Post-replicative nick translation occurs on the lagging strand during prolonged depletion of DNA ligase I in Saccharomyces cerevisiae

During lagging-strand synthesis, strand-displacement synthesis by DNA polymerase delta (Pol ∂), coupled to nucleolytic cleavage of DNA flap structures, produces a nick-translation reaction that replaces the DNA at the 5’ end of the preceding Okazaki fragment. Previous work following depletion of DNA ligase I in Saccharomyces cerevisae suggests that DNA-bound proteins, principally nucleosomes and the transcription factors Abf1/Rap1/Reb1, pose a barrier to Pol ∂ synthesis and thereby limit the extent of nick translation in vivo. However, the extended ligase depletion required for these experiments could lead to ongoing, non-physiological nick translation. Here, we investigate nick translation by analyzing Okazaki fragments purified after transient nuclear depletion of DNA ligase I in synchronized or asynchronous S. cerevisiae cultures. We observe that, even with a short ligase depletion, Okazaki fragment termini are enriched around nucleosomes and Abf1/Reb1/Rap1 binding sites. However protracted ligase depletion leads to a global change in the location of these termini, moving them towards nucleosome dyads from a more upstream location and further enriching termini at Abf1/Reb1/Rap1 binding sites. Additionally, we observe an under-representation of DNA derived from DNA polymerase alpha – the polymerase that initiates Okazaki fragment synthesis – around the sites of Okazaki termini obtained from very brief ligase depletion. Our data suggest that, while nucleosomes and transcription factors do limit strand-displacement synthesis by Pol ∂ in vivo, post-replicative nick translation can occur at unligated Okazaki fragment termini such that previous analyses represent an overestimate of the extent of nick translation occurring during normal lagging-strand synthesis.

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

ABSTRACTDNA ligase I (LIG1) completes base excision repair (BER) pathway at the last nick sealing step following DNA polymerase (pol) β gap filling DNA synthesis. We previously reported that pol β 8-oxo-2’-deoxyribonucleoside 5’-triphosphate (8-oxodGTP) insertion confounds LIG1 leading to the formation of ligation failure products with 5’-adenylate (AMP) block. Here, we report the mutagenic ligation of pol β 8-oxodGTP insertion products and an inefficient substrate-product channeling from pol β Watson-Crick like dG:T mismatch insertion to DNA ligation by LIG1 mutant with perturbed fidelity (E346A/E592A) in vitro. Moreover, our results revealed that the substrate discrimination of LIG1 for the nicked repair intermediates with preinserted 3’-8-oxodG or mismatches is governed by the mutations at both E346 and E592 residues. Finally, we found that Aprataxin (APTX) and Flap Endonuclease 1 (FEN1), as compensatory DNA-end processing enzymes, can remove 5’-AMP block from the abortive ligation products with 3’-8-oxodG or all possible 12 non-canonical base pairs. These findings contribute to understand the role of LIG1 as an important determinant of faithful BER, and how a multi-protein complex (LIG1, pol β, APTX and FEN1) can coordinate to hinder the formation of mutagenic repair intermediates with damaged or mismatched ends at the downstream steps of the BER pathway.

DNA ligase I variants fail in the ligation of mutagenic repair intermediates with mismatches and oxidative DNA damage

DNA ligase I (LIG1) joins DNA strand breaks during DNA replication and repair transactions and contributes to genome integrity. The mutations (P529L, E566K, R641L and R771W) in LIG1 gene are described in patients with LIG1-deficiency syndrome that exhibit immunodeficiency. LIG1 senses 3’-DNA ends with a mismatch or oxidative DNA base inserted by a repair DNA polymerase. However, the ligation efficiency of the LIG1 variants for DNA polymerase-promoted mutagenesis products with 3’-DNA mismatches or 8-oxo-2’-deoxyguanosine (8-oxodG) remains undefined. Here, we report that R641L and R771W fail in the ligation of nicked DNA with 3’-8-oxodG, leading to an accumulation of 5’-AMP-DNA intermediates in vitro. Moreover, we found that the presence of all possible 12 non-canonical base pairs variously impacts the ligation efficiency by P529L and R771W depending on the architecture at the DNA end, whereas E566K exhibits no activity against all substrates tested. Our results contribute to the understanding of the substrate specificity and mismatch discrimination of LIG1 for mutagenic repair intermediates and the effect of non-synonymous mutations on ligase fidelity.

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

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.