The multifunctional enzyme Flap EndoNuclease (FEN-1 or FEN) and the homologous 5'family nuclease XPG play key but incompletely understood roles in DNA replication, repair, and genome integrity. FEN and XPG activities are furthermore aided by their partner protein, the processivity factor for DNA polymerase, termed proliferating cell nuclear antigen (PCNA). This proposal has three Aims: (1) characterize FEN and XPG substrate interactions, (2) elucidate FEN and XPG nuclease mechanisms and conformational changes, and (3) determine PCNA interactions and roles in FEN, XPG, and ligase activities including handoffs, which we propose avoid the release of toxic DNA intermediates in DNA replication and repair pathways.
The Aims will test our hypotheses on mechanisms for DNA recognition, for structural rearrangements to position the DNA in the active site for catalysis and for PCNA-mediated coordination rather than interference between FEN and ligase bound to PCNA and between XPG and the p21 inhibitor of PCNA binding by polymerase. To accomplish the Aims, we will combine the complementary techniques of small angle X-ray scattering (SAXS) for solution structures and macromolecular X-ray crystallography (MX) for high resolution, with informed biochemical and mutational experiments. Targeted experiments on human and archaeal proteins will allow efficient and systematic analyses fundamental to understanding FEN, XPG, and ligase activities plus their functional PCNA interactions. Quantitative characterizations by SAXS and MX along with biophysical and mutational analyses in the Tainer lab will be aided by ongoing collaborative research on FEN biochemistry, FEN inhibition, and XPG activities plus single molecule fluorescence technologies applied to PCNA complexes. The expected results will clarify the molecular determinants for 5'family DNA interactions and nuclease activities including FEN, XPG, and ligase interactions with PCNA relevant to DNA replication, base repair, and nucleotide excision repair. Overall, this research will provide a fundamental molecular framework for FEN, XPG, ligase and PCNA activities and consequent roles in the regulation of genome fidelity. The results will also clarify mechanisms whereby loss of the functions of these coordinated complexes, which act in DNA replication and repair processes, may lead to inheritable genetic defects and the cancer initiation.

Public Health Relevance

This proposal focuses on the multifunctional enzyme Flap EndoNuclease (FEN-1 or FEN) and the homologous 5'family nuclease XPG, which play key but incompletely understood roles in DNA replication, repair, and genome integrity. The strategy is to combine both low resolution and high resolution structural techniques with targeted biochemical studies to clarify the molecular determinants for DNA interactions and catalytic nuclease activities. Overall, this research will provide a fundamental molecular framework for FEN and XPG function and consequent roles in the regulation of genome fidelity. The results will also clarify mechanisms whereby loss of the functions of these coordinated complexes, which act in DNA replication and repair processes, may lead to inheritable genetic defects and the cancer initiation.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA081967-14
Application #
8474699
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Pelroy, Richard
Project Start
1999-03-01
Project End
2014-05-31
Budget Start
2013-06-01
Budget End
2014-05-31
Support Year
14
Fiscal Year
2013
Total Cost
$320,242
Indirect Cost
$152,829
Name
Lawrence Berkeley National Laboratory
Department
Biophysics
Type
Organized Research Units
DUNS #
078576738
City
Berkeley
State
CA
Country
United States
Zip Code
94720
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
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; Classen, Scott; Chapados, Brian R et al. (2011) Human flap endonuclease structures, DNA double-base flipping, and a unified understanding of the FEN1 superfamily. Cell 145:198-211
Tomlinson, Christopher G; Syson, Karl; Sengerova, Blanka et al. (2011) Neutralizing mutations of carboxylates that bind metal 2 in T5 flap endonuclease result in an enzyme that still requires two metal ions. J Biol Chem 286:30878-87
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
Syson, Karl; Tomlinson, Christopher; Chapados, Brian R et al. (2008) Three metal ions participate in the reaction catalyzed by T5 flap endonuclease. J Biol Chem 283:28741-6
Putnam, Christopher D; Hammel, Michal; Hura, Greg L et al. (2007) X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution. Q Rev Biophys 40:191-285

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