One of the most common lesions induced by oxidative DNA damage is 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG). Replicative DNA polymerases poorly traverse this highly mutagenic lesion, suggesting that the replication fork may switch to a polymerase specialized for translesion DNA synthesis (TLS) to catalyze 8-oxodG bypass in vivo. Here, we systematically compared the 8-oxodG bypass efficiencies and fidelities of the TLS-specialized, human Y-family DNA polymerases eta (hPolη), iota (hPolι), kappa (hPolκ), and Rev1 (hRev1) either alone or in combination. Primer extension assays revealed that the times required for hPolη, hRev1, hPolκ, and hPolι to bypass 50% of the 8-oxodG lesions encountered (t50bypass) were 0.58, 0.86, 108, and 670 s, respectively. Although hRev1 bypassed 8-oxodG efficiently, hRev1 failed to catalyze the extension step of TLS, and a second polymerase was required to extend the lesion bypass products. A high-throughput short oligonucleotide sequencing assay (HT-SOSA) was used to quantify the types and frequencies of incorporation errors produced by the human Y-family DNA polymerases at and near the 8-oxodG site. Although hPolη bypassed 8-oxodG most efficiently, hPolη correctly incorporated dCTP opposite 8-oxodG within only 54.5% of the sequences analyzed. In contrast, hPolι bypassed the lesion least efficiently but correctly incorporated dCTP at a frequency of 65.8% opposite the lesion. The combination of hRev1 and hPolκ was most accurate opposite 8-oxodG (92.3%), whereas hPolκ alone was the least accurate (18.5%). The t50bypass value and correct dCTP incorporation frequency in the presence of an equal molar concentration of all four Y-family enzymes were 0.60 s and 43.5%, respectively. These values are most similar to those of hPolη alone, suggesting that hPolη outcompetes the other three Y-family polymerases to catalyze 8-oxodG bypass in vitro and possibly in vivo.

The X-family DNA polymerases λ (Polλ) and β (Polβ) possess similar 5′-2-deoxyribose-5-phosphatelyase (dRPase) and polymerase domains. Besides these domains, Polλ also possesses a BRCA1 C-terminal (BRCT) domain and a proline-rich domain at its N terminus. However, it is unclear how these non-enzymatic domains contribute to the unique biological functions of Polλ. Here, we used primer extension assays and a newly developed high-throughput short oligonucleotide sequencing assay (HT-SOSA) to compare the efficiency of lesion bypass and fidelity of human Polβ, Polλ and two N-terminal deletion constructs of Polλ during the bypass of either an abasic site or a 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) lesion. We demonstrate that the BRCT domain of Polλ enhances the efficiency of abasic site bypass by approximately 1.6-fold. In contrast, deletion of the N-terminal domains of Polλ did not affect the efficiency of 8-oxodG bypass relative to nucleotide incorporations opposite undamaged dG. HT-SOSA analysis demonstrated that Polλ and Polβ preferentially generated −1 or −2 frameshift mutations when bypassing an abasic site and the single or double base deletion frequency was highly sequence dependent. Interestingly, the BRCT and proline-rich domains of Polλ cooperatively promoted the generation of −2 frameshift mutations when the abasic site was situated within a sequence context that was susceptible to homology-driven primer realignment. Furthermore, both N-terminal domains of Polλ increased the generation of −1 frameshift mutations during 8-oxodG bypass and influenced the frequency of substitution mutations produced by Polλ opposite the 8-oxodG lesion. Overall, our data support a model wherein the BRCT and proline-rich domains of Polλ act cooperatively to promote primer/template realignment between DNA strands of limited sequence homology. This function of the N-terminal domains may facilitate the role of Polλ as a gap-filling polymerase within the non-homologous end joining pathway.

On May 7, 2020, the Coalition for Physician Accountability's released “Medical Students in the Class of 2021: Moving Across Institutions for Post Graduate Training,” which comprises official recommendations on keeping programs and medical students safe during the upcoming match cycle with the challenges posed by COVID-19. In these recommendations, away rotations are discouraged, and all programs are compelled to commit to virtual interviews. Unlike employers and applicants in other industries, orthopaedic residency/fellowship programs and candidates seeking those positions have not routinely conducted virtual interviews. Without in-person interviews, applicants may perceive a limited ability to demonstrate their qualifications, judge program culture, and gauge ultimate program compatibility. Likewise, programs may perceive the inability to evaluate a candidate in real time, physically show program strengths, and ultimately judge applicant compatibility. Careful preparation and execution of a virtual interview can overcome these perceived limitations, whereas benefits, such as decreased cost for both programs and applicants, can make virtual interviews appealing. The purpose of this review was to help define a virtual interview, illustrate the benefits, and offer tips to both programs and applicants on how to prepare and perform optimally on an interview day.