Excision of uracil from DNA by hSMUG1 includes strand incision and processing. Issue 2 (28th November 2018)

• Record Type: Journal Article
• Authors: Alexeeva, Marina; Moen, Marivi N; Grøsvik, Kristin; Tesfahun, Almaz N; Xu, Xiang Ming; Muruzábal-Lecumberri, Izaskun; Olsen, Kristine M; Rasmussen, Anette; Ruoff, Peter; Kirpekar, Finn; Klungland, Arne; Bjelland, Svein
• Is Part Of: Nucleic acids research. Volume 47:Issue 2(2019)
• Page Start: 779
• Page End: 793
• Publication Date: 2018-11-28
• Subjects: Nucleic acids -- Periodicals; Molecular biology -- Periodicals; 572.805
• DOI: 10.1093/nar/gky1184
• Languages: English
• ISSNs: 0305-1048

Abstract

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 18 O- or 16 O-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.