The repair of broken chromosomes is essential for maintenance of chromosome structure and genome stability. This proposal focuses on delineating the mechanisms of repair of double-strand chromosome breaks (DSBs) by break-induced replication (BIR). BIR plays a key role in restarting stalled and broken DNA replication forks, in maintaining telomeres in the absence of the telomerase enzyme and in the newly discovered phenomenon of single chromosome shattering, known as chromothripsis, which is associated with human cancers and developmental diseases. A microhomology-mediated BIR (MM-BIR) process has been hypothesized to account for long-distance template switching events that lead to the joining of distant sequences to create novel gene fusions. Previous work from this laboratory has identified roles for replication proteins Pol32 and PCNA that are essential for BIR but not for normal replication or other types of DSB repair in the model organism, budding yeast. The proposed project to continue this study will focus on two main topics. First, the molecular mechanism of BIR will be pursued, creating site-specific DSBs by an inducible endonuclease. A novel assay has been developed to study surprisingly frequent template jumps during BIR both between homologous sequences in distant locations or between homeologous sequences (highly mismatched sequences that permit study of MM-BIR). This assay permits one to distinguish between the way in which a broken DNA end locates and recombines with a distant sequence by strand invasion and how the subsequent replicating strands in BIR then can jump to a third location. The role of mismatch repair proteins in discouraging BIR between diverged sequences will be investigated. Evidence that template jumping uses a different mechanism from the initial strand invasion step will be pursued. The possibility that the jump does not require the Rad51 recombinase protein will be explored. Special attention will be devoted to the role of Rdh54, the first protein that is specifically required for template jumps but competent for simple BIR and other recombination events. Whether repair of a broken replication fork, created by nicking a specific strand with a CRISPR endonuclease, obeys the same rules as the ectopic model systems and telomere-repair events that have so far been studied is a fundamentally important question. A second major goal will be to examine translocations and rearrangements in which there is little or no homology at the repair junctions. A novel assay involving creation o a functional intron by joining distant sequences will be used. Finally, template jumps into unrelated sequences will be recovered and analyzed by DNA sequencing in order to better define how much homology and adjacent homeology is required for microhomology-mediated events. These studies are highly significant in understanding the origins of chromosome rearrangements associated with human disease, including the loss of heterozygosity caused by the formation of nonreciprocal translocations, segmental duplications and the astonishing rearrangements in chromosomes experiencing chromothripsis.

Public Health Relevance

The repair of broken chromosomes is essential for maintenance of chromosome structure and genome stability. This proposal focuses on delineating the mechanisms of repair by break- induced replication (BIR) that plays a key role in restarting stalled and broken DNA replication forks, in maintaining telomeres in the absence of the telomerase enzyme and in the newly discovered phenomenon of single chromosome shattering, known as chromothripsis that is associated with human cancers and developmental diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM076020-09
Application #
8755011
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Willis, Kristine Amalee
Project Start
2006-08-01
Project End
2018-07-31
Budget Start
2014-08-01
Budget End
2015-07-31
Support Year
9
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Brandeis University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
City
Waltham
State
MA
Country
United States
Zip Code
02453
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Lemos, Brenda R; Kaplan, Adam C; Bae, Ji Eun et al. (2018) CRISPR/Cas9 cleavages in budding yeast reveal templated insertions and strand-specific insertion/deletion profiles. Proc Natl Acad Sci U S A 115:E2040-E2047
Haber, James E (2018) DNA Repair: The Search for Homology. Bioessays 40:e1700229
Anand, Ranjith; Beach, Annette; Li, Kevin et al. (2017) Rad51-mediated double-strand break repair and mismatch correction of divergent substrates. Nature 544:377-380
Mehta, Anuja; Beach, Annette; Haber, James E (2017) Homology Requirements and Competition between Gene Conversion and Break-Induced Replication during Double-Strand Break Repair. Mol Cell 65:515-526.e3
Haber, James E (2016) The rule of three. Nat Rev Mol Cell Biol 17:333
Jain, Suvi; Sugawara, Neal; Mehta, Anuja et al. (2016) Sgs1 and Mph1 Helicases Enforce the Recombination Execution Checkpoint During DNA Double-Strand Break Repair in Saccharomyces cerevisiae. Genetics 203:667-75
Jasin, Maria; Haber, James E (2016) The democratization of gene editing: Insights from site-specific cleavage and double-strand break repair. DNA Repair (Amst) 44:6-16
Yimit, Askar; Kim, TaeHyung; Anand, Ranjith P et al. (2016) MTE1 Functions with MPH1 in Double-Strand Break Repair. Genetics 203:147-57
Jain, Suvi; Sugawara, Neal; Haber, James E (2016) Role of Double-Strand Break End-Tethering during Gene Conversion in Saccharomyces cerevisiae. PLoS Genet 12:e1005976

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