Abstract
DNA ligase I (LIG1) completes the base excision repair (BER) pathway at the last nick-sealing step after DNA polymerase (pol) β gap-filling DNA synthesis. However, the mechanism by which LIG1 fidelity mediates the faithful substrate–product channeling and ligation of repair intermediates at the final steps of the BER pathway remains unclear. We previously reported that pol β 8-oxo-2'-deoxyribonucleoside 5'-triphosphate insertion confounds LIG1, leading to the formation of ligation failure products with a 5'-adenylate block. Here, using reconstituted BER assays in vitro, we report the mutagenic ligation of pol β 8-oxo-2'-deoxyribonucleoside 5'-triphosphate insertion products and an inefficient ligation of pol β Watson–Crick–like dG:T mismatch insertion by the LIG1 mutant with a perturbed fidelity (E346A/E592A). Moreover, our results reveal that the substrate discrimination of LIG1 for the nicked repair intermediates with preinserted 3'-8-oxodG or mismatches is governed by mutations at both E346 and E592 residues. Finally, we found that aprataxin and flap endonuclease 1, as compensatory DNA-end processing enzymes, can remove the 5'-adenylate block from the abortive ligation products harboring 3'-8-oxodG or the 12 possible noncanonical base pairs. These findings contribute to the understanding of the role of LIG1 as an important determinant in faithful BER and how a multiprotein complex (LIG1, pol β, aprataxin, and flap endonuclease 1) can coordinate to prevent the formation of mutagenic repair intermediates with damaged or mismatched ends at the downstream steps of the BER pathway.
Highlights
Human DNA ligases (LIGs) (LIG1, LIG3, and LIG4) catalyze the formation of a phosphodiester bond between the 5’-phosphate (P) and 3’-hydroxyl (OH) termini of a DNA intermediate during DNA replication, repair, and genetic recombination [1,2,3,4]
We previously demonstrated that the nicked repair product of pol β 8-oxodGTP insertion cannot be used as a DNA substrate by LIG1 during substrate–product channeling at the downstream steps of base excision repair (BER) [12,13,14,15,16]
We used the coupled assay that measures the activities of pol β and LIG1 simultaneously in a reaction mixture that includes LIG1 WT or E346 are mutated to alanine (EE/AA) mutant, pol β, 8-oxodGTP, and onenucleotide-gap DNA substrate with template base A or C (Fig. 1A)
Summary
Human DNA ligases (LIGs) (LIG1, LIG3, and LIG4) catalyze the formation of a phosphodiester bond between the 5’-phosphate (P) and 3’-hydroxyl (OH) termini of a DNA intermediate during DNA replication, repair, and genetic recombination [1,2,3,4]. Overall results indicate that (i) the functional coordination between pol β and LIG1 is sensitive to the ligase fidelity and (ii) the cavity that is formed due to the EE/ AA mutation at the MgHiFi of the LIG1 active site facilitates sealing of the nicked pol β repair product including an inserted 8-oxodGMP, while showing slight differences in the efficiency of mutagenic ligation depending on the type of template base to which pol β inserts the damaged nucleotide (Scheme S1).
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