Analysis of uracil DNA glycosylase (UNG2) stimulation by replication protein A (RPA) at ssDNA-dsDNA junctions

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    Abstract

    Replication Protein A (RPA) is a single-stranded DNA binding protein that interacts with DNA repair proteins including Uracil DNA Glycosylase (UNG2). Here, I report DNA binding and activity assays using purified recombinant RPA and UNG2. Using synthetic DNA substrates, RPA was found to promote UNG2's interaction with ssDNA-dsDNA junctions regardless of the DNA strand polarity surrounding the junction. RPA stimulated UNG2's removal of uracil bases paired with adenine or guanine in DNA as much as 17-fold when the uracil was positioned 21 bps from ssDNA-dsDNA junctions, and the largest degree of UNG2 stimulation occurred when RPA was in molar excess compared to DNA. I found that RPA becomes sequestered on ssDNA regions surrounding junctions which promotes its spatial targeting of UNG2 near the junction. However, when RPA concentration exceeds free ssDNA, RPA promotes UNG2's activity without spatial constraints in dsDNA regions. These effects of RPA on UNG2 were found to be mediated primarily by interactions between RPA's winged-helix domain and UNG2's N-terminal domain, but when the winged-helix domain is unavailable, a secondary interaction between UNG2's N-terminal domain and RPA can occur. This work supports a widespread role for RPA in stimulating uracil base excision repair.

    Original languageEnglish (US)
    Article number140347
    JournalBiochimica et Biophysica Acta - Proteins and Proteomics
    Volume1868
    Issue number3
    DOIs
    StatePublished - Mar 2020

    All Science Journal Classification (ASJC) codes

    • Analytical Chemistry
    • Biophysics
    • Biochemistry
    • Molecular Biology

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