from Cooper, 2nd ed', Fig' 530

1
Homologous Recombination
Allows mutations to be separated and tested in
new assortments
Needed to repair damaged DNA
from Cooper, 2nd ed., Fig. 5-30
2
Double-strand-break Repair Model for Homologous
Recombination
Recombination is initiated by ds break
Free 3-end invades the homologous region of the
other duplex
Repair synthesis and branch migration
The structure is resolved by nicking
from Kowalczykowski, Trends Biochem.Sci. 25, 156
(2000)
3
Role of RAD50 in Generating and Processing ds
Breaks
Recombination hot spots are sites which undergo
ds breaks
RAD50 converts the flush end into a
single-stranded protrusion
Recombination hot spots in bacteria correspond to
c sites
4
Initiation of Homologous Recombination in E. coli
RecBCD binds to ds ends, unwinds DNA and
degrades DNA using a 3 to 5 exonuclease
DNA is cleaved at the c site using an
endonuclease, and activation of a 5 to 3
exonuclease generates a 3-overhang
RecBCD loads RecA onto c-containing ssDNA
excluding SSB
RecA promotes invasion of the homologous dsDNA
from Kowalczykowski, Trends Biochem.Sci. 25, 156
(2000)
5
Branch Migration and Resolution of Holliday
Junctions
RuvAB is a helicase that stimulates branch
migration
RuvC is a Holliday junction-specific endonuclease
from Cooper, 2nd ed., Fig. 5-37
6
Causes of and Responses to Double Stranded Breaks
DSBs result from exogenous insults and normal
processes
DSBs can result in cell death or cell cycle
arrest by checkpoint activation
Inaccurate repair leads to mutations and
chromosomal aberrations
from van Gent et al., Nature Rev.Genet. 2, 196
(2001)
7
Generation of Double-stranded DNA Breaks and
Single-stranded DNA Gaps
DNA damage causes replication fork collapse
DNA synthesis is reinitiated in an
oriC-independent manner
from Kowalczykowski, Trends Biochem.Sci. 25, 156
(2000)
8
Mechanism of Recombination-dependent Replication
ds breaks are repaired by homologous recombination
Recombination-dependent replication initiates as
D loops
Recombination confers a great selective advantage
RecA mutants have a high lethality
from Kowalczykowski, Trends Biochem.Sci. 25, 156
(2000)
9
Homologous Recombination Repairs ds Breaks
ssDNAs with 3ends are formed and coated with
Rad51, the RecA homolog
Rad51-coated ssDNA invades the homologous dsDNA
in the sister chromatid
The 3-end is elongated by DNA polymerase, and
base pairs with ss 3-end of the other broken DNA
DNA polymerase and DNA ligase fills in gaps
from Lodish et al., Molecular Cell Biology, 5th
ed. Fig 23-31