DSBs are highly cytotoxic lesions that occur spontaneously during normal cellular processes, by treatment of cells with DNA damaging agents or as intermediates in programmed recombination events. If DSBs are left unrepaired, or repaired inappropriately, they can trigger mutagenic events including chromosome loss, deletions, duplications, inversions and translocations, events associated with tumorigenesis. The long-term objective of our research program is to understand how cells repair DNA double strand breaks (DSBs) to maintain genome integrity. The focus of the current proposal is the mechanism and regulation of 5'-3'end resection, which has emerged as a key regulatory step to govern repair pathway choice, and is the critical first step for homology-directed repair of DSBs. While much progress has been made in the identification of the key components of the end resection machinery, the mechanism of resection initiation is still in question.
The first aim of te proposal is to define the roles of Sae2 and Xrs2 in MRX complex functions. We identified several mre11 gain-of-function alleles, mre11-sas, that can bypass the DNA damage sensitivity of the sae2? mutant by extinguishing the DNA damage checkpoint. We plan to investigate the dynamics of MRX association with DNA ends in vivo and by single-molecule imaging, and determine the dependence on other components of the DNA damage checkpoint for suppression of sae2? by the mre11-sas alleles. The role of Xrs2 in the cellular response to DNA damage will be dissected using an MRE11-NLS fusion that bypasses the requirement for Xrs2 to transport Mre11 to the nucleus. The exonuclease-defective mre11-H59S allele will be used with sgs1? and exo1? mutations to test the model that resection initiates by a nick internal to the DNA ends.
The second aim follows up on our interesting new finding that RPA prevents repair of DSBs by microhomology-mediated end joining (MMEJ). This mutagenic repair mechanism is thought to be responsible for chromosome rearrangements in cancer cells. We will investigate the roles of RPA and Sae2 in suppression of chromosomal inverted duplications mediated by annealing between short inverted repeats.
The final aim i s to understand how the DNA damage checkpoint regulates end resection. The ssDNA formed by end resection is important to activate the DNA damage checkpoint;however, excessive ssDNA is detrimental to genome integrity and several studies indicate that the DNA damage checkpoint prevents extensive resection. We will identify the resection nucleases inhibited by Rad9 during the cell cycle and dissect the functions of Rad9 responsible for inhibition of end resection. Finally, the role of the DNA damage clamp in promoting or inhibiting end resection will be tested in the presence or absence of Rad9.

Public Health Relevance

The repair of DNA double-strand breaks (DSBs) is essential to maintain genome integrity and to guard against cancer in humans. In this proposal, genetic and physical approaches will be used to determine the roles of Sae2/CtIP in regulating the nuclease and checkpoint responses of the Mre11 complex, the role of RPA in suppressing genome rearrangements, and how the DNA damage checkpoint regulates the mechanisms used to repair DSBs.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Molecular Genetics B Study Section (MGB)
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Janes, Daniel E
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Columbia University (N.Y.)
Schools of Medicine
New York
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Gnügge, Robert; Oh, Julyun; Symington, Lorraine S (2018) Processing of DNA Double-Strand Breaks in Yeast. Methods Enzymol 600:1-24
Gnügge, Robert; Symington, Lorraine S (2017) Keeping it real: MRX-Sae2 clipping of natural substrates. Genes Dev 31:2311-2312
Oh, Julyun; Al-Zain, Amr; Cannavo, Elda et al. (2016) Xrs2 Dependent and Independent Functions of the Mre11-Rad50 Complex. Mol Cell 64:405-415
Ruff, Patrick; Donnianni, Roberto A; Glancy, Eleanor et al. (2016) RPA Stabilization of Single-Stranded DNA Is Critical for Break-Induced Replication. Cell Rep 17:3359-3368
Wei, Jia; Zhang, Yixiao; Yu, Tai-Yuan et al. (2016) A unified molecular mechanism for the regulation of acetyl-CoA carboxylase by phosphorylation. Cell Discov 2:16044
Symington, Lorraine S (2016) Mechanism and regulation of DNA end resection in eukaryotes. Crit Rev Biochem Mol Biol 51:195-212
Ciccia, Alberto; Symington, Lorraine S (2016) Stressing Out About RAD52. Mol Cell 64:1017-1019
Chen, Huan; Donnianni, Roberto A; Handa, Naofumi et al. (2015) Sae2 promotes DNA damage resistance by removing the Mre11-Rad50-Xrs2 complex from DNA and attenuating Rad53 signaling. Proc Natl Acad Sci U S A 112:E1880-7
Deng, Sarah K; Chen, Huan; Symington, Lorraine S (2015) Replication protein A prevents promiscuous annealing between short sequence homologies: Implications for genome integrity. Bioessays 37:305-13
Deng, Sarah K; Yin, Yi; Petes, Thomas D et al. (2015) Mre11-Sae2 and RPA Collaborate to Prevent Palindromic Gene Amplification. Mol Cell 60:500-8

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