Higher order organization of chromatin generates structures that are essential for high fidelity chromosome segregation, DNA damage repair, and the regulation of gene expression. Defining the chromatin organization in these structures, the mechanisms for forming these structures, and the molecular functions they play are amongst the most important and daunting tasks in cell biology. This proposal tackles this task through the analysis of cohesin, a member of the SMC (Structural Maintenance of Chromosomes) family of protein complexes. Cohesin tethers together two regions of DNA to mediate sister chromatid cohesin, DNA repair, mitotic chromosome condensation and transcription regulation. The importance of cohesin's biological functions is evident from its emerging roles in cancer progression, age-dependent birth defects, and stem cell pluripotency. By utilizing yeast genetics, cytology, new biochemical and single-molecule imaging assays, we have discovered novel cohesin activities and regulators. These discoveries lead to new models for: 1) the topological entrapment of DNA by cohesin; 2) the roles of cohesin ATPases and oligomerization in cohesin's DNA binding and DNA tethering activities; 3) the spatial-temporal regulation of these activities; and 4) cohesin's coordination with other SMC complexes to promote higher order chromosome structure. Based on these models we propose experiments that will inform on the molecular basis for cohesin's activities, their regulation, and how these activities facilitte chromosome organization and function. Another emerging but poorly understood mediators of chromosome function are R-loops. R-loops result when transcripts hybridize to homologous chromosomal sites, generating an RNA-DNA hybrid and a displaced single-stranded DNA. R-loops can lead to gross chromosomal rearrangements (GCRs) and have been linked to cancer and fragile chromosome sites. R-loops can also regulate gene expression by modulating epigenetic marks and antisense RNA. We developed novel genetic and cytological assays to identify many new inhibitors and enhancers of R-loop formation and new mechanisms for their formation. We also have developed a novel quantitative and high-precision technique to map R-loops genome-wide. With these tools we will address key questions in the field. Which features of RNA, DNA and proteins regulate R-loop formation? What characteristics of R-loops and flanking chromatin determine whether they induce DNA damage, and how does this damage cause GCRs? Do R-loops regulate other aspects of chromosomal processes like homologous recombination and condensation? By answering these questions we will elucidate the molecular mechanism of R-loop formation and provide critical insights into the diverse mechanisms by which R-loops modulate chromosome function.

Public Health Relevance

The long threads of DNA in chromosomes are associated with proteins and RNAs to generate chromosome structures critical for chromosome inheritance during cell division and gamete production, the repair of damaged chromosomes, and the expression of genes on chromosomes. This grant proposes to study how chromosome structure and function is altered by the protein clasp called cohesin and short hybrids of RNA and DNA called R-loops. Cohesin and R-loops have been implicated in cancer, birth defects and stem cell function.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Unknown (R35)
Project #
5R35GM118189-03
Application #
9475220
Study Section
Special Emphasis Panel (ZGM1)
Program Officer
Willis, Kristine Amalee
Project Start
2016-05-01
Project End
2021-04-30
Budget Start
2018-05-01
Budget End
2019-04-30
Support Year
3
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of California Berkeley
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
124726725
City
Berkeley
State
CA
Country
United States
Zip Code
94704
Costantino, Lorenzo; Koshland, Douglas (2018) Genome-wide Map of R-Loop-Induced Damage Reveals How a Subset of R-Loops Contributes to Genomic Instability. Mol Cell 71:487-497.e3
Robison, Brett; Guacci, Vincent; Koshland, Douglas (2018) A role for the Smc3 hinge domain in the maintenance of sister chromatid cohesion. Mol Biol Cell 29:339-355
Kim, Skylar Xantus; Çamdere, Gamze; Hu, Xuchen et al. (2018) Synergy between the small intrinsically disordered protein Hsp12 and trehalose sustain viability after severe desiccation. Elife 7:
Bloom, Michelle S; Koshland, Douglas; Guacci, Vincent (2018) Cohesin Function in Cohesion, Condensation, and DNA Repair Is Regulated by Wpl1p via a Common Mechanism in Saccharomyces cerevisiae. Genetics 208:111-124
Çamdere, Gamze Ö; Carlborg, Kristian K; Koshland, Douglas (2018) Intermediate step of cohesin's ATPase cycle allows cohesin to entrap DNA. Proc Natl Acad Sci U S A 115:9732-9737
Wahba, Lamia; Costantino, Lorenzo; Tan, Frederick J et al. (2016) S1-DRIP-seq identifies high expression and polyA tracts as major contributors to R-loop formation. Genes Dev 30:1327-38
Stigler, Johannes; Çamdere, Gamze Ö; Koshland, Douglas E et al. (2016) Single-Molecule Imaging Reveals a Collapsed Conformational State for DNA-Bound Cohesin. Cell Rep 15:988-998
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Amon, Jeremy D; Koshland, Douglas (2016) RNase H enables efficient repair of R-loop induced DNA damage. Elife 5:
Zimmer, Anjali D; Koshland, Douglas (2016) Differential roles of the RNases H in preventing chromosome instability. Proc Natl Acad Sci U S A 113:12220-12225