Molecular Biology Lecture 10 - DBS break repair NHEJ Translesion DNA synthesis Homologous recombination

Question 1

Functions of homologous recombination

Answer 1

1. Meiosis – chromosome pairing and segregation, crossing over, genetic variation
2. DNA repair
3. Restart stalled replication forks
4. Specialized gene expression systems
   
   • mating-type switching in yeast
   • phase variation in trypanosomes
     
     • VDJ joining in immunoglobulin genes

Question 2

Double-strand breaks initiate homologous recombination

Answer 2

Bacteria  
	- DSB repair - major function of homologous recomb.
	- genetic exchange
	     - conjugation
	     - transduction
Eukaryotes
	- essential part of chromosome pairing and segregation 		during meiosis
	- not just a byproduct of meiosis

Question 3

Holliday model

Answer 3

a. alignment of two homologous chromosomes (non-sister chromatids
b. SS nicks in each chromosome
c. strand invasion and ligation

branch migration and heteroduplex formation

Question 4

Branch Migration

Answer 4

..

Question 5

Holiday Model

Answer 5

1. Two single-strand breaks
2. Single Holliday junction
3. Heteroduplex (mismatch) formation
4. Resolution of Holiday junction
   - 50% of the time no recombination
   - 50% of the time recombination

Question 6

Splice

Answer 6

Cross over product or reassortments of flanking genes

Question 7

Patch

Answer 7

Non crossover

Question 8

DSB-repair model

Answer 8

…

Question 9

Gene conversion and the DSB-repair model

Answer 9

What is gene conversion?
One allele is converted to another allele.
Normal segregation during meiosis is 2:2.
Gene conversion segregation - 3:1.

Question 10

RecBCD pathway - RecBCD

Answer 10

1. E. coli pathway; eukaryotic similarities
2. Bind DSB
   
   • E. coli utilizes naturally occurring DSBs
3. 5’ → 3’ and 3’ → 5’ helicases; nuclease
4. Chi site (χ) found and bound - RecC
5. Inhibit digestion of 3’ end, stimulate digestion of 5’ end
6. Result in 3’ SS for strand invasion

Question 11

Homologous recombination in eukaryotes

Answer 11

Functions
- alignment of homologous chromosomes
- proper segregation of homologous chromosomes
- genetic recombination
Mutations in recombination have segregation defects.

Question 12

Eukaryotes - Spo11 generates DS breaks

Answer 12

1. Homologous recombination started by DSB induction
2. Spo11
   
   • cuts DS DNA at many sites (open chromatin)
   • active in early prophase
   • tyrosine induces SS break
   • two subunits
3. two-base 5’ overhang

Question 13

Eukaryoties - MRX complex processes DS breaks - 5’ to 3’ resection

Answer 13

1. MRX binds to DSB generated by Spo11
2. 5’ to 3’ nuclease
3. leaves 3’ SS for strand exchange proteins

Question 14

MRX complex processes DS breaks

Rad51 and Dmc1 - strand-exchange proteins

Answer 14

1. Strand-exchange proteins, Dmc1 and Rad51 bind
2. Induce strand invasion (strand-exchange)
3. Occurs at 4-strand stage
4. Preferentially between non-sister chromatids (Dmc1)
5. Picture implies that Dmc1 coats one strand, and Rad51 coats the other - not the case
6. Eukaryotic proteins analogous to RuvAB and RuvC are not known for sure

Question 15

RecBCD (DSB) pathway - overview

Answer 15

E. coli Eukaryotes
None Spo11; HO
RecBCD MRX

RecA Rad 51, Dmc1

RuvA,B unknown

RuvC unknown

Question 16

Mating-type switching in yeast -

Unidirectional gene conversion

Answer 16

Mating type is determined by genotype of the MAT locus.
- MATa = a; MATα = α
HML α and HMRa are silent (not transcribed) loci.