A Germline Polymorphism of DNA Polymerase Beta Induces Genomic Instability and Cellular Transformation

Abstract

Several germline single nucleotide polymorphisms (SNPs) have been identified in the POLB gene, but little is known about their cellular and biochemical impact. DNA Polymerase β (Pol β), encoded by the POLB gene, is the main gap-filling polymerase involved in base excision repair (BER), a pathway that protects the genome from the consequences of oxidative DNA damage. In this study we tested the hypothesis that expression of the POLB germline coding SNP (rs3136797) in mammalian cells could induce a cancerous phenotype. Expression of this SNP in both human and mouse cells induced double-strand breaks, chromosomal aberrations, and cellular transformation. Following treatment with an alkylating agent, cells expressing this coding SNP accumulated BER intermediate substrates, including single-strand and double-strand breaks. The rs3136797 SNP encodes the P242R variant Pol β protein and biochemical analysis showed that P242R protein had a slower catalytic rate than WT, although P242R binds DNA similarly to WT. Our results suggest that people who carry the rs3136797 germline SNP may be at an increased risk for cancer susceptibility.

Germ-line variant of human NTH1 DNA glycosylase induces genomic instability and cellular transformation

“…Tagging the proteins with the HA epitope enables us to distinguish endogenous from exogenous protein. We selected two independent pools of MCF10A cells expressing either WT or D239Y hNTH1 as described (12,13). The MCF10A cells are an immortalized but nontransformed cell line that also expresses endogenous WT hNTH1 (Fig.…”

“…We speculated that cells expressing D239Y hNTH1 may not remove Tg as efficiently as cells exclusively expressing WT protein, leading to replication blocks that result in chromosomal aberrations. To test this hypothesis, we scored metaphase spreads, prepared as described (12), from MCF10A cells expressing either D239Y or WT hNTH1. As shown in Fig.…”

“…Focus formation assays with the C127 cells were carried out as described (12). Anchorage-independent growth assays with the MCF10A cells were performed as described (12).…”

“…Metaphase spreads were prepared and chromosomal aberrations were analyzed as described (12). See SI Text for additional details.…”

Pools and Pols: Mechanism of a mutator phenotype

“…This hypothesis has evolved over time, with better understanding of the molecular basis for tumorigenesis, and it is now known that a mutator phenotype results from mutations in genes that maintain genome stability (8)(9)(10)(11)(12)(13). The recently identified germ-line and somatic mutations of POLE and POLD, which encode DNA polymerases e and δ, respectively, and which are associated with hypermutated tumors, provide additional strong evidence for the mutator phenotype hypothesis.…”