The effect of single-stranded DNA binding protein RPA2 on XPD helicase processivity

helicase;XPD;RPA;processivity;optical trapping;optical tweezers;nucleotide excision repair;NER

Stekas, Barbara

关键词: helicase;    XPD;    RPA;    processivity;    optical trapping;    optical tweezers;    nucleotide excision repair;    NER;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/101311/STEKAS-DISSERTATION-2018.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Understanding how proteins work together to perform vital cellular functions, such as replicating and repairing DNA, not only extends our understanding of fundamental biology, but can also lead to important medical interventions. As a necessary first step to understanding larger systems, we focus on a two-protein system involved in DNA repair. Xeroderma pigmentosum group D (XPD) is a helicase protein that plays an important role in nucleotide excision repair (NER). Its function is to unwind double-stranded DNA, allowing access to the bases that connect the strands and code genetic information. Previous work has shown that XPD activity is enhanced by the single-stranded DNA binding protein replication protein A (RPA2). However, the mechanism by which unwinding enhancement occurs is unknown. In single-molecule optical trapping experiments, we monitor – with single base-pair precision – the unwinding of a DNA hairpin by XPD in the presence of RPA. We observe the effect of RPA2 on XPD unwinding in real time and distinguish between proposed models of protein cooperation by analyzing changes in unwinding behavior with added RPA. Our data disfavor mechanisms by which RPA2 melts the duplex ahead of XPD as well as RPA2 sequestering ssDNA behind the helicase. We present our own 2-state kinetic model of XPD unwinding that we believe explains our data best. We propose that XPD has two inherent states of unwinding, high and low processivity, and that RPA2 aids unwinding by increasing the likelihood of XPD being in its more processive state.