Competition between Replicative and Translesion Polymerases during Homologous Recombination Repair in Drosophila
McVey M (2012) Competition between Replicative and Translesion Polymerases during Homologous Recombination
Repair in Drosophila. PLoS Genet 8(4): e1002659. doi:10.1371/journal.pgen.1002659
Competition between Replicative and Translesion Polymerases during Homologous Recombination Repair in Drosophila
Daniel P. Kane 0
Michael Shusterman 0
Yikang Rong 0
Mitch McVey 0
R. Scott Hawley, Stowers Institute for Medical Research, United States of America
0 1 Department of Biology, Tufts University , Medford , Massachusetts, United States of America, 2 National Cancer Institute , Bethesda , Maryland, United States of America, 3 Program in Genetics, Tufts Sackler School of Graduate Biomedical Sciences , Boston, Massachusetts , United States of America
In metazoans, the mechanism by which DNA is synthesized during homologous recombination repair of double-strand breaks is poorly understood. Specifically, the identities of the polymerase(s) that carry out repair synthesis and how they are recruited to repair sites are unclear. Here, we have investigated the roles of several different polymerases during homologous recombination repair in Drosophila melanogaster. Using a gap repair assay, we found that homologous recombination is impaired in Drosophila lacking DNA polymerase zeta and, to a lesser extent, polymerase eta. In addition, the Pol32 protein, part of the polymerase delta complex, is needed for repair requiring extensive synthesis. Loss of Rev1, which interacts with multiple translesion polymerases, results in increased synthesis during gap repair. Together, our findings support a model in which translesion polymerases and the polymerase delta complex compete during homologous recombination repair. In addition, they establish Rev1 as a crucial factor that regulates the extent of repair synthesis.
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Funding: This research was supported by grants from the National Science Foundation (MCB-0643253) and the National Institutes of Health (R01GM092866) to
MM. Research in the YR lab is supported by the intramural research program of the National Cancer Institute. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
DNA double-strand breaks (DSBs) pose a serious threat to cell
viability and genome integrity. DSBs can be repaired either by
non-homologous end joining, in which the DSB ends are
processed and directly ligated, potentially leading to loss of
information and mutagenesis (reviewed in [1]), or by a group of
repair mechanisms collectively known as homologous
recombination (HR). During HR, DNA sequence that is lost due to the
original damage event or during subsequent processing is
recovered through invasion of a nearby template and copying of
this sequence into the break site. Because HR makes use of an
intact, homologous template, it is generally considered to be a
conservative process. However, several studies have shown that
HR repair can also be mutagenic, resulting in an increased
mutation frequency both at the original break site [2] and at
nearby sequences [3].
The initial events of HR involve the creation of single-stranded
39 DNA ends, which are then coated with the Rad51 protein to
form a nucleoprotein filament that conducts a genome-wide
homology search (reviewed in [4]). Upon identification of a
homologous template, a displacement loop (D-loop) is formed in
which the duplex template is unwound and the invading broken
strand pairs with its complement. This D-loop extends and/or
migrates as repair synthesis continues. In one model of HR,
termed synthesis-dependent strand annealing, the invading strand
dissociates and anneals to single-stranded DNA on the broken
duplex [5]. Single-stranded gaps are then filled in and the broken
ends are ligated to complete repair.
Two general types of polymerases are potentially available for
DNA synthesis during HR repair. Replicative polymerases are
highly processive and replicate the bulk of DNA during S phase
(reviewed in [6]). In contrast, translesion synthesis (TLS)
polymerases are specialized for replication of damaged or
abnormal templates (reviewed in [7,8,9]). Previous studies have
provided conflicting results with regard to whether replicative or
translesion DNA polymerases are predominantly used during HR
repair synthesis.
In the budding yeast Saccharomyces cerevisiae, the catalytic subunits
of the replicative polymerases (pol) delta and epsilon play
important roles in repair synthesis during HR [2,10,11,12].
Recently, purified pol delta from budding yeast was shown to
efficiently extend D-loops in the presence of the polymerase clamp
PCNA [13], confirming the in vivo findings. In addition, a
nonessential subunit of pol delta, Pol32, is required for break-induced
replication, a form of HR that requires extensive DNA synthesis
[14].
TLS polymerases have also been implicated in HR repair. In
chicken (...truncated)