Excision of uracil from DNA by hSMUG1 includes strand incision and processing

Abstract

Uracil arises in DNA by hydrolytic deamination of cytosine (C) and by erroneous incorporation of deoxyuridine monophosphate opposite adenine, where the former event is devastating by generation of C → thymine transitions. The base excision repair (BER) pathway replaces uracil by the correct base. In human cells two uracil-DNA glycosylases (UDGs) initiate BER by excising uracil from DNA; one is hSMUG1 (human single-strand-selective mono-functional UDG). We report that repair initiation by hSMUG1 involves strand incision at the uracil site resulting in a 3′-α,β-unsaturated aldehyde designated uracil-DNA incision product (UIP), and a 5′-phosphate. UIP is removed from the 3′-end by human apurinic/apyrimidinic (AP) endonuclease 1 preparing for single-nucleotide insertion. hSMUG1 also incises DNA or processes UIP to a 3′-phosphate designated uracil-DNA processing product (UPP). UIP and UPP were indirectly identified and quantified by polyacrylamide gel electrophoresis and chemically characterised by matrix-assisted laser desorption/ionisation time-of-flight mass-spectrometric analysis of DNA from enzyme reactions using 18O- or 16O-water. The formation of UIP accords with an elimination (E2) reaction where deprotonation of C2′ occurs via the formation of a C1′ enolate intermediate. A three-phase kinetic model explains rapid uracil excision in phase 1, slow unspecific enzyme adsorption/desorption to DNA in phase 2 and enzyme-dependent AP site incision in phase 3.

Defective ALC1 nucleosome remodeling confers PARPi sensitization and synthetic lethality with HRD

“…While the exact nature of these lesions remains unclear, our data suggest that toxicity does not arise simply from the generation of AP sites alone, as excision of dU by UNG did not result in toxicity in either ALC1 +/+ or ALC1 −/− cells ( Figures 6 A). Interestingly, SMUG1 has been proposed to catalyze base incision, resulting in a 3′-α,β unsaturated aldehyde and a 5′ phosphate ( Alexeeva et al., 2019 ). This incision intermediate is subsequently removed from the 3′ end by APEX1.…”

A novel use of random priming-based single-strand library preparation for whole genome sequencing of formalin-fixed paraffin-embedded tissue samples

“…To do this, we modified the DDAT protocol to include an initial enzyme digestion step using commercially available SMUG1 (excises deoxyuracil and deoxyuracil derivatives) and Fpg (an N-glycosylase and an AP-lyase that removes damaged based such as 8-oxoguanine). These enzymes create an abasic site in ssDNA and dsDNA, and the AP-lyase activity of Fpg creates a nick in the DNA backbone (13–15). In the standard method, a polymerase would then repair the gap in a dsDNA fragment by adding the missing complementary base; in contrast, in the DDAT method the missing base is not added and a heat denaturation separates the DNA strands, creating shorter ssDNA fragments where a damaged base has been removed.…”

RNA Metabolism Guided by RNA Modifications: The Role of SMUG1 in rRNA Quality Control

“…hSMUG1 has a substrate binding pocket formed by the C-terminal ends and the β strands ( Figure 5 A,B) [ 167 ]. hSMUG1 appears to interact rather non-specifically with DNA [ 170 ]. After initial binding, DNA glycosylases largely utilize base flipping to insert a damaged base into their active site binding pocket, where it is positioned for cleavage of the N -glycosylic bond.…”