Bentzen 4 - Molecular Basis of Recombination

Question 1

What is homologous recombination? When does it occur?

What is the progeny result and what does it maintain?

Answer 1

The exchange of genetic material between two homologous DNA molecules. Commonly occurs as crossing over during prophase I of meiosis. Can also occur during mitosis.

Results in diversity among the progeny derived from common parents. Maintains somatic stability by promoting repair of double strand breaks caused by ionizing radiation and other agents.

Question 2

What is the Holliday of homologous recombination? What results?

Answer 2

Proposes 5 steps for homologous recombination.

1. Homologous chromosomes are aligned
2. Sigle strands break (not at base pairs, but between backbone)
3. Strand invasion occurs, where one single strand attacks another on the other chromosome
4. A Holliday junction is formed
5. Branch migration moves the Holliday junction down the molecules.

This results in heteroduplex DNA, which consists of one nucleotide strand from each homologous chromosome. Resolution (splitting) of the Hollidary junction leads to either crossover recombinants (vertical plane) or noncrossover recombinants (horizontal plane)

Question 3

What is the resolution of the Holliday Junction?

Answer 3

Cleavage through the horizontal plane or the vertical plane of the Hollidary Junction. This results in noncrossover recombinants (horizontal plane) or crossover recombinants (vertical plane)

Question 4

What is the Double Strand Break model of homologous recombination?

Answer 4

• See animation -

1. Two DNA duplex molecules align
2. One duplex experiences a double stranded break (both strands in duplex break)
3. 5’ Ends at the break are degraded to produce 3’ overhangs.
4. Strand invasion into homologous sequence
5. Extension of 3’ end of invading strand
6. Displaces strand serves as template
7. New synthesized DNA is made to fill in gaps, which results in the formation of two Holliday junctions
8. The resolution of the Holliday junctions can result in either noncrossover (horizontal -H plane) or crossover products (vertical -V plane)

Question 5

What enzymes are required for recombination in E. coli? What pathway are they involved in?

Answer 5

Enzymes involved in the RecBCD pathway:

RecBCD complex - an enzyme complex coded by 3 genes (RecB, RecC and RecD).

RecA - assembles on the single stranded DNA and promotes strand invasion and pairing with homologous DNA

RuvAB complex - promotoes branch migration and heteroduplex formation

RuvC resolvase - Is the endonuclease that resolves the Holliday junction. Nicks strands for either horizontal or vertical plane resolution.

Question 6

What does RecBCD do in the RecBCD pathway? What type of activity does it have? What does it require? When does it occur?

Answer 6

Binds to a free duplex at the double strand break.

Combines both helicase and nuclease activity.

Requires ATP

Serves in recombinational repair

Occurs following conjugation

Question 7

What are the 6 steps for generation of a 3’ single stranded terminus by RecBCD?

Answer 7

1. RecBCD binds at double strand break
2. Initiates unwinding of DNA duplex
3. Continues unwinding, cleaving 3’ strand much more frequently than 5’ strand
4. Encounters Chi sequence; stops digesting 3’, increases cleavage of 5’ strand
5. Loads RecA onto (now protruding) 3’ strand
6. RecBCD unbinds from DNA, leaving RecA nucleo-protein filament on 3’

Question 8

What does RecA do in the RecBCD pathway?

Answer 8

After it is unloaded onto the 3’ strand by RecBCD, it assembles on the single stranded DNA and promotes strand invasion and pairing with homologous DNA.

Question 9

What does the RuvAB complex do in the RecBCD pathway? What do RuvA and RuvB do?

Answer 9

Promotes the branch migration and heteroduplex formation.

RuvA recognizes the Holliday junnction, RuvB then binds to RuvA/DNA complex

RuvB drives DNA unwinding and rewinding that is necessary for branch migration

Question 10

What does the RuvC resolvase do in the RecBCD pathway?

Answer 10

It is the endonuclease that resolves the Holliday junction. It Nicks strands for either horizontal or vertical plane resolution.

Question 11

What is gene conversion?

Answer 11

It is a related phenomenon to recombination where genes are transferred between homologous chromosomes. It results in abnormal ratios of games following meiosis.

Question 12

What causes gene conversion?

Answer 12

Heteroduplex formation during some recombination events. Heteroduplexes with mismatched bases are repaired, using one strand or the other as template for correction.

Question 13

What is the result of gene conversion?

Answer 13

Conversion of one copy of the gene to the alternate allele.

Question 14

Why do haploids make the best organisms to study for recombination?

Answer 14

All genes are expressed, so alternative alleles are easy to track. In some species tetrads are preserved in order and can be analyzed for crossing over and gene conversion.

Question 15

What ration of A:a alleles would you expect in a first-division segregation pattern (MI)? Does crossing over occur?

Answer 15

There is no crossing over, would expect to see an octad ration of A:a of 4:4

Question 16

What ratio of octad alleles would you expect in second-division segregation pattern (MII)? Is there crossing over?

Answer 16

4:4

There is crossing over (recombination and separation)

Question 17

How can you detect gene conversion with tetrad analysis?

Answer 17

Looking for aberrant segregation (deviation from the expected 4:4 ratio).

Question 18

What are two aberrant segregation types in tetrad analysis? What are the ratios of each?

Answer 18

Gene conversion (6:2 and 2:6)

Post-meiotic segregation (5:3 and 3:5)
- Failure to repair mismatch leads to post-meiotic segregation.