Cell survival requires genomic stability through the conservation of DNA sequences. If DNA is damaged, most DNA polymerases will stall during DNA replication. The specialized Y-family DNA polymerases rescue stalled DNA replication sites and avoid cell death. Hence, gaining knowledge on the molecular basis of DNA polymerase’s nucleotide selectivity and fidelity during DNA lesion bypass improves our understanding of this process. However, the multiple bypass mechanisms that Y-family DNA polymerases employ are poorly understood. Sulfolobus solfataricus DNA Polymerase IV (Dpo4), a model Y-family member, has a fidelity range of 10-3-10-4 at 37 °C, which does not significantly change between 26 °C and 56 °C. However, the physiological temperature of the S. solfataricus is approximately 80 °C. To determine the kinetic and structural relevance of data collected below 80 °C, we employed a circular dichroism spectroscopic investigation to observe the secondary structural changes of Dpo4 over a large temperature range. We discovered that Dpo4 displayed a three-state cooperative unfolding trend with a hyperthermophilic melting temperature, and exhibited secondary structural stability until 87 °C. We also established that the Dpo4 unfolding intermediate originated from ionic interactions between the linker region and Palm domain. These interactions are considered important for DNA binding during binary complex formation and possibly nucleotide incorporations.
Utilizing transient kinetics, we demonstrated that an active site mutation (Y12A) within Dpo4 caused an average 220-fold increase in matched ribonucleotide incorporation efficiency and an average 9-fold decrease in correct deoxyribonucleotide incorporation efficiency, leading to an average reduction of 2,000-fold in sugar selectivity. Therefore, the bulky side chain of Tyr12 is important for both ribonucleotide discrimination and efficient deoxyribonucleotide incorporation.
To examine mutagenic outcomes of lesion bypass, we employed short oligonucleotide sequencing assay (SOSA) with all human Y-family members and DNA containing a non-informational abasic (AP) site. We observed complex mutagenic patterns of AP site bypass catalyzed by human Y-family enzymes including rare mutagenic events. This data suggested that human DNA Polymerase η (hPolη) is the likely enzyme to bypass AP sites in vivo. Furthermore, our other SOSA studies using DNA substrates containing a cis-syn cyclobutane pyrimidine dimer (a product of UV-exposure) or cisplatin-dGpG (a product of anticancer drug cisplatin) depicted each Y-family DNA polymerase performing lesion bypass uniquely for each DNA lesion and differently from other Y-family members.
Both our pre-steady state kinetic and SOSA investigations on the bypass of N-(deoxyguanosin-8-yl)-1-aminopyrene (dGAP), a product of incomplete fuel combustion, confirmed this hypothesis as Dpo4 was the most error-free while hPolη was the most efficient during lesion bypass. Our work also indicated that all Y-family enzymes utilized different dGAP bypass kinetic mechanisms, and that the dGAP presence decreased nucleotide incorporation efficiencies and accuracies upstream, downstream and opposite the lesion site. Beyond these findings, we elucidated the minimal dGAP bypass mechanism for Dpo4 and hPolη, as well as added more details to the kinetic mechanism employed by hPolη during DNA synthesis with undamaged DNA. Overall, our data contributes to the understanding of lesion bypass and potentially mutagenic outcomes of this vital biological process in vivo.