The eukaryotic genome is synthesized by replication forks in which one strand is made as short segments, called Okazaki fragments, which are then joined. Each segment is initiated with an RNA primer, and then DNA is added to about 100 nucleotides. Polymerization from an upstream segment displaces the initiator RNA on the downstream segment into a flap. Flap endonuclease (FEN1) removes the flap, leaving a nick for ligation. Some flaps are thought to require additional cleavage by the Dna2 nuclease/helicase. Recent evidence also suggests that the Pif1 and Bloom (BLM) helicases have a role. Using genetic information as a guide, we have developed a biochemical system that reconstitutes Okazaki fragment joining. Early results show that while some flaps are readily processed by FEN1, others become long and require the action of Dna2. We will test whether Dna2 promotes correct processing of long flaps. Pif1 mutations suppress Dna2 mutations, suggesting that Pif1 creates a need for Dna2. BLM over expression suppresses Dna2 mutations, suggesting that BLM removes a need for Dna2. The functional relationships of these proteins will be determined. DNA added just after the RNA is thought to have frequent mismatch errors. We will determine whether strand displacement synthesis proceeds further into mismatch containing fragments, and is aided by Pif1, to allow 5'proofreading. Both FEN1 and Dna2 track onto the 5'ends of flaps and move to their points of cleavage, yet neither interferes with the action of the other. We will analyze motions of these two proteins to determine how this coordination is possible. BLM is a strong stimulator of DNA ligase I. The physical and functional interactions of the two proteins will be examined. Long patch base excision repair (LP BER) strongly resembles Okazaki fragment processing and also uses FEN1 and DNA ligase I. The 911 checkpoint complex, AP endonuclease and PCNA all coordinate and stimulate LP BER proteins. We will employ immunoprecipitation, substrate binding and activity stimulation assays to distinguish the roles of these three proteins in facilitation of the LP BER pathway. We will expand on preliminary results suggesting that DNA damage induces more effective interactions between 911 and LP BER proteins. Overall, results will clarify how fundamental DNA replication reactions are regulated to protect genome stability and participate in DNA repair. Human cells have developed a means of replicating the genetic information in their DNA in a very accurate manner. They use proteins that are specifically designed to prevent errors from forming in DNA and to correct any errors after they occur. This allows us to have a long lifespan and slows the onset of cancers. Understanding DNA replication and repair could provide us means to further delay aging and cancers, and to develop innovative cancer therapy. The proteins that are responsible for replicating our DNA have built-in properties that protect the information encoded in the DNA. We are working to understand the mechanisms that they employ to maintain the integrity of our genomes and delay the onset of aging-related diseases and cancers. Our results will clarify this process and also help identify protein functions that could be targets of agents designed to slow genome deterioration.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM024441-30
Application #
7743060
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Hagan, Ann A
Project Start
1979-01-01
Project End
2011-12-31
Budget Start
2010-01-01
Budget End
2010-12-31
Support Year
30
Fiscal Year
2010
Total Cost
$354,470
Indirect Cost
Name
University of Rochester
Department
Biochemistry
Type
Schools of Dentistry
DUNS #
041294109
City
Rochester
State
NY
Country
United States
Zip Code
14627
Balakrishnan, Lata; Bambara, Robert A (2013) Okazaki fragment metabolism. Cold Spring Harb Perspect Biol 5:
Balakrishnan, Lata; Bambara, Robert A (2013) Flap endonuclease 1. Annu Rev Biochem 82:119-38
Miller, Adam S; Balakrishnan, Lata; Buncher, Noah A et al. (2012) Telomere proteins POT1, TRF1 and TRF2 augment long-patch base excision repair in vitro. Cell Cycle 11:998-1007
Gloor, Jason W; Balakrishnan, Lata; Campbell, Judith L et al. (2012) Biochemical analyses indicate that binding and cleavage specificities define the ordered processing of human Okazaki fragments by Dna2 and FEN1. Nucleic Acids Res 40:6774-86
Kantartzis, Athena; Williams, Gregory M; Balakrishnan, Lata et al. (2012) Msh2-Msh3 interferes with Okazaki fragment processing to promote trinucleotide repeat expansions. Cell Rep 2:216-22
Balakrishnan, Lata; Bambara, Robert A (2011) Eukaryotic lagging strand DNA replication employs a multi-pathway mechanism that protects genome integrity. J Biol Chem 286:6865-70
Fortini, Barbara K; Pokharel, Subhash; Polaczek, Piotr et al. (2011) Characterization of the endonuclease and ATP-dependent flap endo/exonuclease of Dna2. J Biol Chem 286:23763-70
Balakrishnan, Lata; Stewart, Jason; Polaczek, Piotr et al. (2010) Acetylation of Dna2 endonuclease/helicase and flap endonuclease 1 by p300 promotes DNA stability by creating long flap intermediates. J Biol Chem 285:4398-404
Stewart, Jason A; Campbell, Judith L; Bambara, Robert A (2010) Dna2 is a structure-specific nuclease, with affinity for 5'-flap intermediates. Nucleic Acids Res 38:920-30
Balakrishnan, Lata; Polaczek, Piotr; Pokharel, Subhash et al. (2010) Dna2 exhibits a unique strand end-dependent helicase function. J Biol Chem 285:38861-8

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