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Title:Direct observation of XPD helicase base-pair stepping and regulation by RPA2
Author(s):Qi, Zhi
Director of Research:Chemla, Yann R.
Doctoral Committee Chair(s):Chemla, Yann R.
Doctoral Committee Member(s):Myong, Su-A; Gruebele, Martin; Ha, Taekjip
Department / Program:School of Molecular & Cell Bio
Discipline:Biophysics & Computnl Biology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Helicase
base-pair
high-resolution dual-trap optical tweezers
Ferroplasma acidarmanus XPD helicase
FeS domain
non-processive unwinding
forward and backward steps
Abstract:Although it is known that single-stranded DNA binding proteins (SSB) can stimulate helicase activity, the mechanism by which this occurs may be more complex than sequestering ssDNA products of duplex separation. Here, we present a singlemolecule helicase assay with base-pair sensitivity, which utilizes high-resolution optical tweezers combined with microfluidics and fluorescence microscopy to decipher how FacXPD helicase is modulated by FacRPA2. FacXPD is the archaeal homolog of yeast Rad3 and human xeroderma pigmentosum group D protein (XPD) helicase from the organism Ferroplasma acidarmanus. This enzyme serves as a model for understanding the molecular mechanism of human Superfamily 2B helicase XPD involved in transcription initiation and nucleotide excision repair and related helicases FANCJ, RTEL and CHLR1 involved in maintenance of the genomic integrity. First, we examined DNA unwinding by XPD helicase in isolation to understand the basic physicochemical process of DNA base pair (bp) separation. We demonstrated that monomeric XPD unwinds duplex DNA in single base-pair steps, yet is non-processive, unwinding for short distances (~12 bp) and displaying a strong dependence on DNA sequence. Second, we investigated how RPA2 by itself interacts with DNA. We show that RPA2 can unwind duplex 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, we performed the experiments in which XPD and RPA2 were sequentially assembled on a DNA substrate in a controlled order. RPA2 molecules increase XPD processivity, so we propose two scenarios: either RPA2 forms a complex with XPD, or it alters its interaction with DNA upon binding, activating it for processive unwinding. We discuss the biological implications of our findings.
Issue Date:2013-05-24
URI:http://hdl.handle.net/2142/44318
Rights Information:Copyright 2013 Zhi Qi
Date Available in IDEALS:2013-05-24
Date Deposited:2013-05


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