A calf DNA polymerase having 3' to 5' exonuclease activity and aphidicolin sensitivity that is not stimulable by proliferating cell nuclear antigen has been described in this and other laboratories, and is now designated polymerase epsilon. This polymerase and a calf enzyme with similar properties designated epsilon* have been isolated. Preliminary photocrosslinking results demonstrate that the active site subunit molecular weights of calf epsilon and epsilon* polymerases are 220 and 145 kDa respectively, further distinguishing these enzymes from other calf DNA polymerases. Preliminary Western blot analysis suggests that calf DNA polymerase epsilon forms are analogues of forms of yeast DNA polymerase II. DNA polymerase II is essential and genetically distinct from other yeast polymerases, suggesting by analogy that calf polymerase epsilon is not derived from another calf polymerase. The above analysis will be completed. Also monoclonal antibodies against forms of calf DNA polymerase epsilon will be raised, and used for further structural analysis and to develop an immunoaffinity purification procedure. Tryptic peptide analysis should reveal whether polymerase epsilon* is derived from polymerase epsilon. Purified calf polymerases epsilon and epsilon* are highly processive. The mechanism of polymerase transfer from one primer-template to another, which may involve the exonuclease active site, will be investigated. Recently a helicase activity has been detected in this laboratory that co- purifies with both DNA polymerases epsilon and epsilon*, but can be resolved from each polymerase. The helicase in both fractions translocates in the 3' to 5' direction on the strand to which it is bound, and uses ATP and dATP for strand separation. These properties suggest that the same helicase protein may associate with both polymerases epsilon and epsilon*. Further information that could suggest the biological role of this helicase will be obtained. Specifically the NTPs or dNTPs that either drive or regulate the helicase on RNA or DNA, and the polynucleotide cofactors and substrates will be identified. Furthermore, the processivity and kinetics of the strand displacement reaction catalyzed either alone by the helicase, or in conjunction with DNA synthesis by the helicase together wit either polymerase epsilon or epsilon* will be measured. The effect of adducts to the DNA that could block helicase motion will also be examined. Elucidation of the basic mechanism and coordinated activities of the enzymes involved in mammalian chromosomal DNA replication and repair provides a basis for solving medical problems, particularly in the areas of cell growth regulation and cancer.
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| Balakrishnan, Lata; Bambara, Robert A (2013) Okazaki fragment metabolism. Cold Spring Harb Perspect Biol 5: |
| 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 |
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| 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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