1. Excision of uracil from DNA by hSMUG1 includes strand incision and processing
- Author
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Marina Alexeeva, Peter Ruoff, Izaskun Muruzábal-Lecumberri, Kristin Grøsvik, Xiang Ming Xu, Almaz Nigatu Tesfahun, Arne Klungland, Anette Rasmussen, Marivi N. Moen, Finn Kirpekar, Kristine M. Olsen, and Svein Bjelland
- Subjects
DNA Repair ,Stereochemistry ,Deamination ,Biology ,Genome Integrity, Repair and Replication ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Genetics ,Humans ,AP site ,DNA Cleavage ,Uracil ,Uracil-DNA Glycosidase ,030304 developmental biology ,0303 health sciences ,Temperature ,Base excision repair ,DNA ,Thymine ,Kinetics ,chemistry ,DNA glycosylase ,030217 neurology & neurosurgery ,Cytosine - 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.
- Published
- 2018