Colon Cancer-associated DNA Polymerase β Variant Induces Genomic Instability and Cellular Transformation

Antonia A Nemec,Katherine A Donigan,Drew L Murphy,Joachim Jaeger,Joann B Sweasy

doi:10.1074/jbc.m112.362111

Antonia A Nemec, Katherine A Donigan + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m112.362111

Copy DOI

Abstract

Rapidly advancing technology has resulted in the generation of the genomic sequences of several human tumors. We have identified several mutations of the DNA polymerase β (pol β) gene in human colorectal cancer. We have demonstrated that the expression of the pol β G231D variant increased chromosomal aberrations and induced cellular transformation. The transformed phenotype persisted in the cells even once the expression of G231D was extinguished, suggesting that it resulted as a consequence of genomic instability. Biochemical analysis revealed that its catalytic rate was 140-fold slower than WT pol β, and this was a result of the decreased binding affinity of nucleotides by G231D. Residue 231 of pol β lies in close proximity to the template strand of the DNA. Molecular modeling demonstrated that the change from a small and nonpolar glycine to a negatively charged aspartate resulted in a repulsion between the template and residue 231 leading to the distortion of the dNTP binding pocket. In addition, expression of G231D was insufficient to rescue pol β-deficient cells treated with chemotherapeutic agents suggesting that these agents may be effectively used to treat tumors harboring this mutation. More importantly, this suggests that the G231D variant has impaired base excision repair. Together, these data indicate that the G231D variant plays a role in driving cancer.

Highlights

Mutations in the POLB gene are present in 40% of human colorectal tumors
Expression of G231D Induces Cellular Transformation—Because the G231D variant was initially identified in a colon tumor, we queried whether its expression in mammalian cells
The G231D variant was recently identified in a colon carcinoma [24]

Summary

Background

Results: The G231D variant is a slow polymerase that induces genomic instability and cellular transformation. Expression of G231D was insufficient to rescue pol ␤-deficient cells treated with chemotherapeutic agents suggesting that these agents may be effectively used to treat tumors harboring this mutation This suggests that the G231D variant has impaired base excision repair. In an Ape1-independent pathway, bifunctional glycosylases with ␤,␦-elimination remove the damaged base and incise the backbone leaving 3Ј- and 5Ј-phosphates. The G231D variant of pol ␤ was identified in a colon tumor diagnosed as a stage 3 adenocarcinoma as described in the accompanying article [24] This variant was predicted to be damaging [24] and shown to catalyze DNA synthesis with a rate that is much lower than that of WT pol ␤.

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION