Abstract
Uracil occurs at replication forks via misincorporation of deoxyuridine monophosphate (dUMP) or via deamination of existing cytosines, which occurs 2–3 orders of magnitude faster in ssDNA than in dsDNA and is 100% miscoding. Tethering of UNG2 to proliferating cell nuclear antigen (PCNA) allows rapid post-replicative removal of misincorporated uracil, but potential ‘pre-replicative’ removal of deaminated cytosines in ssDNA has been questioned since this could mediate mutagenic translesion synthesis and induction of double-strand breaks. Here, we demonstrate that uracil-DNA glycosylase (UNG), but not SMUG1 efficiently excises uracil from replication protein A (RPA)-coated ssDNA and that this depends on functional interaction between the flexible winged-helix (WH) domain of RPA2 and the N-terminal RPA-binding helix in UNG. This functional interaction is promoted by mono-ubiquitination and diminished by cell-cycle regulated phosphorylations on UNG. Six other human proteins bind the RPA2-WH domain, all of which are involved in DNA repair and replication fork remodelling. Based on this and the recent discovery of the AP site crosslinking protein HMCES, we propose an integrated model in which templated repair of uracil and potentially other mutagenic base lesions in ssDNA at the replication fork, is orchestrated by RPA. The UNG:RPA2-WH interaction may also play a role in adaptive immunity by promoting efficient excision of AID-induced uracils in transcribed immunoglobulin loci.
Highlights
INTRODUCTION Recent research indicates thatDNA replication and deamination of cytosine and 5-methylcytosine (5-mC) are the major sources of cancer-associated mutations [1,2]
We demonstrate that UNG2 mediates highly efficient uracil excision from replication protein A (RPA)-bound single-stranded DNA (ssDNA), while the same substrate is protected against attack from the uracil-DNA glycosylase SMUG1
UNG2-mediated excision of uracil from dsDNA was recently shown to be stimulated by the presence of an RPAcoated ssDNA junction [31]
Summary
DNA replication and deamination of cytosine and 5-methylcytosine (5-mC) are the major sources of cancer-associated mutations [1,2]. This is supported by analysis of mutational signatures associated with C>T transition across a wide spectrum of human cancers [3]. One potential source of these C>T transitions is deamination of cytosine to uracil within single-stranded DNA (ssDNA) regions at the replication fork. Spontaneous and enzymatic cytosine deamination occurs 2–3 orders of magnitude faster in ssDNA than in dsDNA [5,6,7] and unless corrected before encounter of replicative polymerases, these would lead to C>T mutations after two replicative cycles. Spontaneous and enzymatic cytosine deamination occurs 2–3 orders of magnitude faster in ssDNA than in dsDNA [5,6,7] and unless corrected before encounter of replicative polymerases, these would lead to C>T mutations after two replicative cycles. ssDNA regions continuously form at the lagging strand and could be
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