Project 2 (Replication-Associated Repair and Replication Fork Maintenance) of the SBDR Program Project focuses on the integration of multiple DNA repair pathways at replication forks and their roles in the maintenance of genomic stability. Cells devote significant resources to detecting and repairing DNA damage prior to replication and to protecting active replication forks in the presence of replisome-stalling lesions. Failure in these processes or in their coordination can lead to cancer and aging. The goal of this research is to use a combination of structural and functional approaches to investigate the protein-protein and protein- DNA interfaces required for coordinated damage recognition and repair in association with replication. We propose four Aims to examine keystone proteins involved in excision repair pathways that remove damage prior to or in coordination with the replisome, and with proteins involved in fork stability.
Aim 1 will investigate the structural and functional basis for roles of XPG in NER, BER, and replication-associated repair by atomic resolution studies of the structured endonuclease domain and by structural characterization of the relatively unstructured R- and C-terminus domains via their interaction with partner proteins RPA, ubiquitinated PCNA, and DNA Ligase I.
Aim 2 will investigate the structural and functional basis for the role of the annealing helicase, SMARCAL1, with RPA at stalled replication forks. Since SMARCAL1 is the first annealing helicase demonstrated to act in maintaining genome integrity at stalled forks, it is critically important to understand its function at a mechanistic level.
Aim 3 will investigate the structural biology of NEIL1-initiated BER of oxidized bases, through interactions with FEN-1 and XPG, XRCC1/Ligase III, and with RPA.
Aim 4 will focus on PNKP, which has kinase and phosphatase activities critical for both single-strand and double-strand break repair processes and which is also an essential component of NEIL-directed BER. We will investigate PNKP phosphatase domain substrate binding, structurally characterize PNKP interaction with a specific inhibitor of its phosphatase activity, and interrogate the interaction of PNKP with XRCC1/Ligase III. The proposed studies are built upon major findings and collaborations generated during the previous funding period in SBDR2. They include experimental interactions and substantial synergy with Projects 1, 3, 4, and 6, as well as with the EMB and SCB Cores. The anticipated outcome of these studies is a much more detailed molecular picture of the protein-protein and protein-DNA complexes involved in replication-associated repair. The information generated will elucidate the mode of action of a promising DNA repair inhibitor and contribute to its optimization, and will define new potential targets for novel cancer therapies.
Cancer cells have elevated levels of replication-stress due to abnormal cell division cycles, and many cancers have mutations that alter replication-associated repair mechanisms. These properties make cancer cells particularly dependent upon replication checkpoint and repair pathways. Project 2 aims to define the structural and functional interactions and interfaces between multi-functional protein-protein and protein-DNA complexes required for replication-associated repair, with the goal of providing new potential targets for novel cancer therapies.
|Sishc, Brock J; Davis, Anthony J (2017) The Role of the Core Non-Homologous End Joining Factors in Carcinogenesis and Cancer. Cancers (Basel) 9:|
|Dungrawala, Huzefa; Bhat, Kamakoti P; Le Meur, Rémy et al. (2017) RADX Promotes Genome Stability and Modulates Chemosensitivity by Regulating RAD51 at Replication Forks. Mol Cell 67:374-386.e5|
|Jiang, Bingcheng; Glover, J N Mark; Weinfeld, Michael (2017) Neurological disorders associated with DNA strand-break processing enzymes. Mech Ageing Dev 161:130-140|
|Yang, Chunying; Sengupta, Shiladitya; Hegde, Pavana M et al. (2017) Regulation of oxidized base damage repair by chromatin assembly factor 1 subunit A. Nucleic Acids Res 45:739-748|
|Sugitani, Norie; Voehler, Markus W; Roh, Michelle S et al. (2017) Analysis of DNA binding by human factor xeroderma pigmentosum complementation group A (XPA) provides insight into its interactions with nucleotide excision repair substrates. J Biol Chem 292:16847-16857|
|Aceytuno, R Daniel; Piett, Cortt G; Havali-Shahriari, Zahra et al. (2017) Structural and functional characterization of the PNKP-XRCC4-LigIV DNA repair complex. Nucleic Acids Res 45:6238-6251|
|Ma, Chu Jian; Kwon, Youngho; Sung, Patrick et al. (2017) Human RAD52 interactions with replication protein A and the RAD51 presynaptic complex. J Biol Chem 292:11702-11713|
|Tsutakawa, Susan E; Thompson, Mark J; Arvai, Andrew S et al. (2017) Phosphate steering by Flap Endonuclease 1 promotes 5'-flap specificity and incision to prevent genome instability. Nat Commun 8:15855|
|Shi, Yuqian; Hellinga, Homme W; Beese, Lorena S (2017) Interplay of catalysis, fidelity, threading, and processivity in the exo- and endonucleolytic reactions of human exonuclease I. Proc Natl Acad Sci U S A 114:6010-6015|
|Woodrick, Jordan; Gupta, Suhani; Camacho, Sharon et al. (2017) A new sub-pathway of long-patch base excision repair involving 5' gap formation. EMBO J 36:1605-1622|
Showing the most recent 10 out of 449 publications