a concerted cutting (strands that cut simultaneously and exchange)?

A
  • exchange takes place sequentially.
  • Using suicide substrates to halt the recombination reaction at intermediate stages and then determining the structures of these intermediates demonstrates sequential exchange.
  • If attP and attB undergo the same staggered cleavage, this will generate single strand ends that are complementary to one another and then would be available for cross hybridization
85
Q

How are integrases related to topoisomerase in site specific recombination

A

-integrators are related to topoisomerase in that the break is made in one DNA strand at a time.

HOWEVER,
-Topoisomerase manipulate the ends and then rejoins the original ends, while the integrase will reconnect the nicked ends from different duplexes in a cross-wise fashion.

86
Q

Describe the reaction of integrase that is similar to topoisomerase activity

A

1) Two enzyme subunits bind to each duplex DNA.
2) 4 molecules of the recombinase is needed b/c each one is used to cute one of the four strands of the two duplexes (generationg P-Tyr bond and an -OH end)
3) A catalytic tyrosine in the enzyme is used to break the phosphodiester bond. This results in the 3’ end of the nucleotide chain, now forming a phosphodiester bond w/the tyrosine and the other fragment now has the free 3’-OH.
4) The 3’-OH attacks the P-tyrosine bond of the other strand. This first exchange generates a holliday junction
5) this process is repeated on the other strands by the other two recombinase subunits that were not involved in the first cycle of breakage
6) reunion finally resolve the structure

87
Q

What is the structure of the cre-lox complex?

A
  • Two cre molecules bound to a 15bp length of DNA w/the DNA bent at 100 degrees angel at the center. There is another complex just like this on the other duplex.
  • The two complexes are lined up in an antiparallel manner to for a tetrameric structure. The strand exchange takes place in the central cavity of the protein structure
88
Q

What is a trypanosome?

A

a parasite that causes African sleeping disease and chagas disease

-This parasite is able to evade the immune system in a manner thats called antigenic variation

89
Q

What is antigenic variation?

A

This is how trypanosome attacks the immune system. Trypanosome has a variant surface glycoprotein (VSG) which is recognized by the immune system and will produce antibodies to it. However once the antibodies are produced for that sepcific VSG antigen, the organism can swithc the expression of that particular VSG to anyone of the hundreds of other VSG genes in it s genomes

  • As a result, the antibodies produced against the previous VSG antigen will not work.
  • The organism can do the switching through homologous recombination to remove the VSG to the specific expression sites.
  • gene conversion of the active VSG gene using the information from one of the silent genes in the array results in a change in sequence of the active gene and hence an altered surface antigen. this is what is shown on the left side of the figure. the other way variation can be introduced is through telomere exchange. VSG genes are also located in telomeric arrays on mini chromosomes. so in this recombination event an inactive telomeric VSG gene from the mini chromosome is switched w/the active VSG gene. both mechanisms used homologous recombination factors but the precise mechanism is still to be elucidated.
90
Q

Which two systems allow for targeted recombination and gene knockout construction

A
  • Cre-lox system

- Flp/FRT system

91
Q

What is one of the most common uses of the cre-lox system experimentally?

A

to conditionally turn on or turn off genes in mice

92
Q

In experimental systems, how does the cre-lox system work?

A

-

93
Q

In experiments, how does the Flp/FRT system work?

A

-

94
Q

Describe how circular phage is converted integrated prophage by reciprocal recombination between attP and attB

A

the attachment site on the bacteria is labeled att B with the sequence component BOB’. the attachment site on the phage is identified as att P and the sequence is POP prime. in the BOB prime and the POP prime sequence the O sequence portion is common and is called the core sequence. This is where the recombination event occurs, the flanking regions B and B prime and P and P prime are called the arms, and are distinct in this sequence. and the Lambda phage is circular and integrates into the host DNA as a linear sequence. so this results in the creation of two new att sites on either side of the inserted region, att L and attR. so now for excision to occur the reacting sequences are no longer att B and att P but rather att L an att R. Thus we see that the integration and excision do not involve the same pair of reacting sequences. The recombination event is reversible but the direction of the reaction is controlled by different conditions.

What are these different conditions? well it depends on the proteins that are around. The integration reaction requires the presence of the integrase INT and an integration host factor ihf. The integrase is a phage enzyme while IHF is a bacterial protein. The excision requires the phage integrase enzyme INT. the bacterial integration host factor IHF but also another phage enzyme called XIS inhibits integration

95
Q

Phage lambda int codes for what?

A

for integrase that catalyzes the integratiion reactoin

96
Q

In mitotic cells how are the double strand breaks produced?

A

in mitotic cells, the double strand breaks are produced by either chemical treatment or radiation or by topoisomerases and Nicks on the template strand. the ends of the brakes are degraded by exonucleases generating the single strand free prime ends.

97
Q

Once the 3’-oh strand is generated in doubles strand break what removes any secondary structures?

A

Once the 3’-OH single strand is generated the single strand binding protein RPA binds to remove any secondary structures. The RPA is then displaced by rad51 with the help of mediator proteins. rad51 is required for all homologous recombination processes except single strand annealing. rad 51 is related to recA with 30% identity. Rad51 forms a right handed nuclear filament. the mediator proteins are also related to rad51 and are called the rad paralogs deletion of these parallel genes in mice proved to be lethal

98
Q

stand invasion is dependent on which genes in yeast and mammals?

A

the next step is synapsis when the single strand DNA that is generated and bound by rad 51 filament locates a homologous DNA molecule. once this is found strand invasion commences leading to the formation of the D loop. This step requires Rad54 and RDH54 in yeast and rad 54 and rad54B in mammalian cells. mutations in these genes result in increased sensitivity to radiation. in mouse and chicken cells the deletion of both rad54 and rad54B is not lethal like the deletion of other genes involved in homologous recombination. the proteins involved in the next step of heteroduplex extension and branch migration have not been identified. The D loop extension is an important part of the recombination and strand annealing process. DNA pol delta is believed to be the polymerase that carries out the repair synthesis although recent study suggests it could be DNA pol eta (theta?) or rad30 that carries out the extension from the strand invasion intermediate terminus. Finally, the resolution of the holliday junctions is thought to involve BLM, MUS81, or EME1 in humans, while in yeast it is SGS1 and MUS81 Mms4. The resolution of holliday junctions to generate non crossover products is shown in this figure. The two holliday junctions converge by branch migration to form the middle structure in this figure that is called a hemicatenane, which is basically the DNA strands twisted around each other. This structure is then resolved by a DNA topoisomerase (TopIII) associated with SGS1 in the case of yeast, and Htuple3alpha associated with BLM in the case of mammalian cells. the meiotic resolvases that generate crossover product from the dissolution of the holliday junctions have not been identified although recently Gen 1 in humans and YenN1 in yeast have been shown to resolve holliday junctions in vitro but not in vivo

MBIOS 503 Unit 2 (6 decks)

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