Publications by Year: 2019

2019
Logan R Myler, Michael M Soniat, Xiaoming Zhang, Rajashree A Deshpande, Tanya T Paull, and Ilya J Finkelstein. 2AD. “Purification and Biophysical Characterization of the Mre11-Rad50-Nbs1 Complex.” Methods Mol Biol, 2004, Pp. 269-287. Abstract
The Mre11-Rad50-Nbs1 (MRN) complex coordinates the repair of DNA double-strand breaks, replication fork restart, meiosis, class-switch recombination, and telomere maintenance. As such, MRN is an essential molecular machine that has homologs in all organisms of life, from bacteriophage to humans. In human cells, MRN is a >500 kDa multifunctional complex that encodes DNA binding, ATPase, and both endonuclease and exonuclease activities. MRN also forms larger assemblies and interacts with multiple DNA repair and replication factors. The enzymatic properties of MRN have been the subject of intense research for over 20 years, and more recently, single-molecule biophysics studies are beginning to probe its many biochemical activities. Here, we describe the methods used to overexpress, fluorescently label, and visualize MRN and its activities on single molecules of DNA.
Tanya T Paull. 2AD. “RNA-DNA hybrids and the convergence with DNA repair.” Crit Rev Biochem Mol Biol, 54, 4, Pp. 371-384. Abstract
The repair of DNA double-strand breaks occurs through a series of defined steps that are evolutionarily conserved and well-understood in most experimental organisms. However, it is becoming increasingly clear that repair does not occur in isolation from other DNA transactions. Transcription of DNA produces topological changes, RNA species, and RNA-dependent protein complexes that can dramatically influence the efficiency and outcomes of DNA double-strand break repair. The transcription-associated history of several double-strand break repair factors is reviewed here, with an emphasis on their roles in regulating R-loops and the emerging role of R-loops in coordination of repair events. Evidence for nucleolytic processing of R-loops is also discussed, as well as the molecular tools commonly used to measure RNA-DNA hybrids in cells.
Michael M Soniat, Logan R Myler, Hung-Che Kuo, Tanya T Paull, and Ilya J Finkelstein. 2AD. “RPA Phosphorylation Inhibits DNA Resection.” Mol Cell, 75, 1, Pp. 145-153.e5. Abstract
Genetic recombination in all kingdoms of life initiates when helicases and nucleases process (resect) the free DNA ends to expose single-stranded DNA (ssDNA) overhangs. Resection regulation in bacteria is programmed by a DNA sequence, but a general mechanism limiting resection in eukaryotes has remained elusive. Using single-molecule imaging of reconstituted human DNA repair factors, we identify phosphorylated RPA (pRPA) as a negative resection regulator. Bloom's syndrome (BLM) helicase together with exonuclease 1 (EXO1) and DNA2 nucleases catalyze kilobase-length DNA resection on nucleosome-coated DNA. The resulting ssDNA is rapidly bound by RPA, which further stimulates DNA resection. RPA is phosphorylated during resection as part of the DNA damage response (DDR). Remarkably, pRPA inhibits DNA resection in cellular assays and in vitro via inhibition of BLM helicase. pRPA suppresses BLM initiation at DNA ends and promotes the intrinsic helicase strand-switching activity. These findings establish that pRPA provides a feedback loop between DNA resection and the DDR.
Jae Jin Kim, Seo Yun Lee, Fade Gong, Anna M Battenhouse, Daniel R Boutz, Aarti Bashyal, Samantha T Refvik, Cheng-Ming Chiang, Blerta Xhemalce, Tanya T Paull, Jennifer S Brodbelt, Edward M Marcotte, and Kyle M Miller. 2AD. “Systematic bromodomain protein screens identify homologous recombination and R-loop suppression pathways involved in genome integrity.” Genes Dev, 33, 23-24, Pp. 1751-1774. Abstract
Bromodomain proteins (BRD) are key chromatin regulators of genome function and stability as well as therapeutic targets in cancer. Here, we systematically delineate the contribution of human BRD proteins for genome stability and DNA double-strand break (DSB) repair using several cell-based assays and proteomic interaction network analysis. Applying these approaches, we identify 24 of the 42 BRD proteins as promoters of DNA repair and/or genome integrity. We identified a BRD-reader function of PCAF that bound TIP60-mediated histone acetylations at DSBs to recruit a DUB complex to deubiquitylate histone H2BK120, to allowing direct acetylation by PCAF, and repair of DSBs by homologous recombination. We also discovered the bromo-and-extra-terminal (BET) BRD proteins, BRD2 and BRD4, as negative regulators of transcription-associated RNA-DNA hybrids (R-loops) as inhibition of BRD2 or BRD4 increased R-loop formation, which generated DSBs. These breaks were reliant on topoisomerase II, and BRD2 directly bound and activated topoisomerase I, a known restrainer of R-loops. Thus, comprehensive interactome and functional profiling of BRD proteins revealed new homologous recombination and genome stability pathways, providing a framework to understand genome maintenance by BRD proteins and the effects of their pharmacological inhibition.