Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, US National Institutes of Health, Department of Health and Human Services, 111 TW Alexander Drive, Research Triangle Park, NC, 27709, USA.
Office of Environmental Science Cyberinfrastructure, National Institute of Environmental Health Sciences, US National Institutes of Health, Department of Health and Human Services, 111 TW Alexander Drive, Research Triangle Park, NC, 27709, USA.
Nat Commun. 2024 Oct 9;15(1):8730. doi: 10.1038/s41467-024-53063-1.
Finalization of eukaryotic nuclear DNA replication relies on DNA ligase 1 (LIG1) to seal DNA nicks generated during Okazaki Fragment Maturation (OFM). Using a mutational reporter in Saccharomyces cerevisiae, we previously showed that mutation of the high-fidelity magnesium binding site of LIG1 strongly increases the rate of single-base insertions. Here we show that this rate is increased across the nuclear genome, that it is synergistically increased by concomitant loss of DNA mismatch repair (MMR), and that the additions occur in highly specific sequence contexts. These discoveries are all consistent with incorporation of an extra base into the nascent lagging DNA strand that can be corrected by MMR following mutagenic ligation by the Cdc9-EEAA variant. There is a strong preference for insertion of either dGTP or dTTP into 3-5 base pair mononucleotide sequences with stringent flanking nucleotide requirements. The results reveal unique LIG1-dependent mutational motifs where high fidelity DNA ligation of a subset of OFs is critical for preventing mutagenesis across the genome.
真核生物核 DNA 复制的最终完成依赖于 DNA 连接酶 1（LIG1）来封闭在冈崎片段成熟（OFM）过程中产生的 DNA 缺口。我们之前在酿酒酵母中使用突变报告基因表明，LIG1 高保真镁结合位点的突变会强烈增加单碱基插入的速率。在这里，我们表明这种速率在整个核基因组中都增加了，并且与 DNA 错配修复（MMR）的同时缺失协同增加，并且添加发生在高度特异性的序列背景中。这些发现都与在引发的滞后 DNA 链中掺入额外碱基一致，该碱基可以在 Cdc9-EEAA 变体引发诱变连接后通过 MMR 进行校正。对于具有严格侧翼核苷酸要求的 3-5 个碱基单核苷酸序列，插入 dGTP 或 dTTP 的倾向非常强。这些结果揭示了独特的依赖 LIG1 的突变基序，其中 OF 的亚组的高保真 DNA 连接对于防止整个基因组的诱变至关重要。
