All Publications

Icon for HighWire Icon for PubMed Central Related Articles

Meiosis-specific recombinase Dmc1 is a potent inhibitor of the Srs2 antirecombinase.

Proc Natl Acad Sci U S A. 2018 10 23;115(43):E10041-E10048

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

Abstract
Cross-over recombination products are a hallmark of meiosis because they are necessary for accurate chromosome segregation and they also allow for increased genetic diversity during sexual reproduction. However, cross-overs can also cause gross chromosomal rearrangements and are therefore normally down-regulated during mitotic growth. The mechanisms that enhance cross-over product formation upon entry into meiosis remain poorly understood. In Saccharomyces cerevisiae, the Superfamily 1 (Sf1) helicase Srs2, which is an ATP hydrolysis-dependent motor protein that actively dismantles recombination intermediates, promotes synthesis-dependent strand annealing, the result of which is a reduction in cross-over recombination products. Here, we show that the meiosis-specific recombinase Dmc1 is a potent inhibitor of Srs2. Biochemical and single-molecule assays demonstrate that Dmc1 acts by inhibiting Srs2 ATP hydrolysis activity, which prevents the motor protein from undergoing ATP hydrolysis-dependent translocation on Dmc1-bound recombination intermediates. We propose a model in which Dmc1 helps contribute to cross-over formation during meiosis by antagonizing the antirecombinase activity of Srs2.

PMID: 30301803 [PubMed – indexed for MEDLINE]


Icon for Silverchair Information Systems Icon for PubMed Central Related Articles

Mechanistic insights into the role of Hop2-Mnd1 in meiotic homologous DNA pairing.

Nucleic Acids Res. 2014 Jan;42(2):906-17

Authors: Zhao W, Saro D, Hammel M, Kwon Y, Xu Y, Rambo RP, Williams GJ, Chi P, Lu L, Pezza RJ, Camerini-Otero RD, Tainer JA, Wang HW, Sung P

Abstract
The Hop2-Mnd1 complex functions with the DMC1 recombinase in meiotic recombination. Hop2-Mnd1 stabilizes the DMC1-single-stranded DNA (ssDNA) filament and promotes the capture of the double-stranded DNA partner by the recombinase filament to assemble the synaptic complex. Herein, we define the action mechanism of Hop2-Mnd1 in DMC1-mediated recombination. Small angle X-ray scattering analysis and electron microscopy reveal that the heterodimeric Hop2-Mnd1 is a V-shaped molecule. We show that the protein complex harbors three distinct DNA binding sites, and determine their functional relevance. Specifically, the N-terminal double-stranded DNA binding functions of Hop2 and Mnd1 co-operate to mediate synaptic complex assembly, whereas ssDNA binding by the Hop2 C-terminus helps stabilize the DMC1-ssDNA filament. A model of the Hop2-Mnd1-DMC1-ssDNA ensemble is proposed to explain how it mediates homologous DNA pairing in meiotic recombination.

PMID: 24150939 [PubMed – indexed for MEDLINE]


Icon for HighWire Icon for PubMed Central Related Articles

Mechanistic insights into RAD51-associated protein 1 (RAD51AP1) action in homologous DNA repair.

J Biol Chem. 2012 Apr 06;287(15):12343-7

Authors: Dunlop MH, Dray E, Zhao W, San Filippo J, Tsai MS, Leung SG, Schild D, Wiese C, Sung P

Abstract
Homologous recombination catalyzed by the RAD51 recombinase is essential for maintaining genome integrity upon the induction of DNA double strand breaks and other DNA lesions. By enhancing the recombinase activity of RAD51, RAD51AP1 (RAD51-associated protein 1) serves a key role in homologous recombination-mediated chromosome damage repair. We show here that RAD51AP1 harbors two distinct DNA binding domains that are both needed for maximal protein activity under physiological conditions. We have finely mapped the two DNA binding domains in RAD51AP1 and generated mutant variants that are impaired in either or both of the DNA binding domains. Examination of these mutants reveals that both domains are indispensable for RAD51AP1 function in cells. These and other results illuminate the mechanistic basis of RAD51AP1 action in homologous DNA repair.

PMID: 22375013 [PubMed – indexed for MEDLINE]


Icon for PubMed Central Related Articles

Investigations of homologous recombination pathways and their regulation.

Yale J Biol Med. 2013 Dec 13;86(4):453-61

Authors: Daley JM, Kwon Y, Niu H, Sung P

Abstract
The DNA double-strand break (DSB), arising from exposure to ionizing radiation or various chemotherapeutic agents or from replication fork collapse, is among the most dangerous of chromosomal lesions. DSBs are highly cytotoxic and can lead to translocations, deletions, duplications, or mutations if mishandled. DSBs are eliminated by either homologous recombination (HR), which uses a homologous template to guide accurate repair, or by nonhomologous end joining (NHEJ), which simply rejoins the two broken ends after damaged nucleotides have been removed. HR generates error-free repair products and is also required for generating chromosome arm crossovers between homologous chromosomes in meiotic cells. The HR reaction includes several distinct steps: resection of DNA ends, homologous DNA pairing, DNA synthesis, and processing of HR intermediates. Each occurs in a highly regulated fashion utilizing multiple protein factors. These steps are being elucidated using a combination of genetic tools, cell-based assays, and in vitro reconstitution with highly purified HR proteins. In this review, we summarize contributions from our laboratory at Yale University in understanding HR mechanisms in eukaryotic cells.

PMID: 24348209 [PubMed – indexed for MEDLINE]


Icon for HighWire Icon for PubMed Central Related Articles

Introduction to the Thematic Minireview Series: DNA double-strand break repair and pathway choice.

J Biol Chem. 2018 07 06;293(27):10500-10501

Authors: Sung P

Abstract
Environmental agents and reactive metabolites induce myriad chromosomal lesions that challenge the integrity of our genome. In particular, the DNA double-strand break (DSB) has the highest potential to cause the types of chromosome aberrations and rearrangements found in transformed and cancer cells. Several conserved pathways of DSB repair exist in eukaryotes, and these have been the subject of intense studies in recent years. In this Thematic Minireview Series, four leading research groups review recent progress in deciphering DSB repair mechanisms and the intricate regulatory network that helps determine the preferential engagement of one pathway over others.

PMID: 29853635 [PubMed – indexed for MEDLINE]


Icon for HighWire Icon for PubMed Central Related Articles

Interplay between Ku and Replication Protein A in the Restriction of Exo1-mediated DNA Break End Resection.

J Biol Chem. 2015 Jul 24;290(30):18806-16

Authors: Krasner DS, Daley JM, Sung P, Niu H

Abstract
DNA double-strand breaks can be eliminated via non-homologous end joining or homologous recombination. Non-homologous end joining is initiated by the association of Ku with DNA ends. In contrast, homologous recombination entails nucleolytic resection of the 5′-strands, forming 3′-ssDNA tails that become coated with replication protein A (RPA). Ku restricts end access by the resection nuclease Exo1. It is unclear how partial resection might affect Ku engagement and Exo1 restriction. Here, we addressed these questions in a reconstituted system with yeast proteins. With blunt-ended DNA, Ku protected against Exo1 in a manner that required its DNA end-binding activity. Despite binding poorly to ssDNA, Ku could nonetheless engage a 5′-recessed DNA end with a 40-nucleotide (nt) ssDNA overhang, where it localized to the ssDNA-dsDNA junction and efficiently blocked resection by Exo1. Interestingly, RPA could exclude Ku from a partially resected structure with a 22-nt ssDNA tail and thus restored processing by Exo1. However, at a 40-nt tail, Ku remained stably associated at the ssDNA-dsDNA junction, and RPA simultaneously engaged the ssDNA region. We discuss a model in which the dynamic equilibrium between Ku and RPA binding to a partially resected DNA end influences the timing and efficiency of the resection process.

PMID: 26067273 [PubMed – indexed for MEDLINE]


Icon for Elsevier Science Related Articles

Importance of homo-dimerization of Fanconi-associated nuclease 1 in DNA flap cleavage.

DNA Repair (Amst). 2018 04;64:53-58

Authors: Rao T, Longerich S, Zhao W, Aihara H, Sung P, Xiong Y

Abstract
Fanconi-associated nuclease 1 (FAN1) removes interstrand DNA crosslinks (ICLs) through its DNA flap endonuclease and exonuclease activities. Crystal structures of human and bacterial FAN1 bound to a DNA flap have been solved. The Pseudomonas aeruginosa bacterial FAN1 and human FAN1 (hFAN1) missing a flexible loop are monomeric, while intact hFAN1 is homo-dimeric in structure. Importantly, the monomeric and dimeric forms of FAN1 exhibit very different DNA binding modes. Here, we interrogate the functional differences between monomeric and dimeric forms of FAN1 and provide an explanation for the discrepancy in oligomeric state between the two hFAN1 structures. Specifically, we show that the flexible loop in question is needed for hFAN1 dimerization. While monomeric and dimeric bacterial or human FAN1 proteins cleave a short 5′ flap strand with similar efficiency, optimal cleavage of a long 5′ flap strand is contingent upon protein dimerization. Our study therefore furnishes biochemical evidence for a role of hFAN1 homodimerization in biological processes that involve 5′ DNA Flap cleavage.

PMID: 29518739 [PubMed – indexed for MEDLINE]


Icon for HighWire Icon for PubMed Central Related Articles

Human RAD52 interactions with replication protein A and the RAD51 presynaptic complex.

J Biol Chem. 2017 07 14;292(28):11702-11713

Authors: Ma CJ, Kwon Y, Sung P, Greene EC

Abstract
Rad52 is a highly conserved protein involved in the repair of DNA damage. Human RAD52 has been shown to mediate single-stranded DNA (ssDNA) and is synthetic lethal with mutations in other key recombination proteins. For this study, we used single-molecule imaging and ssDNA curtains to examine the binding interactions of human RAD52 with replication protein A (RPA)-coated ssDNA, and we monitored the fate of RAD52 during assembly of the presynaptic complex. We show that RAD52 binds tightly to the RPA-ssDNA complex and imparts an inhibitory effect on RPA turnover. We also found that during presynaptic complex assembly, most of the RPA and RAD52 was displaced from the ssDNA, but some RAD52-RPA-ssDNA complexes persisted as interspersed clusters surrounded by RAD51 filaments. Once assembled, the presence of RAD51 restricted formation of new RAD52-binding events, but additional RAD52 could bind once RAD51 dissociated from the ssDNA. Together, these results provide new insights into the behavior and dynamics of human RAD52 during presynaptic complex assembly and disassembly.

PMID: 28551686 [PubMed – indexed for MEDLINE]


Icon for PubMed Central Related Articles

Guidelines for DNA recombination and repair studies: Mechanistic assays of DNA repair processes.

Microb Cell. 2019 Jan 07;6(1):65-101

Authors: Klein HL, Ang KKH, Arkin MR, Beckwitt EC, Chang YH, Fan J, Kwon Y, Morten MJ, Mukherjee S, Pambos OJ, El Sayyed H, Thrall ES, Vieira-da-Rocha JP, Wang Q, Wang S, Yeh HY, Biteen JS, Chi P, Heyer WD, Kapanidis AN, Loparo JJ, Strick TR, Sung P, Van Houten B, Niu H, Rothenberg E

Abstract
Genomes are constantly in flux, undergoing changes due to recombination, repair and mutagenesis. In vivo, many of such changes are studies using reporters for specific types of changes, or through cytological studies that detect changes at the single-cell level. Single molecule assays, which are reviewed here, can detect transient intermediates and dynamics of events. Biochemical assays allow detailed investigation of the DNA and protein activities of each step in a repair, recombination or mutagenesis event. Each type of assay is a powerful tool but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies.

PMID: 30652106 [PubMed]


Icon for HighWire Icon for PubMed Central Related Articles

Functions and regulation of the multitasking FANCM family of DNA motor proteins.

Genes Dev. 2015 Sep 01;29(17):1777-88

Authors: Xue X, Sung P, Zhao X

Abstract
Members of the conserved FANCM family of DNA motor proteins play key roles in genome maintenance processes. FANCM supports genome duplication and repair under different circumstances and also functions in the ATR-mediated DNA damage checkpoint. Some of these roles are shared among lower eukaryotic family members. Human FANCM has been linked to Fanconi anemia, a syndrome characterized by cancer predisposition, developmental disorder, and bone marrow failure. Recent studies on human FANCM and its orthologs from other organisms have provided insights into their biological functions, regulation, and collaboration with other genome maintenance factors. This review summarizes the progress made, with the goal of providing an integrated view of the functions and regulation of these enzymes in humans and model organisms and how they advance our understanding of genome maintenance processes.

PMID: 26341555 [PubMed – indexed for MEDLINE]


Filter results

Secured By miniOrange