Chapter 11: Homologous Recombination at the Molecular Level

After introduction of the double strand break, what happens?

A DNA-cleaving enzyme degrades the broken DNA molecule to generate regions of single-stranded DNA

What is the result of the change in activity spurred by the Chi site?

A single-stranded DNA is formed that terminates in the Chi site on its 3' end

What is the mechanism of Spo11 DNA cleavage?

A specific tyrosine in the side chain of Spo11 attacks the phosphodiester backbone to cut the DNA and form a protein-DNA covalent complex

What enzyme does RuvA recruit? What is the role of this molecule?

After binding to the Holliday junction, RuvA recruits RuvB to this site RuvB fuels the movement of the DNA branch which drives the exchange of bases

The RecBCD complex binds to DNA at what site? How does it track along the DNA?

At the site of the DSB It tracks along the DNA using the energy of ATP hydrolysis

In addition to promoting DNA pairing, DNA repair, and genetic exchange, homologous recombination can also serve to ____________ the DNA sequence at a specific chromosomal location. What is this type of recombination sometimes used to accomplish?

Change Sometimes used to accomplish regulation of gene activity

The ssDNA tail generated by RecBCD must be ____________ by ____________ for recombination to occur.

Coated RecA

Meiotic recombination frequently gives rise to ____________ ____________ between genes on the two homologous ____________ ____________.

Crossing over Parental chromosomes

What is the result of cleavage by RuvC?

DNA ends that terminate with 5' phosphates and 3' OH groups that can be joined by DNA ligase

What enzymes facilitate strand exchange during meiotic recombination?

Dmc1 and Rad51

Recombination is initiated by the presence of a ____________ in one of the DNA molecules.

Double strand break

Double-junction dissolution prevents the exchange of ____________ ____________.

Flanking sequences

The resulting Spo11-linked 5' DNA ends are the initial sites for what?

For DNA processing to create the ssDNA tails required for strand invasion

How do Chi sites protect bacterial cells from foreign DNA?

Foreign DNA will lack a Chi site which will lead to its degradation rather than activation for recombination

____________ ____________ occurs when an allele of a gene is lost and replaced by an alternative allele.

Gene conversion

If the DNA sequence at MAT is a, then invasion will occur with ____________. If the DNA sequence at MAT is alpha, then invasion will occur with ____________.

HML HMR

If the DNA sequence at MAT is alpha, then invasion will occur with ____________.

HMR

Strand invasion generates a ____________ ____________ that can then move along the DNA by ____________ ____________.

Holliday junction Branch migration

What is the major mechanism used to repair DSBs in most cells?

Homologous recombination

Branch migration ____________ the length of the DNA exchanged.

Increases

What sequence does RuvC cleave? Is this sequence seen frequently in the DNA?

5'-A/T-T-T-G/C-3' RuvC cleaves after the second T in the sequence Yes, it appears an average of every 64 nucleotides

Does RecA bind to double stranded or single stranded DNA more quickly?

It binds to single-stranded DNA more quickly

What is the role of Spo11?

It cuts eukaryotic DNA at many chromosomal locations

Upon RecA binding what happens to the length of the DNA?

It is extended 1.5 fold

What is the specificity of Spo11?

It is not sequence specific, but it cuts at a very specific time during meiosis

What is the unique role homologous recombination plays in eukaryotic cells?

It is required for proper chromosome pairing during meiosis

What would happen if RuvC was not sequence specific?

It may cleave the Holliday junction too soon which would restriction branch migration and limit exchange of homologous DNA

What is the function of Rad52?

It promotes the assemble of Rad51 filaments and the annealing and base pairing of complementary ssDNA molecules

Does RuvC regonize structure or sequence?

It recognizes structure

Mating-type switch is initiated by the introduction of a DSB at the ____________ ____________. What enzyme performs this?

MAT locus HO endonuclease

During meiotic recombination, what enzyme complex is responsible for DNA processing?

MRX-enzyme complex

How many RecA subunits does a filament have? How many nucleotides of DNA?

May have 100 subunits of RecA and 300 nucleotides of DNA

The homologous recombination events that occur during meiosis are called ____________ ____________.

Meiotic recombination

What are the two ways gene conversion occurs in homologous recombination?

Mismatch base repair and if a gene is very close to the site of the DSB

Is the order of genes changed in the patch product?

No

Why is mate-type switching different than traditional homologous recombination?

No gene crossover occurs

Are locations of Spo11 cleavage random? Where are the most common sites of cleavage?

No, it's not random Cut sites are most commonly located at sites that are not tightly packed by nucleosomes such as promotor regions

Dmc1-dependent recombination is preferential between...

Non-sister homologous chromatids

The improper segregation of chromosomes is called ____________.

Nondisjunction

When is DMC1 expressed?

Only as cells enter meiosis

In preparation for the first nuclear division, duplicated homologous chromosomes must ____________ and ____________ at the ____________ of the cell.

Pair and align Center

RuvC selectively nicks two of the homologous DNA strands that have the same _____________.

Polarity

What happens when there is a defect in the BRCA2 gene?

Rad51 does not assemble, so the double strand break is not repaired.

In eukaryotes, what enzymes promote Holliday Junction resolvase activity?

Rad51C and and XRCC3

After meiotic recombination, the alleles present on the parents DNA molecules are ____________ for the next generation.

Reassorted

What are the roles of RecB, RecC, and RecD?

RecB and D are both helicases that move along the DNA sequence RecC recognizes the Chi site

How do RecB and RecD move in relation to eachother?

RecB and RecD move independently at different speeds RecB is slow, RecD is fast

In E. coli, what enzyme processes the broken DNA molecules to generate regions of ssDNA? What is the other role of this enzyme?

RecBCD It helps load the RecA strand-exchange protein onto ssDNA ends

Why does RecA bind to the ssDNA instead of other SSB proteins?

RecBCD interacts directly with the RecA and promotes its assembly Direct protein-protein interaction

What is the DSB-repair pathway referred to in E.coli?

RecBCD pathway

Does RecB or RecD move faster?

RecD moves faster

In eukaryotes, what helicase is involved in Holliday junction resolution?

RecQ

The MAT locus is always the ____________ partner during recombination.

Recipient

What must be complete for the first nuclear division to occur?

Recombination

In bacteria, the major biological role of homologous recombination is to ____________.

Repair DSBs

When is homologous recombination required in eukaryotes?

Required for DNA repair Required for restarting a stalled replication fork

When does Spo11 cleavage occur?

Right around the time when the replication homologous chromosomes begin to pair

What is the specificity of RuvA?

RuvA recognizes the structure of the DNA junction, regardless of its sequence

What is the major Holliday junction resolving endonuclease in bacteria?

RuvC

During what phase of meiosis are the sister chromatids replicated? What is the total DNA content after this replication?

S phase 4n

In Holliday junction resolution, strands with the same ____________ and ____________ must be cleaved.

Sequence and polarity

Ho is a ____________-____________ endonuclease that only cuts at the ____________ locus.

Sequence specific MAT

Why must the invading ssDNA tail have a 3' end?

So that they can serve as primers for new DNA synthesis

What is the product of Holliday junction resolution if the cut is made through the strand that contains all parental DNA?

Splice product

The enzymes that catalyze strand invasion are called ____________-____________ ____________.

Strand-exchange proteins

A central feature of homologous recombination is that it can occur between any two regions of DNA, regardless of the sequence, provided these regions are.... Why is this true?

Sufficiently similair None of the steps in homologous recombination require recognition of a highly specialized DNA sequence

What strand of DNA is resected by the MRX-enzyme complex?

The 5' terminating strand that contains the Spo11 This results in a 3' tail being formed on the opposite strand

What needs to happen for homologous recombination to occur?

The DNA must have a DSB

Frequency of recombination between any two genes is generally proportional too...

The distance between those genes

What model of recombination is applicable when a double-strand break initiates homologous recombination?

The double strand break repair pathway

What is the product of Holliday junction resolution if the cut is made through the strand that contains regions from both parental strands?

The patch product or the noncrossover product

What Holliday junction cleaving product results in the reassortment of genes? What is another name for this product?

The splice product The crossover product

What effect does the absence of recombination have on meiosis?

There is a high incidence of chromosome loss because chromosomes often fail to align properly

What happens once the 3' ssDNA strands have been formed?

They are coated in Rad51 and Rad52 proteins and search homologous regions to start strand invasion

What happens to the ssDNA tails generated by degradation of the parent molecule?

They invade the unbroken homologous DNA duplex

How is recombination finished?

Through resolution of the Holliday junction by cutting the DNA strands near the site of the cross

Meiosis involves ____________ rounds of nuclear division, resulting in the reduction of DNA content from normal content of ____________ cells to content present in ____________.

Two Diploid Gametes

Two subunits of Spo11 cleave the DNA ____________ nucleotides apart to form a ____________ DSB.

Two Staggered

What end of DNA is most likely to be coated by RecA?

the 3' end because the RecA filament is added in the 5' to 3' direction

Moveable DNA segments called ____________ occasionally "jump" around chromosomes and DNA rearrangements.

transposons

ssDNA tails terminate with ____________ ends.

3'

What are the key steps of homologous recombination?

1) Alignment of two homologous DNA molecules: DNA sequences that are identical or nearly identical line up 2) Introduction of breaks in the DNA: once the breaks are formed, the ends are processed to form regions of single-stranded DNA 3) Strand invasion: short regions of base pairing are formed when a single-stranded region of DNA from one parental molecule pairs with its complementary strand in the homologous duplex DNA molecule 4) Formation of the Holliday junction: after strand invasion, two DNA molecules become connected by crossing DNA strands that form the Holliday Junction. This moves along the DNA through a process called branch migration 5) Resolution of the Holliday junction: DNA molecules are regenerated once the genetic exchange is completed

What are three characteristics of DNA molecules that allow RecA to work?

1) DNA sequence complementarity between the two partner molecules 2) A region of ssDNA on at least one molecule to allow RecA to bind 3) The presence of a DNA end within the region of complementarity that allows the strands to intertwine

What three events happen when the RecBCD complex comes in contact with a Chi site?

1) The ssDNA loop in front of RecB is pulled through and RecB becomes the leading helicase 2) The RecD subunit dissociates 3) The nuclease activity is ended on the strand containing the chi site and heightened on other strand

Chi sites increase the frequency of recombination ____________-fold.

10

How is RecBCD activity controlled?

By Chi sites They increase the frequency of homologous recombination

How are double strand breaks formed?

By exposure to ionizing radiation of damage to DNA that interferes with the replication fork

How does the double strand break repair pathway start?

By introduction of a double strand break in one of two homologous duplex DNA molecules

How does recombination control the mating type of budding yeast?

By switching which mating type genes are present at a specific location that is being expressed in the organism's genome

How do double-strand breaks arise in E. coli?

DNA damage or collapse of a replication fork

Recombination involves the physical exchange of ____________ between ____________.

DNA Chromosomes

Before division in meiosis, the cell has how many copies of each chromosome? What are these copies called? Where do these come from?

Two copies Called homologs One each was inherited from two parents

What happens after strand invasion by the RecA-DNA complex? What does movement of this feature require?

Two recombining DNA molecules are connected by a Holliday junction Movement of the Holliday junction requires exchange of DNA base pairs between the two homologous DNA duplexes

When does branch migration create DNA duplexes carrying one or a few sequence mismatches?

When the two parental strands have slight sequence difference

Does RecC contribute to the efficiency of the helicase activity?

Yes

Does the Holliday junction require energy to move? If so, where does it get it from?

Yes, energy is needed to move the branch rapidly in one direction This energy comes from the ATPase activity of RuvB

Can the cut made by Spo11 be easily reversed? How?

Yes, they can be easily reversed due to the energy stored in the covalent bond between the Spo11 and the DNA

Are genes shuffled when homologous recombination takes place during meiosis?

Yes, this ensures that there is a variation in the set of genes passed to the next generation

Strand exchange is the ____________ step in homologous recombination because it is this invasion and then pairing of complementary strands between the two homologous duplexes that establishes the stable pairing between these molecules.

central