Background: DNA Pol ␤ participates in base excision repair by choosing correct dNTP to fill single-nucleotide gaps in DNA. Results: Pol ␤ experiences a non-covalent step with correct dNTP selection. Conclusion: Correct and incorrect dNTP incorporation by Pol ␤ are different. Significance: FRET-based system of Pol ␤ elucidates a mechanism of substrate choice necessary for understanding the molecular basis of human disease.

Background: Human DNA polymerase ␤ is mutated in a high percentage of cancers with specific variants impacting enzymatic activity and/or fidelity. Results: In the E295K carcinoma variant, dCTP competes with cognate dTTP with dA as templating base. Conclusion: Structures verify that the E295K variant favors the mismatch over cognate dNTP. Significance: Relevant mutations may have the potential to lower the mismatch energy barrier to catalysis.

DNA polymerases synthesize new DNA during DNA replication and repair, and their ability to do so faithfully is essential to maintaining genomic integrity. DNA polymerase beta (Pol β) functions in Base Excision Repair (BER) to fill in single-nucleotide gaps, and variants of Pol β have been associated with cancer. Specifically, the E288K Pol β variant has been found in colon tumors and has been shown to display sequence-specific mutator activity. In order to probe the mechanism that may underlie E288K’s loss of fidelity, a fluorescence resonance energy transfer (FRET) system was employed which utilizes a fluorophore on the fingers domain of Pol β and a quencher on the DNA substrate. Our results show that E288K utilizes an overall mechanism similar to wild type (WT) Pol β when incorporating correct dNTP. However, when inserting the correct dNTP, E288K exhibits a faster rate of fingers closing combined with a slower rate of nucleotide release compared to WT Pol β. We also detect enzyme closure upon mixing with the incorrect dNTP for E288K but not WT Pol β. Taken together, our results suggest that E288K Pol β incorporates all dNTPs more readily than WT due to an inherent defect that results in rapid isomerization of dNTPs within its active site. Structural modeling implies that this inherent defect is due to interaction of E288K with DNA, resulting in a stable closed enzyme structure.

Repair of DNA damage is critical for maintaining the genomic integrity of cells. DNA polymerase lambda (POLL/Pol λ) is suggested to function in base excision repair (BER) and non-homologous end-joining (NHEJ), and is likely to play a role in damage tolerance at the replication fork. Here, using next-generation sequencing, it was discovered that the POLL rs3730477 single nucleotide polymorphism (SNP) encoding R438W Pol λ was significantly enriched in the germlines of breast cancer patients. Expression of R438W Pol λ in human breast epithelial cells induces cellular transformation and chromosomal aberrations. The role of estrogen was assessed as it is commonly used in hormone replacement therapies and is a known breast cancer risk factor. Interestingly, the combination of estrogen treatment and the expression of the R438W Pol λ SNP drastically accelerated the rate of transformation. Estrogen exposure produces 8-oxoguanine lesions that persist in cells expressing R438W Pol λ compared to WT Pol λexpressing cells. Unlike WT Pol λ, which performs error-free bypass of 8-oxoguanine lesions, expression of R438W Pol λ leads to an increase in mutagenesis and replicative stress in cells treated with estrogen. Together, these data suggest that individuals who carry the rs3730477 POLL germline variant have an increased risk of estrogen-associated breast cancer.

DNA polymerase β (pol β) fills single nucleotide gaps in DNA during base excision repair and non-homologous end-joining. Pol β must select the correct nucleotide from among a pool of four nucleotides with similar structures and properties in order to maintain genomic stability during DNA repair. Here, we use a combination of X-ray crystallography, fluorescence resonance energy transfer and nuclear magnetic resonance to show that pol β‘s ability to access the appropriate conformations both before and upon binding to nucleotide substrates is integral to its fidelity. Importantly, we also demonstrate that the inability of the I260Q mutator variant of pol β to properly navigate this conformational landscape results in error-prone DNA synthesis. Our work reveals that precatalytic conformational rearrangements themselves are an important underlying mechanism of substrate selection by DNA pol β.