Abstract
Mammalian DNA polymerase (pol) lambda is a member of the X-family of DNA polymerases and has striking enzymatic and structural similarities to mammalian DNA pol beta. Because pol beta provides two important enzymatic activities for base excision repair (BER), we examined whether pol lambda might also contribute to BER. We used extracts from mouse embryonic fibroblasts representing wild-type and null genotypes for pol beta and pol lambda. In combination with neutralizing antibodies against pol beta and pol lambda, our results show a BER deficiency in the pol lambda -/- cell extract compared with extract from isogenic wild-type cells. In addition, the pol lambda antibody strongly reduced in vitro BER in the pol beta -/- cell extract. These data indicate that pol lambda is able to contribute to BER in mouse fibroblast cell extract.
Highlights
DNA Polymerase Mediates a Back-up Base Excision Repair Activity in Extracts of Mouse Embryonic Fibroblasts*
In Vitro base excision repair (BER) Assay with Uracil-containing Oligonucleotide Duplex DNA Substrate—Because cell extracts support a variety of biochemical activities, they can provide a window into the events within a cell and represent a more biologically relevant model for studying the biochemical activity of a given protein
In an effort to further examine pol in the context of these processes, a series of mouse embryonic fibroblast (MEF) cell extract-mediated BER reactions were performed with uracil-containing oligonucleotides
Summary
Vol 280, No 18, Issue of May 6, pp. 18469 –18475, 2005 Printed in U.S.A. DNA Polymerase Mediates a Back-up Base Excision Repair Activity in Extracts of Mouse Embryonic Fibroblasts*. Pol  contributes both gap-filling DNA synthesis and 5Ј-dRP lyase activities to the. Pol shares two key BER characteristics with pol , gap-filling DNA synthesis activity and dRP lyase activity [16, 17]. These activities allow pol to substitute for pol  in a single-nucleotide BER system reconstituted with purified human proteins [17]. The idea that the BRCA1 C-terminal domain may facilitate protein-protein interactions is supported by a previous study showing that the BRCA1 C-terminal domain is required for efficient gap-filling activity by pol and S. cerevisiae DNA pol IV during non-homologous end-joining in vitro (18 –20). In an effort to further clarify the role of this enzyme in BER, we performed a series of in vitro BER assays with extracts from genetically modified mouse cells lacking functional pol alleles
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