The ligation of pol β mismatch insertion products governs the formation of promutagenic base excision DNA repair intermediates

Abstract

DNA ligase I and DNA ligase III/XRCC1 complex catalyze the ultimate ligation step following DNA polymerase (pol) β nucleotide insertion during base excision repair (BER). Pol β Asn279 and Arg283 are the critical active site residues for the differentiation of an incoming nucleotide and a template base and the N-terminal domain of DNA ligase I mediates its interaction with pol β. Here, we show inefficient ligation of pol β insertion products with mismatched or damaged nucleotides, with the exception of a Watson–Crick-like dGTP insertion opposite T, using BER DNA ligases in vitro. Moreover, pol β N279A and R283A mutants deter the ligation of the promutagenic repair intermediates and the presence of N-terminal domain of DNA ligase I in a coupled reaction governs the channeling of the pol β insertion products. Our results demonstrate that the BER DNA ligases are compromised by subtle changes in all 12 possible noncanonical base pairs at the 3′-end of the nicked repair intermediate. These findings contribute to understanding of how the identity of the mismatch affects the substrate channeling of the repair pathway and the mechanism underlying the coordination between pol β and DNA ligase at the final ligation step to maintain the BER efficiency.

DNA ligase I fidelity mediates the mutagenic ligation of pol β oxidized nucleotide insertion products and base excision repair intermediates with mismatches

“…Oligodeoxyribonucleotides with and without a 6-carboxyfluorescein (FAM) label and the 5'adenylate (AMP) were obtained from Integrated DNA Technologies. The DNA substrates used in this study were prepared as described previously (12)(13)(14)(15)(16)(34)(35)(36)38,39). The one nucleotide gapped DNA substrates were used for coupled assays (Supplementary Table 1).…”

“…The constructs for DNA ligase I (LIG1) mutants (E346A, E592A and E346A/E592A) were prepared using the wild-type full-length DNA ligase I (pET-24b) and site-directed mutagenesis with synthetic primers and confirmed by sequencing of the coding region. The His-tag recombinant for LIG1 low fidelity mutants were purified as described previously for wild-type LIG1 with slight modifications (12)(13)(14)(15)(16). Briefly, the protein was overexpressed in Recombinant (GST-tagged pGEX4T3) wild-type full-length human DNA polymerase β was purified as previously described (12)(13)(14)(15)(16).…”

“…The His-tag recombinant for LIG1 low fidelity mutants were purified as described previously for wild-type LIG1 with slight modifications (12)(13)(14)(15)(16). Briefly, the protein was overexpressed in Recombinant (GST-tagged pGEX4T3) wild-type full-length human DNA polymerase β was purified as previously described (12)(13)(14)(15)(16). Briefly, the protein was overexpressed in One Shot BL21(DE3)pLysS E. coli cells (Invitrogen) and grown at 37 °C with 0.5 mM IPTG induction.…”

“…Human DNA polymerases and DNA ligases have been considered as key determinants of genome integrity (11). In our previous studies, we demonstrated the importance of the coordination between DNA polymerase (pol) β and DNA ligase I (LIG1) during the repair of single DNA base lesions through base excision repair (BER) (9,10,(12)(13)(14)(15)(16). The BER is a critical process for preventing the mutagenic and lethal consequences of DNA damage that arises from endogenous and environmental agents and underlies disease and aging (17,18).…”

“…In our prior studies, we demonstrated that pol β 8-oxodGTP insertion confounds the DNA ligation step of BER pathway and leads to the formation of ligation failure product with 5'-adenylate (AMP) block (12)(13)(14). We also reported the effect of pol β mismatch nucleotide insertion on the substrate-product channeling to LIG1 during the final steps of BER and the fidelity of this mechanism in the presence of epigenetically important 5methylcytosine base modifications (15,16).…”