Kinetic Studies of the DNA Lesion Bypass Polymerase DNA polymerase IV

Abstract

High fidelity replication of DNA is a requisite for maintaining genome stability. However, cellular DNA is continually being damaged. In response to such damage, organisms have evolved lesion bypass polymerases. The three million basepair genome of Sulfolobus solfataricus, a thermophilic crenarchaeon, likely undergoes base loss frequently in vivo. The unrepaired abasic lesions are expected to be bypassed by DNA polymerase IV (Dpo4), the only lesion bypass polymerase from Sulfolobus solfataricus. Here, we use Dpo4 as a model lesion bypass polymerase to establish the mechanistic basis for DNA lesion bypass using pre-steady state kinetic techniques. While showing efficient bypass, Dpo4 pauses when incorporating nucleotides directly opposite and one position downstream from an abasic lesion due to a drop of several orders of magnitude in catalytic efficiency. Moreover, Dpo4-catalyzed abasic bypass, which is governed by the local DNA sequence, involves robust competition between the A-rule and the lesion loop-out mechanism. Analysis of the strong pause sites revealed biphasic kinetics for incorporation indicating that Dpo4 primarily formed a nonproductive complex with DNA. These strong pause sites are mutational hot spots with the embedded lesion affecting the efficiency of downstream incorporations. Our results suggest that abasic lesion bypass requires tight regulation to maintain genomic stability.