N-terminal domain of human uracil DNA glycosylase (hUNG2) promotes targeting to uracil sites adjacent to ssDNA-dsDNA junctions

Brian P. Weiser, Gaddiel Rodriguez, Philip A. Cole, James Stivers

Research output: Contribution to journalArticle

Abstract

The N-terminal domain (NTD) of nuclear human uracil DNA glycosylase (hUNG2) assists in targeting hUNG2 to replication forks through specific interactions with replication protein A (RPA). Here, we explored hUNG2 activity in the presence and absence of RPA using substrates with ssDNA-dsDNA junctions that mimic structural features of the replication fork and transcriptional R-loops. We find that when RPA is tightly bound to the ssDNA overhang of junction DNA substrates, base excision by hUNG2 is strongly biased toward uracils located 21 bp or less from the ssDNA-dsDNA junction. In the absence of RPA, hUNG2 still showed an 8-fold excision bias for uracil located <10 bp from the junction, but only when the overhang had a 5 end. Biased targeting required the NTD and was not observed with the hUNG2 catalytic domain alone. Consistent with this requirement, the isolated NTD was found to bind weakly to ssDNA. These findings indicate that the NTD of hUNG2 targets the enzyme to ssDNA-dsDNA junctions using RPA-dependent and RPA-independent mechanisms. This structure-based specificity may promote efficient removal of uracils that arise from dUTP incorporation during DNA replication, or additionally, uracils that arise from DNA cytidine deamination at transcriptional R-loops during immunoglobulin class-switch recombination.

Original languageEnglish (US)
Pages (from-to)7169-7178
Number of pages10
JournalNucleic Acids Research
Volume46
Issue number14
DOIs
StatePublished - Jan 1 2018

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Replication Protein A
Uracil-DNA Glycosidase
Uracil
Cytidine
Deamination
Immunoglobulin Isotypes
DNA
DNA Replication
Genetic Recombination
Catalytic Domain
Enzymes

ASJC Scopus subject areas

  • Genetics

Cite this

N-terminal domain of human uracil DNA glycosylase (hUNG2) promotes targeting to uracil sites adjacent to ssDNA-dsDNA junctions. / Weiser, Brian P.; Rodriguez, Gaddiel; Cole, Philip A.; Stivers, James.

In: Nucleic Acids Research, Vol. 46, No. 14, 01.01.2018, p. 7169-7178.

Research output: Contribution to journalArticle

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