Uracil-excising activity of hSMUG1 in bubble and R-loop DNA
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Uracil arises in cellular DNA by hydrolytic cytosine (C) deamination and by erroneous repli-cative incorporation of deoxyuridine monophosphate opposite adenine. The former event is devastating by generation of C thymine transition mutations, causing cancer, aging and neurodegenerative diseases, if uracil is not removed by uracil-DNA glycosylase (UDG) and replaced by cytosine through downstream base excision repair (BER) proteins before replica-tion. The most important human UDG is hUNG, with hSMUG1 as back-up. During immuno-globulin gene diversification in activated B cells, targeted deamination by activation-induced cytidine deaminase (AID) followed by uracil excision by hUNG is important for class switch recombination (CSR) and somatic hypermutation (SHM) by providing DNA double strand breaks and mutagenesis, respectively. hSMUG1 may substitute for hUNG in CSR, but its role in CSR and SHM is largely unclear. The aim of this investigation was to determine hSMUG1 excision activity for uracil in R-loop DNA, the product of AID and formed during transcription, and compare it to that in bubble U-DNA, which is formed tran-siently in cellular processes as e.g. DNA replication. The results show that hSMUG1 excises uracil in bubble more efficient than in R-loop DNA, indicating a back-up UDG function ra-ther than a specific function during CSR/SHM and/or transcription. hSMUG1 also function in ribosomal and telomerase RNA quality control by e.g. regulating the presence of base modifications, and binds the major pseudouridine synthase in mammals DKC1, where the (non-catalytic) Ser26 and Glu35 residues participate. The results show that hSMUG1 S26R/E35D mutant protein excises uracil in R-loop slightly more efficient than in DNA bubble, i.e., close to the opposite of wild type hSMUG1, suggesting altered enzyme function. hSMUG1 Pro240 is part of the His239–Lys249 intercalating loop being inserted into the damaged site in the DNA double helix. The hSMUG1 P240G protein excised uracil in R-loop much more efficient than in DNA bubble, demonstrating significantly altered enzyme func-tion. Thus, while wild type hSMUG1 excised uracil in DNA bubble significantly more effi-cient than in R-loop, this was completely reversed regarding the hSMUG1 P240G mutant protein, indicating a role of Pro240 in stabilizing the hSMUG1–bubble U-DNA complex, or inhibiting the hSMUG1–R-loop U-DNA complex. When compared to results on hUNG from another research group member, hSMUG1 excises uracil in R-loop DNA more than two or-ders of magnitude less efficient than hUNG.