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 activities of the Sgs1/BLM and Exo1 DNA processing enzymes on chromatin substrates, and we also identified pathways that control recruitment of chromatin regulators to DSBs. In addition, the conserved INO80-C chromatin remodeling enzyme was shown to regulate the stability of stalled replisomes and to exhibit histone dimer exchange activity. 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 chromatin in the early steps of HR.
This aim exploits both ensemble biochemical studies as well as single molecule, fluorescent DNA curtains to monitor how the Sgs1/Dna2 machinery disrupts nucleosome structures. Here we also develop yeast strains that allow for conditional depletion of multiple chromatin regulators, and such strains will be used in live cell imaging studies to investigate roles for chromatin regulators in he nuclear mobility of DSBs and the completion of the homology search step of HR.
Aim 2 dissects the function of the Ies6/Arp5 module of the INO80-C remodeling enzyme, employing a combination of EM, chemical cross-linking coupled to mass spectrometry, and in vitro chromatin remodeling assays to dissect its role in the dimer exchange reaction. Studies described in Aim 3 investigate recruitment and function of INO80-C at replication origins and stalled forks. A combination of conditional depletion of preRC components, nucleosome mapping, ChIP, and 2D gel analyses are used.
Aim 4 describes a combination of analytical ultracentrifugation, AFM, and EM studies to analyze how the Sir2/Sir4 complex interacts with nucleosomal arrays, and we probe the structure and solution dynamics of a reconstituted Sir2/Sir3/Sir4 heterochromatin fiber.

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 #
5R01GM054096-18
Application #
8987574
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Carter, Anthony D
Project Start
1997-01-01
Project End
2018-12-31
Budget Start
2016-01-01
Budget End
2016-12-31
Support Year
18
Fiscal Year
2016
Total Cost
$392,558
Indirect Cost
$140,585
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
Azmi, Ishara F; Watanabe, Shinya; Maloney, Michael F et al. (2017) Nucleosomes influence multiple steps during replication initiation. Elife 6:
Adkins, Nicholas L; Swygert, Sarah G; Kaur, Parminder et al. (2017) Nucleosome-like, Single-stranded DNA (ssDNA)-Histone Octamer Complexes and the Implication for DNA Double Strand Break Repair. J Biol Chem 292:5271-5281
Xue, Yong; Pradhan, Suman K; Sun, Fei et al. (2017) Mot1, Ino80C, and NC2 Function Coordinately to Regulate Pervasive Transcription in Yeast and Mammals. Mol Cell 67:594-607.e4
Watanabe, Shinya; Tan, Dongyan; Lakshminarasimhan, Mahadevan et al. (2015) Structural analyses of the chromatin remodelling enzymes INO80-C and SWR-C. Nat Commun 6:7108
Bennett, Gwendolyn; Peterson, Craig L (2015) SWI/SNF recruitment to a DNA double-strand break by the NuA4 and Gcn5 histone acetyltransferases. DNA Repair (Amst) 30:38-45
Zhao, Huaying; Ghirlando, Rodolfo; Alfonso, Carlos et al. (2015) A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation. PLoS One 10:e0126420
Xue, Yong; Van, Christopher; Pradhan, Suman K et al. (2015) The Ino80 complex prevents invasion of euchromatin into silent chromatin. Genes Dev 29:350-5
Swygert, Sarah G; Peterson, Craig L (2014) Chromatin dynamics: interplay between remodeling enzymes and histone modifications. Biochim Biophys Acta 1839:728-36
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
Papamichos-Chronakis, Manolis; Peterson, Craig L (2013) Chromatin and the genome integrity network. Nat Rev Genet 14:62-75

Showing the most recent 10 out of 34 publications