Our overall objective is to determine how chromosome structure and chromatin remodeling enzymes influence genome stability. In particular, we are interested in how these factors regulate the repair of DNA double strand breaks (DSBs) by homologous recombination (HR) and how they control the progression and stability of replication forks. Defects in either of these pathways directly impact cell survival and maintenance of genome integrity, leading to mutations, gene translocations, gross chromosomal rearrangements, or cellular lethality. During the past budget period, biochemical assays were developed to dissect the early steps of HR on chromatin substrates, and heterochromatin-like structures were reconstituted that repress recombination and impose a requirement for ATP-dependent chromatin remodeling. In addition, the conserved Ino80.com chromatin remodeling enzyme was shown to be a key regulator of replication fork stability in vivo. Our general strategy is to continue to exploit a powerful combination of biochemical and molecular genetic approaches to dissect the dynamics of chromatin structure during the repair of DSBs and during the replication process, using budding yeast as the experimental system. Experiments described in this proposal address four aims.
The first aim i nvestigates the role of the Ino80.com chromatin remodeling enzyme in DSB processing.
This aim uses genetic approaches to dissect how Ino80 is recruited to a DSB and how it contributes to processing. Biochemical studies are also described which will reconstitute DSB processing in vitro on nucleosomal substrates.
Aim 2 describes biochemical studies that investigate changes in chromatin structure that occur during formation of the initial joint molecule during early steps of homologous recombination. Studies described in Aim 3 will use in vivo and in vitro methods to investigate functional interactions between Ino80.com and the Htz1 histone variant.
Aim 4 describes a novel combination of single molecule, analytical ultracentrifugation, histone-histone and histone-DNA crosslinking methods to dissect the structural features of Sir heterochromatin.

Public Health Relevance

Chromosome structure plays a central role in regulating the repair of DNA and the faithful copying of DNA during cell division. Consequently, defects in these pathways can impact cell survival and maintenance of genome integrity. The studies described here will investigate the role of key enzymes and structural proteins that regulate these events.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular Genetics A Study Section (MGA)
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Carter, Anthony D
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University of Massachusetts Medical School Worcester
Other Basic Sciences
Schools of Medicine
United States
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Clapier, Cedric R; Iwasa, Janet; Cairns, Bradley R et al. (2017) Mechanisms of action and regulation of ATP-dependent chromatin-remodelling complexes. Nat Rev Mol Cell Biol 18:407-422
Azmi, Ishara F; Watanabe, Shinya; Maloney, Michael F et al. (2017) Nucleosomes influence multiple steps during replication initiation. Elife 6:
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