Direct observation of XPD helicase base-pair stepping and regulation by RPA2

Helicase;base-pair;high-resolution dual-trap optical tweezers;Ferroplasma acidarmanus XPD helicase;FeS domain;non-processive unwinding;forward and backward steps

Qi, Zhi

关键词: Helicase;    base-pair;    high-resolution dual-trap optical tweezers;    Ferroplasma acidarmanus XPD helicase;    FeS domain;    non-processive unwinding;    forward and backward steps;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/44318/Zhi_Qi.pdf?sequence=1&isAllowed=y

美国|英语

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

【 摘 要 】

Although it is known that single-stranded DNA binding proteins (SSB) canstimulate helicase activity, the mechanism by which this occurs may be more complexthan sequestering ssDNA products of duplex separation. Here, we present a singlemoleculehelicase assay with base-pair sensitivity, which utilizes high-resolution opticaltweezers combined with microfluidics and fluorescence microscopy to decipher howFacXPD helicase is modulated by FacRPA2. FacXPD is the archaeal homolog of yeastRad3 and human xeroderma pigmentosum group D protein (XPD) helicase from theorganism Ferroplasma acidarmanus. This enzyme serves as a model for understandingthe molecular mechanism of human Superfamily 2B helicase XPD involved intranscription initiation and nucleotide excision repair and related helicases FANCJ,RTEL and CHLR1 involved in maintenance of the genomic integrity. First, we examinedDNA unwinding by XPD helicase in isolation to understand the basic physicochemicalprocess of DNA base pair (bp) separation. We demonstrated that monomeric XPDunwinds duplex DNA in single base-pair steps, yet is non-processive, unwinding for shortdistances (~12 bp) and displaying a strong dependence on DNA sequence. Second, weinvestigated how RPA2 by itself interacts with DNA. We show that RPA2 can unwindduplex DNA in steps of ~5-8 bp in the presence of an assisting force of 12 pN. Finally,we examined the effect of RPA2 on XPD activity. Using our microfluidic platform, weperformed the experiments in which XPD and RPA2 were sequentially assembled on aDNA substrate in a controlled order. RPA2 molecules increase XPD processivity, so wepropose two scenarios: either RPA2 forms a complex with XPD, or it alters its interactionwith DNA upon binding, activating it for processive unwinding. We discuss thebiological implications of our findings.