Abstract
Base excision repair (BER) is a DNA repair pathway designed to correct small base lesions in genomic DNA. While DNA polymerase beta (pol β) is known to be the main polymerase in the BER pathway, various studies have implicated other DNA polymerases in back-up roles. One such polymerase, DNA polymerase lambda (pol λ), was shown to be important in BER of oxidative DNA damage. To further explore roles of the X-family DNA polymerases λ and β in BER, we prepared a mouse embryonic fibroblast cell line with deletions in the genes for both pol β and pol λ. Neutral red viability assays demonstrated that pol λ and pol β double null cells were hypersensitive to alkylating and oxidizing DNA damaging agents. In vitro BER assays revealed a modest contribution of pol λ to single-nucleotide BER of base lesions. Additionally, using co-immunoprecipitation experiments with purified enzymes and whole cell extracts, we found that both pol λ and pol β interact with the upstream DNA glycosylases for repair of alkylated and oxidized DNA bases. Such interactions could be important in coordinating roles of these polymerases during BER.
Highlights
Cells are constantly exposed to environmental and endogenous stressors such as reactive oxygen and nitrogen species, alkylating molecules and other reactive metabolites that are capable of damaging DNA
By using a neutral red viability assay and extracts prepared from these double knockout cell lines in combination with an in vitro Base excision repair (BER) assay, we revealed an increase in cellular hypersensitivity to DNA damaging agents and a decrease in BER capacity when compared to extract from cells containing a targeted deletion in one of the polymerases
Generation of the pol l 2/2 and pol b 2/2 double knockout mouse embryonic fibroblast cell line To further characterize the contributions of pol l and pol b in mammalian BER, we bred pol l +/2/pol b +/2 mice together
Summary
Cells are constantly exposed to environmental and endogenous stressors such as reactive oxygen and nitrogen species, alkylating molecules and other reactive metabolites that are capable of damaging DNA. DNA lesions induced by genotoxic compounds can encode for alternate nucleotides, potentially leading to permanent modifications in the genetic material. If these changes alter the function of key proteins required to regulate cell cycle progression or cellular defense mechanisms, adverse consequences for the cell may result. Base excision repair (BER) is considered the predominant defense system for eliminating DNA lesions generated by alkylating agents, reactive oxygen species and spontaneous base loss or strand breakage in mammalian cells. DNA polymerase-mediated DNA synthesis and 59-deoxyribose phosphate group (dRP) removal leads to a substrate for DNA ligase that completes SN BER. Since several mutagenic and cytotoxic intermediates are formed during BER, it is important that the process proceed efficiently to completion once the pathway is initiated [1,2,3]
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