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

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

Flashcards in Molecular Biology Lecture 10 - DBS break repair NHEJ Translesion DNA synthesis Homologous recombination Deck (16):
1

Functions of homologous recombination

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

2

Double-strand breaks initiate homologous recombination

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

3

Holliday model

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

4

Branch Migration

..

5

Holiday Model

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

6

Splice

Cross over product or reassortments of flanking genes

7

Patch

Non crossover

8

DSB-repair model

...

9

Gene conversion and the DSB-repair model

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

10

RecBCD pathway - RecBCD

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

11

Homologous recombination in eukaryotes

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

12

Eukaryotes - Spo11 generates DS breaks

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

13

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

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

14

MRX complex processes DS breaks
Rad51 and Dmc1 - strand-exchange proteins

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

15

RecBCD (DSB) pathway - overview

E. coli Eukaryotes
None Spo11; HO
RecBCD MRX

RecA Rad 51, Dmc1


RuvA,B unknown

RuvC unknown

16

Mating-type switching in yeast -
Unidirectional gene conversion

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