Characterization of the p125 subunit of human DNA polymerase delta and its deletion mutants. Interaction with cyclin-dependent kinase-cyclins.

The Journal of Biological Chemistry
S M WuM Y Lee

Abstract

The catalytic subunit of human DNA polymerase (pol) delta was overexpressed in an active, soluble form by the use of a baculovirus system in insect cells. The recombinant enzyme was separated from endogenous DNA polymerases by phosphocellulose, Mono Q-Sepharose, and single-stranded DNA-cellulose chromatography. Recombinant DNA pol delta was also purified by immunoaffinity chromatography. The enzymatic properties of the purified catalytic subunit were characterized. The enzyme was active and possessed both DNA polymerase and associated 3' to 5' exonuclease activities. NH2-terminal deletion mutants retained polymerase activity, whereas the core and COOH-terminal deletion mutants were devoid of any measurable activities. Coinfection of Sf9 cells with recombinant baculovirus vectors for pol delta and cyclin-dependent kinase (cdk)-cyclins followed by metabolic labeling with 32Pi showed that the recombinant catalytic subunit of pol delta could be hyperphosphorylated by G1 phase-specific cdk-cyclins. When cdk2 was coexpressed with pol delta in Sf9 cells, pol delta was found to coimmunoprecipitate with antibodies against cdk2. Experiments with deletion mutants of pol delta showed that the NH2-terminal region was essential for this inte...Continue Reading

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Citations

Jun 5, 2002·Annual Review of Biochemistry·Ulrich HubscherSilvio Spadari
May 3, 2000·Proceedings of the National Academy of Sciences of the United States of America·T BashirK Willwand
Oct 11, 2012·Environmental and Molecular Mutagenesis·Marietta Y W T LeeErnest Y C Lee
Jul 3, 1999·The Journal of Biological Chemistry·D J MozzherinP A Fisher
Apr 6, 2000·The Journal of Biological Chemistry·L LiuM Y Lee
Apr 5, 2000·The Journal of Biological Chemistry·H J Einolf, F P Guengerich
Apr 7, 2005·The Journal of Biological Chemistry·Bin XieMarietta Y W T Lee
Mar 3, 2006·The Journal of Biological Chemistry·Hao LiMarietta Y W T Lee

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