Cancer research progress, having beneficially greatly from studies on powerful model systems, strongly supports the hypothesis that specific mutations leading to decreased genome stability are critical early events in tumori-genesis. Experiments in yeast show that eukaryotic genome integrity requires the action of the multi-functional enzyme Flap EndoNuclease (FEN-1), and that mutations in FEN-1 result in DNA duplication defects that occur in human tumors and inherited human diseases. FEN, which cleaves unpaired, over-hanging flaps in double- stranded DNA during repair and the terminal priming RNA base during DNA replication, is a structure-specific nuclease necessary for DNA repair and for processing the 5' ends of Okazaki fragments during lagging strand DNA synthesis. The proposed work aims to understand in atomic detain the structural metallobiochemistry responsible for FEN-1 catalytic activity, substrate specificity, and in vivo function. The structure of FEN-1 from the thermophilic archaebacterium Pyrococcus furiosus (pFEN-1) will be determined by X-ray crystallography and then used to solve pFEN-1 mutant structures as well as possible human, yeast, Archaeglobis fulgidus and Methanococcus jannaschii FEN-1 structures. The availability of multiple FEN-1 structures will identify structurally conserved, functionally important regions, and aim in the structure determinations and interpretations of FEN-1 complexes with DNA and with the processivity factor for DNA polymerase, termed proliferating cell nuclear antigen (PCNA). Based upon the high sequence homology among these enzymes, each FEN-1 structure should provide results applicable to the entire FEN-1 nuclease family. Thus, structures of any FEN-1 enzyme will be useful, and access to five FEN-1 enzymes will increase opportunities for successfully determining structures of FEN-1 complexes with DNA and PCNA. The high temperature optimum for the archael FEN-1 enzymes will assist the formation of stable complexes and allow possible trapping of DNA binding and cleavage intermediates via temperature control during mixing and crystallization steps. Competitive analyses of FEN-1 structures and complexes in the Tainer lab will be integrated with coupled biochemical and mutational analysis in the Shen lab, so mutant structures, biochemistry, and biology will experimentally test structure-based hypotheses and probe features key to FEN-1 structure- function relationships. Overall, this research will provide fundamental knowledge on FEN-1 structure and function relevant to defining its role in the regulation of genome fidelity and the mechanisms whereby loss of FEN-1 functions may lead to inheritable genetic defects and the initiation of cancer.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
1R01CA081967-01
Application #
2869473
Study Section
Radiation Study Section (RAD)
Program Officer
Pelroy, Richard
Project Start
1999-03-01
Project End
2002-12-31
Budget Start
1999-03-01
Budget End
1999-12-31
Support Year
1
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Tsutakawa, Susan E; Yan, Chunli; Xu, Xiaojun et al. (2015) Structurally distinct ubiquitin- and sumo-modified PCNA: implications for their distinct roles in the DNA damage response. Structure 23:724-733
Tsutakawa, Susan E; Tainer, John A (2015) Bending Forks and Wagging Dogs--It's about the DNA 3' Tail. Mol Cell 58:972-3
Tsutakawa, Susan E; Lafrance-Vanasse, Julien; Tainer, John A (2014) The cutting edges in DNA repair, licensing, and fidelity: DNA and RNA repair nucleases sculpt DNA to measure twice, cut once. DNA Repair (Amst) 19:95-107
Shin, David S; Pratt, Ashley J; Tainer, John A (2014) Archaeal genome guardians give insights into eukaryotic DNA replication and damage response proteins. Archaea 2014:206735
Grasby, Jane A; Finger, L David; Tsutakawa, Susan E et al. (2012) Unpairing and gating: sequence-independent substrate recognition by FEN superfamily nucleases. Trends Biochem Sci 37:74-84
Patel, Nikesh; Atack, John M; Finger, L David et al. (2012) Flap endonucleases pass 5'-flaps through a flexible arch using a disorder-thread-order mechanism to confer specificity for free 5'-ends. Nucleic Acids Res 40:4507-19
Finger, L David; Atack, John M; Tsutakawa, Susan et al. (2012) The wonders of flap endonucleases: structure, function, mechanism and regulation. Subcell Biochem 62:301-26
Querol-Audi, Jordi; Yan, Chunli; Xu, Xiaojun et al. (2012) Repair complexes of FEN1 endonuclease, DNA, and Rad9-Hus1-Rad1 are distinguished from their PCNA counterparts by functionally important stability. Proc Natl Acad Sci U S A 109:8528-33
Tsutakawa, Susan E; Tainer, John A (2012) Double strand binding-single strand incision mechanism for human flap endonuclease: implications for the superfamily. Mech Ageing Dev 133:195-202
Tsutakawa, Susan E; Van Wynsberghe, Adam W; Freudenthal, Bret D et al. (2011) Solution X-ray scattering combined with computational modeling reveals multiple conformations of covalently bound ubiquitin on PCNA. Proc Natl Acad Sci U S A 108:17672-7

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