Abstract
Balanced DNA synthesis and repair is significant for cell survival and maintenance of genomic stability. The XP-D family is a large group of Fe-S cluster-containing helicases involving in DNA repair and genomic integrity. As a member of this family, YoaA helicase is induced by DNA damage and plays a role in promoting cell tolerance to nucleoside azidothymidine (AZT), a chain terminator. In this study, YoaA is of great interest because of its interaction with an accessory clamp loader protein HolC in Escherichia coli. This interaction aids to prevent stalling of the replication fork and has been proposed to compete with the interaction of HolC and another component of the DNA polymerase III holoenzyme, HolD. Based on prior genetic analysis and pulldown experiments, C-terminally truncated proteolytic YoaA failed to pulldown with HolC, which indicated that the C-terminus region of YoaA is required to bind with HolC. We utilized C-terminally truncated YoaA (YoaA∆619-637) and two individually mutated YoaA within C-terminus (YoaA R619A and T620A) in this work to determine the expression level of YoaA with biotin binding domain. The effects of the mutations in C-terminus of YoaA and their interactions with HolC were explored via western blot analysis and pulldown assays.