Drosophila DNA polymerase theta utilizes both helicase-like and polymerase domains during microhomology-mediated end joining and interstrand crosslink repair.

Kelly Beagan,Alice Witsell,Robin L. Armstrong,Orlando D. Schärer,Upasana Roy,Mitch McVey,Amy E. Baker,Nikolai Renedo,Lorraine S. Symington

doi:10.1371/journal.pgen.1006813

Abstract

Double strand breaks (DSBs) and interstrand crosslinks (ICLs) are toxic DNA lesions that can be repaired through multiple pathways, some of which involve shared proteins. One of these proteins, DNA Polymerase θ (Pol θ), coordinates a mutagenic DSB repair pathway named microhomology-mediated end joining (MMEJ) and is also a critical component for bypass or repair of ICLs in several organisms. Pol θ contains both polymerase and helicase-like domains that are tethered by an unstructured central region. While the role of the polymerase domain in promoting MMEJ has been studied extensively both in vitro and in vivo, a function for the helicase-like domain, which possesses DNA-dependent ATPase activity, remains unclear. Here, we utilize genetic and biochemical analyses to examine the roles of the helicase-like and polymerase domains of Drosophila Pol θ. We demonstrate an absolute requirement for both polymerase and ATPase activities during ICL repair in vivo. However, similar to mammalian systems, polymerase activity, but not ATPase activity, is required for ionizing radiation-induced DSB repair. Using a site-specific break repair assay, we show that overall end-joining efficiency is not affected in ATPase-dead mutants, but there is a significant decrease in templated insertion events. In vitro, Pol θ can efficiently bypass a model unhooked nitrogen mustard crosslink and promote DNA synthesis following microhomology annealing, although ATPase activity is not required for these functions. Together, our data illustrate the functional importance of the helicase-like domain of Pol θ and suggest that its tethering to the polymerase domain is important for its multiple functions in DNA repair and damage tolerance.

Highlights

DNA double strand breaks (DSBs) and interstrand crosslinks (ICLs) compromise both strands of the DNA duplex and must be repaired to ensure cellular survival
Polymerase θ (Pol θ) is an A-family polymerase with homology to E. coli Pol I and contains an N-terminal helicase-like domain connected to the polymerase domain through a long, unstructured central domain [7,8,9]
We show that purified Pol θ can bypass model unhooked ICL substrates and promote the initial steps of microhomology-mediated end joining (MMEJ) in vitro, with minimal requirement for ATPase activity

Summary

Introduction

DNA double strand breaks (DSBs) and interstrand crosslinks (ICLs) compromise both strands of the DNA duplex and must be repaired to ensure cellular survival. While DSBs disrupt the physical integrity of DNA, ICLs tether DNA strands together through a covalent bond. Both types of lesions can impede critical processes such as replication and transcription. In Drosophila melanogaster, DNA Polymerase θ (Pol θ) has emerged as one of these dualfunction proteins It was first identified in a mutagen sensitivity screen where mutations in mus308, the gene encoding Pol θ, caused hypersensitivity to ICL-inducing agents but not to other DNA alkylating agents [5, 6]. Pol θ is an A-family polymerase with homology to E. coli Pol I and contains an N-terminal helicase-like domain connected to the polymerase domain through a long, unstructured central domain [7,8,9] Both the polymerase and helicase domains are conserved among metazoans. The mechanisms by which Pol θ promotes ICL repair remain uncharacterized

Methods

Results

Discussion

Conclusion