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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM054096-16
Application #
8450139
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Carter, Anthony D
Project Start
1997-01-01
Project End
2014-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
16
Fiscal Year
2013
Total Cost
$386,359
Indirect Cost
$151,491
Name
University of Massachusetts Medical School Worcester
Department
Other Basic Sciences
Type
Schools of Medicine
DUNS #
603847393
City
Worcester
State
MA
Country
United States
Zip Code
01655
Swygert, Sarah G; Manning, Benjamin J; Senapati, Subhadip et al. (2014) Solution-state conformation and stoichiometry of yeast Sir3 heterochromatin fibres. Nat Commun 5:4751
Swygert, Sarah G; Peterson, Craig L (2014) Chromatin dynamics: interplay between remodeling enzymes and histone modifications. Biochim Biophys Acta 1839:728-36
Papamichos-Chronakis, Manolis; Peterson, Craig L (2013) Chromatin and the genome integrity network. Nat Rev Genet 14:62-75
Bennett, Gwendolyn; Papamichos-Chronakis, Manolis; Peterson, Craig L (2013) DNA repair choice defines a common pathway for recruitment of chromatin regulators. Nat Commun 4:2084
Adkins, Nicholas L; Niu, Hengyao; Sung, Patrick et al. (2013) Nucleosome dynamics regulates DNA processing. Nat Struct Mol Biol 20:836-42
Peterson, Craig L (2011) The ins and outs of heterochromatic DNA repair. Dev Cell 20:285-7
Peterson, Craig L (2011) Chromatin: a ubiquitin crowbar opens chromatin. Nat Chem Biol 7:68-9
Papamichos-Chronakis, Manolis; Watanabe, Shinya; Rando, Oliver J et al. (2011) Global regulation of H2A.Z localization by the INO80 chromatin-remodeling enzyme is essential for genome integrity. Cell 144:200-13
Oza, Pranav; Peterson, Craig L (2010) Opening the DNA repair toolbox: localization of DNA double strand breaks to the nuclear periphery. Cell Cycle 9:43-9
Watanabe, Shinya; Resch, Michael; Lilyestrom, Wayne et al. (2010) Structural characterization of H3K56Q nucleosomes and nucleosomal arrays. Biochim Biophys Acta 1799:480-6

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