DNA double-strand breaks associated with replication forks are predominantly repaired by homologous recombination involving an exchange mechanism in mammalian cells
C. Arnaudeau et al., DNA double-strand breaks associated with replication forks are predominantly repaired by homologous recombination involving an exchange mechanism in mammalian cells, J MOL BIOL, 307(5), 2001, pp. 1235-1245
DNA double-strand breaks (DSB) represent a major disruption in the integrit
y of the genome. DSB can be generated when a replication fork encounters a
DNA lesion. Recombinational repair is known to resolve such replication for
k-associated DSB, but the molecular mechanism of this repair process is poo
rly understood in mammalian cells. In the present study, we investigated th
e molecular mechanism by which recombination resolves camptothecin (CPT)-in
duced DSB at DNA replication forks. The frequency of homologous recombinati
on (HR) was measured using V79/SPD8 cells which contain a duplication in th
e endogenous hprt gene that is resolved by HR. We demonstrate that DSB asso
ciated with replication forks induce HR at the hprt gene in early S phase.
Further analysis revealed that these HR events involve an exchange mechanis
m. Both the irs1SF and V3-3 cell lines, which are deficient in HR and non-h
omologous end joining (NHEJ), respectively, were found to be more sensitive
than wild-type cells to DSB associated with replication forks. The irs1SF
cell line was more sensitive in this respect than V3-3 cells, an observatio
n consistent with the hypothesis that DSB associated with replication forks
are repaired primarily by HR. The frequency of formation of DSB associated
with replication forks was not affected in HR and NHEJ deficient cells, in
dicating that the loss of repair, rather than the formation of DSB associat
ed with replication forks is responsible for the increased sensitivity of t
he mutant strains. We propose that the presence of DSB associated with repl
ication forks rapidly induces HR via an exchange mechanism and that HR play
s a more prominent role in the repair of such DSB than does NHEJ. (C) 2001
Academic Press.