DNA ligase I fidelity mediates the mutagenic ligation of pol ? oxidized and mismatch nucleotide insertion products in base excision repair

DNA ligase I (LIG1) completes the base excision repair (BER) pathway at last nick-sealing step after polymerase (pol) ? gap-filling synthesis. However, mechanism by which LIG1 fidelity mediates faithful substrate–product channeling and ligation of intermediates final steps BER remains unclear. We previously reported that pol 8-oxo-2'-deoxyribonucleoside 5'-triphosphate insertion confounds LIG1, leading to formation failure products with a 5'-adenylate block. Here, using reconstituted assays in vitro, we report mutagenic an inefficient Watson–Crick–like dG:T mismatch mutant perturbed (E346A/E592A). Moreover, our results reveal substrate discrimination for nicked preinserted 3'-8-oxodG or mismatches is governed mutations both E346 E592 residues. Finally, found aprataxin flap endonuclease 1, as compensatory DNA-end processing enzymes, can remove block from abortive harboring 12 possible noncanonical pairs. 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Our dGTP:T EE/AA reaction. was be dependent double mutation typically ensure fidelity. roles trimming FEN1, 8-oxodG all 3'-end intermediate. herein situations involving aberrant provide novel insight BER. cannot used present low-fidelity coupled assay measures activities simultaneously mixture WT mutant, 8-oxodGTP, one-nucleotide-gap C (Fig. 1A). A, consistent studies (13Ça?layan Scholar), observed fails 1B, lanes 2–5). feature accumulation (5'-AMP). accompanied (i.e., sealing 3'-damage-containing intermediate, case, 8-oxodG) over time incubation 1C). Conversely, there no insertions impairs only 6–9). amount ?10-fold higher than obtained C, 2A, 2–8). weak 8-oxodGTP:C accumulated later points (2–6 min) compared earlier (10–60 s) case 8-oxodGTP:A 1B versus Fig. 2B). Similarly, reactions include 9–15). ?80-fold more efficient 8-oxodGMP relative initial (30 s 60 common S1). control dGTP, dGTP:C either 3A). complete 3B, 2–8 9–15, respectively). did not observe significant difference 3C). conversion (pol alone) LIG1) separately S2). ligated shown faster decrease same S2, B). Overall indicate (i) sensitive (ii) facilitates inserted 8-oxodGMP, while showing slight differences depending inserts (Scheme (P529L, E566K, R641L, R771W) identified patients LIG1-deficiency syndrome exhibit immunodeficiency cancer predisposition (38Barnes D.E. Lehmann A.R. Webster A.D. Lindahl Mutations individual immunodeficiencies cellular hypersensitivity DNA-damaging agents.Cell. 69: 495-503Abstract (224) 39Webster Barnes Arlett C.F. Growth retardation patient gene.Lancet. 339: 1508-1509Abstract (81) residing different domains located apart S3). altered ?–promoted mutagenesis (14Tang addition mutants, vitro described above. contrast (Figs. 2), tested study 4). P529L, R771W, R641L 4A, 6–9, 10–13, 14–17, respectively), yielded amounts were 4B). suggest disease-associated do interfere level recently published (40Jurkiw Cunningham-Rundles C. remodel cooperative network ligand efficiency.Nucleic 2021; 1619-1630Crossref (3) destabilized Mg2+ cofactor development pathology sugges