1. Molecular Biology Fourth Edition
Lecture PowerPoint to accompany
Molecular Biology Fourth Edition
Robert F. Weaver
Chapter 22
Homologous Recombination
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

2. 22.1 Homologous Recombination Pathways

3. RecBCD Pathway – Initial Binding
RecBDC-sponsored homologous recombination in E. coli:
DNA helicase activity unwinds the DNA toward a Chi-site
Sequence 5’-GCTGGTGG-3’
Chi sites found on average every 5000 bp in E. coli genome
RecBCD protein has
ds- and ss-exonuclease activity
ss-endonuclease activity
Activities permit RecBCD to produce a ss-tail now coated by RecA protein

4. The RecBCD Pathway Schematic
RecBCD pathway is a well-studied homologous recombination pathway used by E. coli

5. RecBCD Pathway – D Loop
Invasion of a duplex DNA by a RecA-coated single-stranded DNA from another duplex that has suffered a double-stranded break
Invading strand forms a D loop (displacement)
Loop is defined by displaced DNA strand
When tail finds homologous region, nick occurs in in D-looped DNA
Nick allows RecA and ss-break create a new tail that can pair with gap in the other DNA
Subsequent degradation of the D-loop strand leads to the formation of a branched intermediate

6. Holliday Junctions
Branch migration in this intermediate yields a Holliday junction with 2 strands exchanging between homologous chromosomes
Branch in the Holliday junction can migrate in either direction by breaking old base pairs and forming new ones in a process called branch migration
This migration process does not occur at a useful rate spontaneously
DNA unwinding required
Unwinding requires helicase activity and energy from ATP

7. Resolving Holliday Junctions
Holliday junctions can be resolved by nicking 2 of its strands
Yielding:
2 noncrossover recombinant DNAs with patches of heteroduplex
2 crossover recombinant DNAs that have traded flanking DNA regions

8. 22.2 Experimental Support for the RecBCD Pathway - RecA
The recA gene has been cloned and overexpressed with abundant RecA protein available for study
It is a 38-kD protein that can promote a variety of strand exchange reactions
There are 3 stages of participation of RecA in strand exchange
Presynapsis – RecA coats the ss-DNA
Synapsis – alignment of complementary sequences in ss- and ds-DNAs
Postsynapsis – ss-DNA replaces the (+) strand in ds-DNA to form a new double helix
Joint molecule is an intermediate in this process

9. Presynapsis In the presynapsis step of recombination:
RecA coats a ss-DNA participating in recombination
SSB accelerates the recombination process
Melting secondary structure
Preventing RecA from trapping any secondary structure that would inhibit strand exchange later in the recombination process

10. Synapsis: Synapsis is the proper alignment of complementary sequences
Synapsis occurs when:
Single-stranded DNA finds a homologous region in a double-stranded DNA
This ss-DNA aligns with the ds-DNA
No intertwining of the 2 DNAs occurs at this point

11. Postsynapsis: Strand Exchange
RecA and ATP collaborate to promote strand exchange between ss- and ds-DNA
ATP is necessary to clear RecA off the synapsing DNAs
This makes way for formation of ds-DNA involving the single strand and one of the strands of the DNA duplex

12. RecBCD RecBCD has a DNA endonuclease activity
Nicks ds-DNA especially near Chi sites
ATPase-driven DNA helicase activity that can unwind ds-DNA from their ends
The activities help RecBCD provide the ss-DNA ends that RecA needs to initiate strand exchange

13. RuvA and RuvB
RuvA and RuvB form a DNA helicase that can drive branch migration
RuvA tetramer with square planar symmetry recognizes the center of a Holliday junction and binds to it
Likely induces the Holliday junction itself:
To adopt a square planar conformation
To promote binding of hexamer rings of RuvB to 2 diametrically opposed branches of the Holliday junction
RuvB uses its ATPase to drive the DNA unwinding and rewinding necessary for branch migration

14. A Synthetic Holliday Junction
Mix oligonucleotides at annealing conditions for complementary base-pairing
5’-end of oligo 2 base-pairs with the 3’-end of oligo 1
5’-end of oligo 1 base-pairs with the 3’-end of oligo 2
Ends cross over in pairing

15. RuvC
Resolution of Holliday junctions is catalyzed by the RuvC resolvase
This protein acts as a dimer to clip 2 DNA strands to yield either patch or splice recombinant products
Clipping occurs preferentially at the consensus sequence 5’-(A/T)TT(G/C)-3’
Branch migration is essential for efficient resolution of Holliday junctions
Essential to reach preferred cutting sites
RuvA, B, and C work together in a complex to locate and cut those sites

16. Resolution of a Holliday Junction
Holliday junction can be resolved in 2 ways:
Cuts 1 and 2 yield 2 duplex DNAs with patches of heteroduplex
Cuts 3 and 4 yield crossover recombinant molecules with the 2 parts joined by a staggered splice

17. 22.3 Meiotic Recombination
Meiosis in most eukaryotes is accompanied by recombination
This process shares many characteristics with homologous recombination in bacteria
This section focuses on meiotic recombination in yeast

18. Mechanism Overview Start with chromosomal lesion: ds-DNA break
Next exonuclease recognizes the break
Digests the 5’-end of the 2 strands
Creates 3’-single strand overhangs
One single-stranded end can invade other DNA duplex, forming a D loop
DNA repair synthesis fills in the gaps in the top duplex expanding the D loop
Branch migration can occur in both directions leading to 2 Holliday junctions
Holliday junctions can be resolved to yield either a noncrossover or a crossover recombinant

19. Model of Yeast Recombination

20. The Double-Stranded DNA Break
DNA cleavage uses 2 Spo11
Active site Tyr as OH
Attack 2 DNA strands at offset positions
Transesterification reaction breaks phosphodiester bonds within DNA strands
Creates new bonds
Nicking DNA strands
Nicking is asymmetric
Yields 2 sizes oligos
Release of Spo11-linked oligos nt long

21. DSB End Resection Resection occurs on both strands using prior nicks
Recombinases load asymmetrically onto the newly created single-stranded regions
One protein tags coated free 3’-end for invasion into homologous duplex
This leads to initiating Holliday complex formation

22. Creation of Single-Stranded Ends at DSBs
Formation of the DSB in meiotic recombination is followed by 5’3’ exonuclease digestion of the 5’-ends at the break
Digestion yields overhanging 3’-ends that can invade another DNA duplex
Rad50 and Mre11 collaborate to carry out this reaction

23. 22.4 Gene Conversion
When 2 similar, non-identical DNA sequences interact, possibility exists for gene conversion
Conversion of one DNA sequence into that of another
Sequences participating in gene conversions can be:
Alleles, as in meiosis
Nonallelic genes, such as the MAT genes that determine mating type in yeast

24. Gene Conversion Model
Strand exchange event with branch migration during sporulation has resolved to yield 2 duplex DNAs with patches of heteroduplex

25. Gene Conversion Without Mismatch Repair
Consider from the middle of the DSB recombination scheme
Invading strand is partially resected
DNA repair synthesis more extensive
Branch migration and resolution do not change nature of the 4 DNA strands