Genetic Recombination

Question 1

Recombination

Answer 1

Breaking and rejoining DNA molecules in new combinations.

Leads to production of offspring with combinations of traits that differ from those found in either parent.

Question 2

Types of Recombination

Answer 2

Homologous recombination: Nucleotide sequences are exchanged between two similar or identical molecules of DNA. e.g. meiotic recombination during meiosis I

Site-specific recombination: DNA strand exchange takes place between segments possessing at least a certain degree of sequence homology.

Question 3

Role of cohesins in genetic recombination

Answer 3

Searching for homologous sequences is facilitated by cohesins.
Cohesins hold sister chromatids together.

Question 4

Holliday model

Answer 4

A model for homologous recombination.

1. Single-strand breaks occur at the same point on one strand of each parental DNA.
2. Free ends of each broken strand then migrate across to the other DNA helix.
3. Forms a Holliday Junction/Intermediate (Like a 4 way intersection) by kinking the duplexes at the centre and rotating the lower half by 180 degrees.
4. Holliday Junction then resolved into the now recombinated DNA sequences.

Question 5

Branch migration

Answer 5

The process by which base pairs on homologous DNA strands are consecutively exchanged at a Holliday junction, moving the branch point up or down the DNA sequence.

Question 6

Double-Strand Gap Repair Model

Answer 6

1. Double Strand Break
2. End resection (Generates a long 3′ single-stranded DNA tail that can invade the homologous DNA strand.)
3. Strand Invasion
4. Repair

Question 7

Factors Leading to the Generation of Holliday Junctions

Answer 7

1. Strand Invasion (Recombination)
2. Double Strand Breaks (DNA repair)
3. Replication Fork Reversion (Replication, Fork Regression Model)

Question 8

Homologous genetic recombination in E. coli (Enzymes and their role)

Answer 8

RecBCD: Creation of Recombinogenic End
RecA: Strand Invasion
RuvAB and RecG: Branch Migration
RuvC: Resolution of Holliday Junction to Recombinated Sequences

Question 9

RecBCD

Answer 9

Creation of Recombinogenic End
A Nuclease and a Helicase.
Enzyme progresses along duplex, driven by bipolar BD helicase motors. During this the 3’ tail is digested.
RecC recognises Chi sequence, stopping digestion of the 3’ tail. 5’ tail is now able to access nuclease, and is degraded.

Question 10

RecA (and Filament)

Answer 10

Strand Invasion
DNA-dependent ATPase (Weak)
RecA binds to ss DNA to generate filament :
Rec A filament is the active species in strand exchange.
It is polar and binds in in a 5’ to 3’ direction (i.e. migrates to 3’ end of molecule).
Filament contains 1 monomer of RecA per 4-9 nucleotides of DNA. Promotes homologous pairing.
ATP not required to form filament but addition of ATP increases rate of dissociation.

Question 11

RuvAB

Answer 11

Branch Migration
Part of the ruv operon of E. coli
RuvA + RuvB jointly act on junction, promoting branch migration:
RuvA: Binds selectively to DNA junctions as a tetramer opening the structure to facilitate branch migration.
RuvB: Binds to DNA as a hexamer and exhibits DNA helicase activity. The helicase ‘pumps’ DNA through, conferring directionality.

Question 12

RuvC

Answer 12

Resolution of Holliday Junction to Recombinated Sequences
Structure-selective nuclease targeted to DNA junctions.
Cleaves the Holliday Junction (Also termed a “resolvase”, and is a junction resolving enzyme). Other junction resolving enzymes exists such as T4 endonuclease VII

Question 13

GEN1

Answer 13

GEN1 is a resolving enzyme in humans, not E.coli
Holliday junction 5’ flap endonuclease
Binds discretely to a DNA junction with high affinity.in dimeric form.
GEN1 is able to “slide” along DNA.