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Role of replication protein A in double holliday junction dissolution mediated by the BLM-Topo IIIα-RMI1-RMI2 protein complex.

J Biol Chem. 2013 May 17;288(20):14221-7

Authors: Xue X, Raynard S, Busygina V, Singh AK, Sung P

The conserved BTR complex, composed of the Bloom’s syndrome helicase (BLM), topoisomerase IIIα, RMI1, and RMI2, regulates homologous recombination in favor of non-crossover formation via the dissolution of the double Holliday Junction (dHJ). Here we show enhancement of the BTR-mediated dHJ dissolution reaction by the heterotrimeric single-stranded DNA binding protein replication protein A (RPA). Our results suggest that RPA acts by sequestering a single-stranded DNA intermediate during dHJ dissolution. We provide evidence that RPA physically interacts with RMI1. The RPA interaction domain in RMI1 has been mapped, and RMI1 mutants impaired for RPA interaction have been generated. Examination of these mutants ascertains the significance of the RMI1-RPA interaction in dHJ dissolution. Our results thus implicate RPA as a cofactor of the BTR complex in dHJ dissolution.

PMID: 23543748 [PubMed – indexed for MEDLINE]

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Role of RAD51AP1 in homologous recombination DNA repair and carcinogenesis.

DNA Repair (Amst). 2017 11;59:76-81

Authors: Pires E, Sung P, Wiese C

Homologous recombination (HR) serves to repair DNA double-strand breaks and damaged replication forks and is essential for maintaining genome stability and tumor suppression. HR capacity also determines the efficacy of anticancer therapy. Hence, there is an urgent need to better understand all HR proteins and sub-pathways. An emerging protein that is critical for RAD51-mediated HR is RAD51-associated protein 1 (RAD51AP1). Although much has been learned about its biochemical attributes, the precise molecular role of RAD51AP1 in the HR reaction is not yet fully understood. The available literature also suggests that RAD51AP1 expression may be relevant for cancer development and progression. Here, we review the efforts that led to the discovery of RAD51AP1 and elaborate on our current understanding of its biochemical profile and biological function. We also discuss how RAD51AP1 may help to promote cancer development and why it could potentially represent a promising new target for therapeutic intervention.

PMID: 28963981 [PubMed – indexed for MEDLINE]

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RIF1 in DNA break repair pathway choice.

Mol Cell. 2013 Mar 07;49(5):840-1

Authors: Daley JM, Sung P

New findings on the RIF1 protein by four research groups, including Chapman et al. (2013) and Escribano-Díaz et al. (2013) in this issue, provide insights into DNA double-strand break repair pathway choice in mammalian cells.

PMID: 23473603 [PubMed]

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Restriction of replication fork regression activities by a conserved SMC complex.

Mol Cell. 2014 Nov 06;56(3):436-45

Authors: Xue X, Choi K, Bonner J, Chiba T, Kwon Y, Xu Y, Sanchez H, Wyman C, Niu H, Zhao X, Sung P

Conserved, multitasking DNA helicases mediate diverse DNA transactions and are relevant for human disease pathogenesis. These helicases and their regulation help maintain genome stability during DNA replication and repair. We show that the structural maintenance of chromosome complex Smc5-Smc6 restrains the replication fork regression activity of Mph1 helicase, but not its D loop disruptive activity. This regulatory mechanism enables flexibility in replication fork repair without interfering with DNA break repair. In vitro studies find that Smc5-Smc6 binds to a Mph1 region required for efficient fork regression, preventing assembly of Mph1 oligomers at the junction of DNA forks. In vivo impairment of this regulatory mechanism compensates for the inactivation of another fork regression helicase and increases reliance on joint DNA structure removal or avoidance. Our findings provide molecular insights into replication fork repair regulation and uncover a role of Smc5-Smc6 in directing Mph1 activity toward a specific biochemical outcome.

PMID: 25439736 [PubMed – indexed for MEDLINE]

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Regulatory control of Sgs1 and Dna2 during eukaryotic DNA end resection.

Proc Natl Acad Sci U S A. 2019 03 26;116(13):6091-6100

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

In the repair of DNA double-strand breaks by homologous recombination, the DNA break ends must first be processed into 3′ single-strand DNA overhangs. In budding yeast, end processing requires the helicase Sgs1 (BLM in humans), the nuclease/helicase Dna2, Top3-Rmi1, and replication protein A (RPA). Here, we use single-molecule imaging to visualize Sgs1-dependent end processing in real-time. We show that Sgs1 is recruited to DNA ends through Top3-Rmi1-dependent or -independent means, and in both cases Sgs1 is maintained in an immoble state at the DNA ends. Importantly, the addition of Dna2 triggers processive Sgs1 translocation, but DNA resection only occurs when RPA is also present. We also demonstrate that the Sgs1-Dna2-Top3-Rmi1-RPA ensemble can efficiently disrupt nucleosomes, and that Sgs1 itself possesses nucleosome remodeling activity. Together, these results shed light on the regulatory interplay among conserved protein factors that mediate the nucleolytic processing of DNA ends in preparation for homologous recombination-mediated chromosome damage repair.

PMID: 30850524 [PubMed – indexed for MEDLINE]

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Regulation of Hed1 and Rad54 binding during maturation of the meiosis-specific presynaptic complex.

EMBO J. 2018 04 03;37(7):

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

Most eukaryotes have two Rad51/RecA family recombinases, Rad51, which promotes recombination during mitotic double-strand break (DSB) repair, and the meiosis-specific recombinase Dmc1. During meiosis, the strand exchange activity of Rad51 is downregulated through interactions with the meiosis-specific protein Hed1, which helps ensure that strand exchange is driven by Dmc1 instead of Rad51. Hed1 acts by preventing Rad51 from interacting with Rad54, a cofactor required for promoting strand exchange during homologous recombination. However, we have a poor quantitative understanding of the regulatory interplay between these proteins. Here, we use real-time single-molecule imaging to probe how the Hed1- and Rad54-mediated regulatory network contributes to the identity of mitotic and meiotic presynaptic complexes. Based on our findings, we define a model in which kinetic competition between Hed1 and Rad54 helps define the functional identity of the presynaptic complex as cells undergo the transition from mitotic to meiotic repair.

PMID: 29444896 [PubMed – indexed for MEDLINE]

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Regulation of FANCD2 and FANCI monoubiquitination by their interaction and by DNA.

Nucleic Acids Res. 2014 May;42(9):5657-70

Authors: Longerich S, Kwon Y, Tsai MS, Hlaing AS, Kupfer GM, Sung P

FANCD2 and FANCI function together in the Fanconi anemia network of deoxyribonucleic acid (DNA) crosslink repair. These proteins form the dimeric ID2 complex that binds DNA and becomes monoubiquitinated upon exposure of cells to DNA crosslinking agents. The monoubiquitinated ID2 complex is thought to facilitate DNA repair via recruitment of specific nucleases, translesion DNA polymerases and the homologous recombination machinery. Using the ubiquitin conjugating enzyme (E2) UBE2T and ubiquitin ligase (E3) FANCL, monoubiquitination of human FANCD2 and FANCI was examined. The ID2 complex is a poor substrate for monoubiquitination, consistent with the published crystal structure showing the solvent inaccessibility of the target lysines. Importantly, FANCD2 monoubiquitination within the ID2 complex is strongly stimulated by duplex or branched DNA, but unstructured single-stranded DNA or chromatinized DNA is ineffective. Interaction of FANCL with the ID2 complex is indispensable for its E3 ligase efficacy. Interestingly, mutations in FANCI that impair its DNA binding activity compromise DNA-stimulated FANCD2 monoubiquitination. Moreover, we demonstrate that in the absence of FANCD2, DNA also stimulates FANCI monoubiquitination, but in a FANCL-independent manner. These results implicate the role of a proper DNA ligand in FANCD2 and FANCI monoubiquitination, and reveal regulatory mechanisms that are dependent on protein-protein and protein-DNA interactions.

PMID: 24623813 [PubMed – indexed for MEDLINE]

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Regulation of DNA pairing in homologous recombination.

Cold Spring Harb Perspect Biol. 2014 Sep 04;6(11):a017954

Authors: Daley JM, Gaines WA, Kwon Y, Sung P

Homologous recombination (HR) is a major mechanism for eliminating DNA double-strand breaks from chromosomes. In this process, the break termini are resected nucleolytically to form 3′ ssDNA (single-strand DNA) overhangs. A recombinase (i.e., a protein that catalyzes homologous DNA pairing and strand exchange) assembles onto the ssDNA and promotes pairing with a homologous duplex. DNA synthesis then initiates from the 3′ end of the invading strand, and the extended DNA joint is resolved via one of several pathways to restore the integrity of the injured chromosome. It is crucial that HR be carefully orchestrated because spurious events can create cytotoxic intermediates or cause genomic rearrangements and loss of gene heterozygosity, which can lead to cell death or contribute to the development of cancer. In this review, we will discuss how DNA motor proteins regulate HR via a dynamic balance of the recombination-promoting and -attenuating activities that they possess.

PMID: 25190078 [PubMed – indexed for MEDLINE]

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Reconstituted System for the Examination of Repair DNA Synthesis in Homologous Recombination.

Methods Enzymol. 2017;591:307-325

Authors: Kwon Y, Daley JM, Sung P

In homologous recombination (HR), DNA polymerase δ-mediated DNA synthesis occurs within the displacement loop (D-loop) that is made by the recombinase Rad51 in conjunction with accessory factors. We describe in this chapter the reconstitution of the D-loop and repair DNA synthesis reactions using purified Saccharomyces cerevisiae HR (Rad51, RPA, and Rad54) and DNA replication (PCNA, RFC, and DNA polymerase δ) proteins and document the role of the Pif1 helicase in DNA synthesis via a migrating DNA bubble intermediate. These reconstituted systems are particularly valuable for understanding the conserved mechanism of repair DNA synthesis dependent on DNA polymerase δ and its cognate helicase in eukaryotic organisms.

PMID: 28645374 [PubMed – indexed for MEDLINE]

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Rad52, Maestro of Inverse Strand Exchange.

Mol Cell. 2017 Jul 06;67(1):1-3

Authors: Kwon Y, Sung P

In this issue of Molecular Cell, Mazina et al. (2017) describe how the Rad52 protein mediates RNA-dependent DNA double-strand break repair via inverse strand exchange. This finding sheds light on how eukaryotes utilize RNA to repair chromosome breaks.

PMID: 28686872 [PubMed – indexed for MEDLINE]

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