All Publications

Yeast Srs2 Helicase Promotes Redistribution of Single-Stranded DNA-Bound RPA and Rad52 in Homologous Recombination Regulation.

Cell Rep. 2017 Oct 17;21(3):570-577

Authors: De Tullio L, Kaniecki K, Kwon Y, Crickard JB, Sung P, Greene EC

Abstract
Srs2 is a super-family 1 helicase that promotes genome stability by dismantling toxic DNA recombination intermediates. However, the mechanisms by which Srs2 remodels or resolves recombination intermediates remain poorly understood. Here, single-molecule imaging is used to visualize Srs2 in real time as it acts on single-stranded DNA (ssDNA) bound by protein factors that function in recombination. We demonstrate that Srs2 is highly processive and translocates rapidly (∼170 nt per second) in the 3’→5′ direction along ssDNA saturated with replication protein A (RPA). We show that RPA is evicted from DNA during the passage of Srs2. Remarkably, Srs2 also readily removes the recombination mediator Rad52 from RPA-ssDNA and, in doing so, promotes rapid redistribution of both Rad52 and RPA. These findings have important mechanistic implications for understanding how Srs2 and related nucleic acid motor proteins resolve potentially pathogenic nucleoprotein intermediates.

PMID: 29045827 [PubMed – indexed for MEDLINE]

Tumor-associated mutations in O⁶ -methylguanine DNA-methyltransferase (MGMT) reduce DNA repair functionality.

Mol Carcinog. 2014 Mar;53(3):201-10

Authors: Lamb KL, Liu Y, Ishiguro K, Kwon Y, Paquet N, Sartorelli AC, Sung P, Rockwell S, Sweasy JB

Abstract
MGMT is the primary vehicle for cellular removal of alkyl lesions from the O-6 position of guanine and the O-4 position of thymine. While key to the maintenance of genomic integrity, MGMT also removes damage induced by alkylating chemotherapies, inhibiting the efficacy of cancer treatment. Germline variants of human MGMT are well-characterized, but somatic variants found in tumors were, prior to this work, uncharacterized. We found that MGMT G132R, from a human esophageal tumor, and MGMT G156C, from a human colorectal cancer cell line, are unable to rescue methyltransferase-deficient Escherichia coli as well as wild type (WT) human MGMT after treatment with a methylating agent. Using pre-steady state kinetics, we biochemically characterized these variants as having a reduced rate constant. G132R binds DNA containing an O⁶ -methylguanine lesion half as tightly as WT MGMT, while G156C has a 40-fold decrease in binding affinity for the same damaged DNA versus WT. Mammalian cells expressing either G132R or G156C are more sensitive to methylating agents than mammalian cells expressing WT MGMT. G132R is slightly resistant to O⁶ -benzylguanine, an inhibitor of MGMT in clinical trials, while G156C is almost completely resistant to this inhibitor. The impared functionality of expressed variants G132R and G156C suggests that the presence of somatic variants of MGMT in a tumor could impact chemotherapeutic outcomes.

PMID: 23065697 [PubMed – indexed for MEDLINE]

Tolerance of DNA Mismatches in Dmc1 Recombinase-mediated DNA Strand Exchange.

J Biol Chem. 2016 Mar 04;291(10):4928-38

Authors: Borgogno MV, Monti MR, Zhao W, Sung P, Argaraña CE, Pezza RJ

Abstract
Recombination between homologous chromosomes is required for the faithful meiotic segregation of chromosomes and leads to the generation of genetic diversity. The conserved meiosis-specific Dmc1 recombinase catalyzes homologous recombination triggered by DNA double strand breaks through the exchange of parental DNA sequences. Although providing an efficient rate of DNA strand exchange between polymorphic alleles, Dmc1 must also guard against recombination between divergent sequences. How DNA mismatches affect Dmc1-mediated DNA strand exchange is not understood. We have used fluorescence resonance energy transfer to study the mechanism of Dmc1-mediated strand exchange between DNA oligonucleotides with different degrees of heterology. The efficiency of strand exchange is highly sensitive to the location, type, and distribution of mismatches. Mismatches near the 3′ end of the initiating DNA strand have a small effect, whereas most mismatches near the 5′ end impede strand exchange dramatically. The Hop2-Mnd1 protein complex stimulates Dmc1-catalyzed strand exchange on homologous DNA or containing a single mismatch. We observed that Dmc1 can reject divergent DNA sequences while bypassing a few mismatches in the DNA sequence. Our findings have important implications in understanding meiotic recombination. First, Dmc1 acts as an initial barrier for heterologous recombination, with the mismatch repair system providing a second level of proofreading, to ensure that ectopic sequences are not recombined. Second, Dmc1 stepping over infrequent mismatches is likely critical for allowing recombination between the polymorphic sequences of homologous chromosomes, thus contributing to gene conversion and genetic diversity.

PMID: 26709229 [PubMed – indexed for MEDLINE]

To Cut or Not to Cut: Discovery of a Novel Regulator of DNA Break Resection.

Mol Cell. 2016 Feb 04;61(3):325-326

Authors: Daley JM, Sung P

Abstract
Nucleolytic resection of DNA double-strand breaks is the crucial first step in their repair via homologous recombination. New findings by Tkáč et al. (2016) published in this issue of Molecular Cell identify HELB as a novel, cell-cycle-specific negative regulator of DNA end resection.

PMID: 26849190 [PubMed – indexed for MEDLINE]

Icon for Silverchair Information Systems

The RecQ helicase Sgs1 drives ATP-dependent disruption of Rad51 filaments.

Nucleic Acids Res. 2019 May 21;47(9):4694-4706

Authors: Crickard JB, Xue C, Wang W, Kwon Y, Sung P, Greene EC

Abstract
DNA helicases of the RecQ family are conserved among the three domains of life and play essential roles in genome maintenance. Mutations in several human RecQ helicases lead to diseases that are marked by cancer predisposition. The Saccharomyces cerevisiae RecQ helicase Sgs1 is orthologous to human BLM, defects in which cause the cancer-prone Bloom’s Syndrome. Here, we use single-molecule imaging to provide a quantitative mechanistic understanding of Sgs1 activities on single stranded DNA (ssDNA), which is a central intermediate in all aspects of DNA metabolism. We show that Sgs1 acts upon ssDNA bound by either replication protein A (RPA) or the recombinase Rad51. Surprisingly, we find that Sgs1 utilizes a novel motor mechanism for disrupting ssDNA intermediates bound by the recombinase protein Rad51. The ability of Sgs1 to disrupt Rad51-ssDNA filaments may explain some of the defects engendered by RECQ helicase deficiencies in human cells.

PMID: 30916344 [PubMed – in process]

The Rad51 paralogs facilitate a novel DNA strand specific damage tolerance pathway.

Nat Commun. 2019 Aug 05;10(1):3515

Authors: Rosenbaum JC, Bonilla B, Hengel SR, Mertz TM, Herken BW, Kazemier HG, Pressimone CA, Ratterman TC, MacNary E, De Magis A, Kwon Y, Godin SK, Van Houten B, Normolle DP, Sung P, Das SR, Paeschke K, Roberts SA, VanDemark AP, Bernstein KA

Abstract
Accurate DNA replication is essential for genomic stability and cancer prevention. Homologous recombination is important for high-fidelity DNA damage tolerance during replication. How the homologous recombination machinery is recruited to replication intermediates is unknown. Here, we provide evidence that a Rad51 paralog-containing complex, the budding yeast Shu complex, directly recognizes and enables tolerance of predominantly lagging strand abasic sites. We show that the Shu complex becomes chromatin associated when cells accumulate abasic sites during S phase. We also demonstrate that purified recombinant Shu complex recognizes an abasic analog on a double-flap substrate, which prevents AP endonuclease activity and endonuclease-induced double-strand break formation. Shu complex DNA binding mutants are sensitive to methyl methanesulfonate, are not chromatin enriched, and exhibit increased mutation rates. We propose a role for the Shu complex in recognizing abasic sites at replication intermediates, where it recruits the homologous recombination machinery to mediate strand specific damage tolerance.

PMID: 31383866 [PubMed – in process]

The Rad50 coiled-coil domain is indispensable for Mre11 complex functions.

Nat Struct Mol Biol. 2011 Sep 04;18(10):1124-31

Authors: Hohl M, Kwon Y, Galván SM, Xue X, Tous C, Aguilera A, Sung P, Petrini JH

Abstract
The Mre11 complex (Mre11, Rad50 and Xrs2 in Saccharomyces cerevisiae) influences diverse functions in the DNA damage response. The complex comprises the globular DNA-binding domain and the Rad50 hook domain, which are linked by a long and extended Rad50 coiled-coil domain. In this study, we constructed rad50 alleles encoding truncations of the coiled-coil domain to determine which Mre11 complex functions required the full length of the coils. These mutations abolished telomere maintenance and meiotic double-strand break (DSB) formation, and severely impaired homologous recombination, indicating a requirement for long-range action. Nonhomologous end joining, which is probably mediated by the globular domain of the Mre11 complex, was also severely impaired by alteration of the coiled-coil and hook domains, providing the first evidence of their influence on this process. These data show that functions of Mre11 complex are integrated by the coiled coils of Rad50.

PMID: 21892167 [PubMed – indexed for MEDLINE]

The MRX complex regulates Exo1 resection activity by altering DNA end structure.

EMBO J. 2018 08 15;37(16):

Authors: Gobbini E, Cassani C, Vertemara J, Wang W, Mambretti F, Casari E, Sung P, Tisi R, Zampella G, Longhese MP

Abstract
Homologous recombination is triggered by nucleolytic degradation (resection) of DNA double-strand breaks (DSBs). DSB resection requires the Mre11-Rad50-Xrs2 (MRX) complex, which promotes the activity of Exo1 nuclease through a poorly understood mechanism. Here, we describe the Mre11-R10T mutant variant that accelerates DSB resection compared to wild-type Mre11 by potentiating Exo1-mediated processing. This increased Exo1 resection activity leads to a decreased association of the Ku complex to DSBs and an enhanced DSB resection in G1, indicating that Exo1 has a direct function in preventing Ku association with DSBs. Molecular dynamics simulations show that rotation of the Mre11 capping domains is able to induce unwinding of double-strand DNA (dsDNA). The R10T substitution causes altered orientation of the Mre11 capping domain that leads to persistent melting of the dsDNA end. We propose that MRX creates a specific DNA end structure that promotes Exo1 resection activity by facilitating the persistence of this nuclease on the DSB ends, uncovering a novel MRX function in DSB resection.

PMID: 29925516 [PubMed – indexed for MEDLINE]

The MHF complex senses branched DNA by binding a pair of crossover DNA duplexes.

Nat Commun. 2014;5:2987

Authors: Zhao Q, Saro D, Sachpatzidis A, Singh TR, Schlingman D, Zheng XF, Mack A, Tsai MS, Mochrie S, Regan L, Meetei AR, Sung P, Xiong Y

Abstract
The conserved MHF1-MHF2 (MHF) complex functions in the activation of the Fanconi anaemia pathway of the DNA damage response, in regulating homologous recombination, and in DNA replication fork maintenance. MHF facilitates the processing of multiple types of branched DNAs by the DNA translocase FANCM. Here we report the crystal structure of a human MHF-DNA complex that reveals the DNA-binding mode of MHF. The structure suggests that MHF prefers branched DNA over double-stranded DNA because it engages two duplex arms. Biochemical analyses verify that MHF preferentially engages DNA forks or various four-way junctions independent of the junction-site structure. Furthermore, genetic experiments provide evidence that the observed DNA-binding interface of MHF is important for cellular resistance to DNA damage. These results offer insights into how the MHF complex recognizes branched DNA and stimulates FANCM activity at such a structure to promote genome maintenance.

PMID: 24390579 [PubMed – indexed for MEDLINE]

The Fanconi anemia proteins FANCD2 and FANCJ interact and regulate each other’s chromatin localization.

J Biol Chem. 2014 Sep 12;289(37):25774-82

Authors: Chen X, Wilson JB, McChesney P, Williams SA, Kwon Y, Longerich S, Marriott AS, Sung P, Jones NJ, Kupfer GM

Abstract
Fanconi anemia is a genetic disease resulting in bone marrow failure, birth defects, and cancer that is thought to encompass a defect in maintenance of genomic stability. Mutations in 16 genes (FANCA, B, C, D1, D2, E, F, G, I, J, L, M, N, O, P, and Q) have been identified in patients, with the Fanconi anemia subtype J (FA-J) resulting from homozygous mutations in the FANCJ gene. Here, we describe the direct interaction of FANCD2 with FANCJ. We demonstrate the interaction of FANCD2 and FANCJ in vivo and in vitro by immunoprecipitation in crude cell lysates and from fractions after gel filtration and with baculovirally expressed proteins. Mutation of the monoubiquitination site of FANCD2 (K561R) preserves interaction with FANCJ constitutively in a manner that impedes proper chromatin localization of FANCJ. FANCJ is necessary for FANCD2 chromatin loading and focus formation in response to mitomycin C treatment. Our results suggest not only that FANCD2 regulates FANCJ chromatin localization but also that FANCJ is necessary for efficient loading of FANCD2 onto chromatin following DNA damage caused by mitomycin C treatment.

PMID: 25070891 [PubMed – indexed for MEDLINE]

All Publications

Filter results