This application focuses on iron-sulfur containing (FeS) helicases, a prominent DNA helicase family whose deficiency or dysregulation is linked to human diseases ranging from cancer predisposition to hypertension. In addition to the Superfamily II motor core, FeS helicases possess two family specific auxiliary domains: an FeS cluster domain and an ARCH domain. The secondary DNA binding site formed with the help of the auxiliary domains which positions the helicase in an orientation to unwind duplex, controls the helicase rate, and verifies the integrity of the translocating strand. I propose that the frequency of ARCH domain opening and closing in FeS helicases modulates their activities. We will use this helicase family to test for the first time how the exogenous factors affect the mechano-chemistry of the helicases through modulating the frequency of its core and auxiliary domains motions. Our objective is to determine the mechanism by which the domain mobility controls the activities of three FeS helicases, XPD, FANCJ and RTEL1. To achieve this objective we will use a synergistic set of biochemical reconstitutions and novel single-molecule methodologies developed in my lab.
Aim1 : Determine the role of ARCH domain mobility in controlling XPD activities. We will build on our preliminary data showing that the cognate DNA lesions stabilize the closed conformation of the ARCH. Using single-molecule total internal reflection fluorescence microscopy (TIRFM), we will observe domain motions of individual fluorescently labeled XPD molecules as they interact with DNA. We will learn how ARCH domain motions control activities of XPD helicase and its malfunction in disease.
Aim2 : Determine the role of ARCH domain mobility in FANCJ and RTEL1 mediated DNA unwinding and remodeling of G-quadruplexes. Upon completion of this aim we will learn how the helicase and G- quadruplex remodeling activities of FANCJ and RTEL1 correlate with ARCH domain motions. We will also learn how FANCJ mutations associated with breast cancer and Fanconi Anemia perturb FANCJ activities, ARCH domain mobility and the ability to discriminate between damaged and damage-free DNA.
Aim3 : Determine how protein partners tune the activities of FANCJ and RTEL1. We will test the hypothesis that interactions with key protein partners (BRCA1 tumor suppressor protein, hMLH1 mismatch repair protein and PCNA clamp) govern helicase and translocase activities by modifying domain mobility of FANCJ and RTEL1. Together, the anticipated results of the three proposed aims will not only close the gaps in the mechanistic understanding of how helicases'distinct biochemical activities are regulated, but also identify explicit strategies to selectively modulate them. This information will pave the way for the design of inhibitors of FANCJ or RTEL1 to be used to target specific aspects of cancer and aging related diseases.

Public Health Relevance

A synergistic set of biochemical reconstitutions and innovative single-molecule methodologies will be employed to decipher the role of domain dynamics in the activities and regulation of DNA helicases, the molecular motors of DNA repair and maintenance machines. Successful completion of the proposed studies will increase our understanding of the fundamental life processes and may lay the foundation for advances in understanding, diagnosing, and treating genomic instability disorders, cancer and aging.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function B Study Section (MSFB)
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Preusch, Peter
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University of Iowa
Schools of Medicine
Iowa City
United States
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Subramanyam, Shyamal; Kinz-Thompson, Colin D; Gonzalez Jr, Ruben L et al. (2018) Observation and Analysis of RAD51 Nucleation Dynamics at Single-Monomer Resolution. Methods Enzymol 600:201-232
Subramanyam, Shyamal; Spies, Maria (2018) Expression, Purification, and Biochemical Evaluation of Human RAD51 Protein. Methods Enzymol 600:157-178
Caldwell, Colleen C; Spies, Maria (2017) Helicase SPRNTing through the nanopore. Proc Natl Acad Sci U S A 114:11809-11811
Spies, Maria; Smith, Brian O (2017) Protein-nucleic acids interactions: new ways of connecting structure, dynamics and function. Biophys Rev 9:289-291
Hengel, Sarah R; Spies, M Ashley; Spies, Maria (2017) Small-Molecule Inhibitors Targeting DNA Repair and DNA Repair Deficiency in Research and Cancer Therapy. Cell Chem Biol 24:1101-1119
Xu, Zhen; Gakhar, Lokesh; Bain, Fletcher E et al. (2017) The Tiam1 guanine nucleotide exchange factor is auto-inhibited by its pleckstrin homology coiled-coil extension domain. J Biol Chem 292:17777-17793
Spies, Maria (2017) A time for promiscuity in a eukaryotic recombinase. J Biol Chem 292:11136-11137
Chen, Ran; Subramanyam, Shyamal; Elcock, Adrian H et al. (2016) Dynamic binding of replication protein a is required for DNA repair. Nucleic Acids Res 44:5758-72
Subramanyam, Shyamal; Ismail, Mohammed; Bhattacharya, Ipshita et al. (2016) Tyrosine phosphorylation stimulates activity of human RAD51 recombinase through altered nucleoprotein filament dynamics. Proc Natl Acad Sci U S A 113:E6045-E6054
Boehm, E M; Subramanyam, S; Ghoneim, M et al. (2016) Quantifying the Assembly of Multicomponent Molecular Machines by Single-Molecule Total Internal Reflection Fluorescence Microscopy. Methods Enzymol 581:105-145

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