Genome instability in the form of chromosome rearrangements is a hallmark of cancer cells and a driver of tumorigenesis. Mounting evidence indicates that an error-prone alternative form of double-strand break repair called microhomology-mediated end joining (MMEJ) promotes chromosome rearrangements associated with DNA deletions by utilizing sequence microhomology to recombine broken DNA. MMEJ is distinct from the classical non-homologous end joining (NHEJ) pathway since it functions in a Ku and Ligase IV independent manner and is therefore referred to as alternative end joining (alt-EJ). Although MMEJ appears to be the major form of alt-EJ, the central mechanism of this elusive pathway remains unknown. Genetic studies in C. elegans and Drosophila, however, suggest a central role for the atypical A-family DNA polymerase theta (Pol?). In preliminary studies, we demonstrate for the first time that the polymerase domain expressed by human POLQ - herein referred to as Pol?-performs MMEJ of DNA containing 3' single-strand DNA (ssDNA) overhangs with two or more base-pairs of homology, including DNA modeled after telomeres. We show that MMEJ is specific to Pol?, is facilitated by hydrogen bond formation between opposing overhangs, and is dependent on Pol? in vivo. Remarkably, we find that Pol? exhibits DNA end joining and microhomology annealing activities separately from its replication function. Yet, the polymerase utilizes the opposing overhang as a template in trans to stabilize the DNA synapse. We further find that Pol? preferentially performs MMEJ of DNA containing a 5'-terminal phosphate, which demonstrates a functional similarity to X-family polymerases involved in NHEJ. Additionally, we identify a conserved insertion loop domain in Pol? that is essential for MMEJ and higher-order structures of the polymerase which likely facilitate DNA tethering. Lastly, we present data suggesting that Pol? exhibits terminal transferase activity, which is thought to contribute to MMEJ. We propose to further characterize the biochemical mechanisms of Pol?and its involvement in MMEJ by developing the following specific aims: 1. To elucidate the mechanism of MMEJ promoted by Pol?; 2. To investigate and characterize terminal transferase activity of Pol?; 3. To characterize the activities of full-lenth Pol? . In summary, these studies will provide new insight into the activities of the atypical A-family DNA polymerase theta, in particular its role in MMEJ of double-strand breaks, and therefore significantly contribute to the DNA repair research community.

Public Health Relevance

Mounting evidence indicates that an error-prone alternative form of double-strand break repair called microhomology-mediated end joining (MMEJ) promotes chromosome rearrangements associated with DNA deletions by utilizing sequence microhomology to recombine broken DNA. Guided by genetic studies in invertebrates, we identify a central role for the atypical DNA polymerase theta (Pol?) in human MMEJ. Elucidating the mechanism of MMEJ promoted by Pol? will provide significant insight into this elusive pathway in human cells, and provide a strong foundation for further research into the overall pathway of mammalian MMEJ.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM115472-04
Application #
9518972
Study Section
Radiation Therapeutics and Biology Study Section (RTB)
Program Officer
Willis, Kristine Amalee
Project Start
2015-07-15
Project End
2020-06-30
Budget Start
2018-07-01
Budget End
2019-06-30
Support Year
4
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Temple University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
057123192
City
Philadelphia
State
PA
Country
United States
Zip Code
19122
McDevitt, Shane; Rusanov, Timur; Kent, Tatiana et al. (2018) How RNA transcripts coordinate DNA recombination and repair. Nat Commun 9:1091
Ozdemir, Ahmet Y; Rusanov, Timur; Kent, Tatiana et al. (2018) Polymerase ?-helicase efficiently unwinds DNA and RNA-DNA hybrids. J Biol Chem 293:5259-5269
Hoang, Trung M; Kent, Tatiana; Pomerantz, Richard T (2017) Modification of 3' Terminal Ends of DNA and RNA Using DNA Polymerase ? Terminal Transferase Activity. Bio Protoc 7:
Mateos-Gomez, Pedro A; Kent, Tatiana; Deng, Sarah K et al. (2017) The helicase domain of Pol? counteracts RPA to promote alt-NHEJ. Nat Struct Mol Biol 24:1116-1123
Black, Samuel J; Kashkina, Ekaterina; Kent, Tatiana et al. (2016) DNA Polymerase ?: A Unique Multifunctional End-Joining Machine. Genes (Basel) 7:
Kent, Tatiana; Mateos-Gomez, Pedro A; Sfeir, Agnel et al. (2016) Polymerase ? is a robust terminal transferase that oscillates between three different mechanisms during end-joining. Elife 5:
Kent, Tatiana; Chandramouly, Gurushankar; McDevitt, Shane Michael et al. (2015) Mechanism of microhomology-mediated end-joining promoted by human DNA polymerase ?. Nat Struct Mol Biol 22:230-7