During mammalian nuclear DNA replication, discontinuous synthesis of the lagging strand of the replication fork involves an important group of reactions. RNA primers are laid down on the parental DNA strand about 100 nucleotides apart and extended with DNA until each is separated by a nick. We have previously found that the RNA primers are removed by the action of ribonuclease HI and a remarkable enzyme called flap endonuclease or FEN1. The segments are then joined to make the full- length replicated DNA. The preferred substrate of FEN1 is double stranded but with a single-stranded 5'-tail of flap. We have mechanistic evidence, supported by structural evidence from other researchers, suggesting that the nuclease threads over the 5'-end of the flap through a loop structure in the protein and tracks down the flap cleaving at its base. One reason for the preference for flap substrates is that initiator RNA may be removed in reactions involving a flap intermediate. Genetic evidence indicates that defects in FEN1 lead to repeat sequence expansions often seen in human genetic diseases. Current proposed mechanisms for expansion involve defects in flap removal. Another reason why the flap cleavage mechanism may have evolved is to allow FEN1 to participate in a frequently employed process called long patch base excision repair. This critical pathway corrects base damage that contributes to cellular aging and carcinogenesis. A significant intermediate is a flap with the damaged nucleotide at the 5'-end. We propose to analyze the FEN1 cleavage mechanism by determining its exact substrate preference, and the mechanism by which it enters and moves on its substrates. We will probe its biological function by reconstituting both discontinuous DNA replication and long patch base excision repair using purified human enzymes. These processes also will be examine din extracts of yeast having mutations in FEN1 and other pathway components. PCNA, the sliding clamp for polymerases, stimulates FEN1, whereas the regulator protein p21/Cip1 inhibits their interaction. The mammalian single-stranded DNA-binding protein RPA alters the substrate specificity of FEN1. We will investigate these processes with pure enzymes and in re-constituted systems. The mechanisms of RNase H will also be examined to determine how it works with FEN1 in RNA primer removal. Results will clarify reaction mechanisms central to DNA replication and repair.
| 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 |
| 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; Bambara, Robert A (2011) Eukaryotic lagging strand DNA replication employs a multi-pathway mechanism that protects genome integrity. J Biol Chem 286:6865-70 |
| 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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