Dual Roles for DNA Polymerase Theta in Alternative End-Joining Repair of Double-Strand Breaks in Drosophila

Dual Roles for DNA Polymerase Theta in Alternative End-Joining Repair of Double-Strand Breaks in Drosophila

Sze Ham Chan1#, Amy Marie Yu1#, Mitch McVey1,2*
1 Department of Biology, Tufts University, Medford, Massachusetts, United States of America, 2 Program in Genetics, Tufts Sackler School of Graduate Biomedical Sciences, Boston, Massachusetts, United States of America

Abstract
DNA double-strand breaks are repaired by multiple mechanisms that are roughly grouped into the categories of homology-directed repair and non-homologous end joining. End-joining repair can be further classified as either classical non-homologous end joining, which requires DNA ligase 4, or “alternative” end joining, which does not. Alternative end joining has been associated with genomic deletions and translocations, but its molecular mechanism(s) are largely uncharacterized. Here, we report that Drosophila melanogaster DNA polymerase theta (pol theta), encoded by the mus308 gene and previously implicated in DNA interstrand crosslink repair, plays a crucial role in DNA ligase 4-independent alternative end joining. In the absence of pol theta, end joining is impaired and residual repair often creates large deletions flanking the break site. Analysis of break repair junctions from flies with mus308 separation-of-function alleles suggests that pol theta promotes the use of long microhomologies during alternative end joining and increases the likelihood of complex insertion events. Our results establish pol theta as a key protein in alternative end joining in Drosophila and suggest a potential mechanistic link between alternative end joining and interstrand crosslink repair.

Author Summary
DNA double-strand breaks, in which both strands of the DNA double helix are cut, must be recognized and accurately repaired in order to promote cell survival and prevent the accumulation of mutations. However, error-prone repair occasionally occurs, even when accurate repair is possible. We have investigated the genetic requirements of an error-prone break-repair mechanism called alternative end joining. We have previously shown that alternative end joining is frequently used in the fruit fly, Drosophila melanogaster. Here, we demonstrate that a fruit fly protein named DNA polymerase theta is a key player in this inaccurate repair mechanism. Genetic analysis suggests that polymerase theta may be important for two processes associated with alternative end joining: (1) annealing at short, complementary DNA sequences, and (2) DNA synthesis that creates small insertions at break-repair sites. In the absence of polymerase theta, a backup repair mechanism that frequently results in large chromosome deletions is revealed. Because DNA polymerase theta is highly expressed in many types of human cancers, our findings lay the groundwork for further investigations into how polymerase theta is involved in repair processes that may promote the development of cancer.

Citation: Chan SH, Yu AM, McVey M (2010) Dual Roles for DNA Polymerase Theta in Alternative End-Joining Repair of Double-Strand Breaks in Drosophila. PLoS Genet 6(7): e1001005. doi:10.1371/journal.pgen.1001005

Editor: R. Scott Hawley, Stowers Institute for Medical Research, United States of America

Received: September 15, 2009; Accepted: May 27, 2010; Published: July 1, 2010

Copyright: © 2010 Chan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by a grant from the NSF (MCB-0643253) and by a New Scholar in Aging award to MM from the Ellison Medical Foundation (www.ellisonfoundation.org). 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.

* E-mail: mitch.mcvey@tufts.edu

# These authors contributed equally to this work.

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