Tumbale Percy P, Jurkiw Thomas J, Krahn Juno M, Bokil Neha V, Admiraal Suzanne J, Pedersen Lars C, Williams Jessica S, Kunkel Tom A, O'Brien Patrick J, Williams R Scott
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, United States.
Biological Chemistry, University of Michigan, 1150 W. Medical Center Drive, Ann Arbor, MI 48109, United States.
Nucleic Acids Res. 2025 Apr 10;53(7). doi: 10.1093/nar/gkaf299.
DNA ligase 1 (LIG1) finalizes DNA replication and repair by catalyzing the joining of DNA nicks. LIG1 is highly specific for DNA-DNA junctions over DNA-RNA junctions, discriminating strongly against a single ribonucleotide at the 5' side of the nick. This selectivity of LIG1 prevents futile and potentially mutagenic DNA-RNA cleavage and re-ligation cycles during Okazaki fragment maturation or ribonucleotide excision repair of genome-embedded ribonucleotide monophosphates (rNMPs), but the determinants of LIG1 rNMP discrimination are ill-defined. We report structural and kinetic analysis of LIG1 DNA-RNA complexes showing that LIG1 employs an aromatic steric gate to stabilize the enzyme-substrate complex and directly exclude rNMP-containing polynucleotides. Mutation of this RNA gate compromises the adenylyl-transfer and nick-sealing reactions but decreases the discrimination against an rNMP-containing substrate by ∼3600-fold. Our results establish the role of the conserved steric gate in ribonucleotide discrimination by high-fidelity (HiFi) DNA ligases at each step of the ligation reaction, which has parallels to the ribonucleotide discrimination by HiFi DNA polymerases.
DNA连接酶1（LIG1）通过催化DNA切口的连接来完成DNA复制和修复。LIG1对DNA-DNA连接比对DNA-RNA连接具有高度特异性，强烈排斥切口5'侧的单个核糖核苷酸。LIG1的这种选择性可防止在冈崎片段成熟或基因组嵌入的单磷酸核糖核苷酸（rNMP）的核糖核苷酸切除修复过程中发生无效且可能致突变的DNA-RNA切割和重新连接循环，但LIG1对rNMP的识别决定因素尚不明确。我们报告了LIG1 DNA-RNA复合物的结构和动力学分析，结果表明LIG1利用芳香族空间门来稳定酶-底物复合物并直接排除含rNMP的多核苷酸。这个RNA门的突变会损害腺苷酸转移和切口封闭反应，但会使对含rNMP底物的识别降低约3600倍。我们的结果确定了保守的空间门在高保真（HiFi）DNA连接酶在连接反应的每个步骤中对核糖核苷酸识别中的作用，这与HiFi DNA聚合酶对核糖核苷酸的识别类似。
