DNA-binding motor proteins play critical roles in the promotion and regulation of homologous recombination (HR), and are indispensable for cancer avoidance in mammals. For instance, mutations in the motor proteins BLM and RECQ5 lead to aberrant recombination events and are associated with tumorigenesis, and mutations in Rad54 and Rad54B are found in a variety of tumor types. Understanding the properties of these motor proteins, and delineating their roles in genome maintenance, will be essential for revealing the molecular basis for defects that produce the disease phenotypes. The Rad54 protein is a key factor in homologous recombination and damage avoidance, and has been postulated to act at pre-synaptic, synaptic and post- synaptic steps in double strand break repair through homologous recombination. Despite this central role for Rad54 in the process, its exact function and regulation in vivo remain unknown, despite the wealth of biochemical studies characterizing its dsDNA-dependent ATPase activity and translocation on dsDNA. srs2 rad54 double mutants are lethal, due to action of Rad51 to promote HR, indicating an overlap in an essential step in HR. Our hypothesis is that the Rad54 DNA motor protein contributes to genome maintenance through a dynamic cycle of phosphorylation/dephosphorylation and this affects its ability to interact with Rad51 recombinase and function in HR. The essential step performed by Srs2 or Rad54 involves setting up the synaptic intermediate after a search for homology has been initiated. To test this hypothesis we will take a multidisciplinary approach that integrates our expertise in genetics (H. Klein) complemented by the biochemistry (P. Sung), and single-molecule biophysics (E. Greene) expertise of our collaborators, in order to conduct a detailed analysis of the S. cerevisiae Rad54 and Srs2 DNA motor proteins. Together, we are well positioned to decipher the mechanisms of these motor proteins and provide a valuable experimental framework for understanding the genome maintenance and tumor suppression roles of their mammalian counterparts.

Public Health Relevance

Genome stability is maintained through the recognition of DNA damage and mounting a response appropriate to the damage to repair the lesions. Failure to correctly repair DNA damage is characteristic of cancers. We propose a new mechanism for regulating genome maintenance.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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Radiation Therapeutics and Biology Study Section (RTB)
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Witkin, Keren L
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New York University
Schools of Medicine
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Mansisidor, Andrés; Molinar Jr, Temistocles; Srivastava, Priyanka et al. (2018) Genomic Copy-Number Loss Is Rescued by Self-Limiting Production of DNA Circles. Mol Cell 72:583-593.e4
Klein, Hannah L (2017) Genome instabilities arising from ribonucleotides in DNA. DNA Repair (Amst) 56:26-32
Niu, Hengyao; Klein, Hannah L (2017) Multifunctional roles of Saccharomyces cerevisiae Srs2 protein in replication, recombination and repair. FEMS Yeast Res 17:
Epshtein, Anastasiya; Potenski, Catherine J; Klein, Hannah L (2016) Increased Spontaneous Recombination in RNase H2-Deficient Cells Arises From Multiple Contiguous rNMPs and Not From Single rNMP Residues Incorporated by DNA Polymerase Epsilon. Microb Cell 3:248-254
Niu, Hengyao; Potenski, Catherine J; Epshtein, Anastasiya et al. (2016) Roles of DNA helicases and Exo1 in the avoidance of mutations induced by Top1-mediated cleavage at ribonucleotides in DNA. Cell Cycle 15:331-6
Qi, Zhi; Redding, Sy; Lee, Ja Yil et al. (2015) DNA sequence alignment by microhomology sampling during homologous recombination. Cell 160:856-869
Lee, Ja Yil; Terakawa, Tsuyoshi; Qi, Zhi et al. (2015) DNA RECOMBINATION. Base triplet stepping by the Rad51/RecA family of recombinases. Science 349:977-81
Silverstein, Timothy D; Gibb, Bryan; Greene, Eric C (2014) Visualizing protein movement on DNA at the single-molecule level using DNA curtains. DNA Repair (Amst) 20:94-109
Gibb, Bryan; Ye, Ling F; Gergoudis, Stephanie C et al. (2014) Concentration-dependent exchange of replication protein A on single-stranded DNA revealed by single-molecule imaging. PLoS One 9:e87922
Potenski, Catherine J; Klein, Hannah L (2014) How the misincorporation of ribonucleotides into genomic DNA can be both harmful and helpful to cells. Nucleic Acids Res 42:10226-34

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