Centro de Biologia Molecular Severo Ochoa (CSIC-UAM), 28049 Madrid, Spain.
Nucleic Acids Res. 2013 Oct;41(19):9105-16. doi: 10.1093/nar/gkt681. Epub 2013 Aug 8.
Human DNA polymerases mu (Polµ) and lambda (Polλ) are X family members involved in the repair of double-strand breaks in DNA during non-homologous end joining. Crucial abilities of these enzymes include bridging of the two 3' single-stranded overhangs and trans-polymerization using one 3' end as primer and the other as template, to minimize sequence loss. In this context, we have studied the importance of a previously uncharacterised sequence ('brooch'), located at the N-terminal boundary of the Polß-like polymerase core, and formed by Tyr(141), Ala(142), Cys(143), Gln(144) and Arg(145) in Polµ, and by Trp(239), Val(240), Cys(241), Ala(242) and Gln(243) in Polλ. The brooch is potentially implicated in the maintenance of a closed conformation throughout the catalytic cycle, and our studies indicate that it could be a target of Cdk phosphorylation in Polµ. The brooch is irrelevant for 1 nt gap filling, but of specific importance during end joining: single mutations in the conserved residues reduced the formation of two ended synapses and strongly diminished the ability of Polµ and polymerase lambda to perform non-homologous end joining reactions in vitro.
人类 DNA 聚合酶 μ（Polμ）和 λ（Polλ）是 X 家族成员，参与非同源末端连接过程中双链 DNA 断裂的修复。这些酶的关键能力包括桥接两个 3'单链突出端，以及使用一个 3'末端作为引物和另一个作为模板进行反式聚合，以最小化序列丢失。在这种情况下，我们研究了位于 Polβ样聚合酶核心 N 端边界的一个以前未被表征的序列（“胸针”）的重要性，该序列由 Polμ 中的 Tyr（141）、Ala（142）、Cys（143）、Gln（144）和 Arg（145）组成，由 Polλ 中的 Trp（239）、Val（240）、Cys（241）、Ala（242）和 Gln（243）组成。胸针可能参与维持整个催化循环中的封闭构象，我们的研究表明，它可能是 Polμ 中 Cdk 磷酸化的靶标。胸针与 1 nt 缺口填充无关，但在末端连接中具有特异性重要性：保守残基的单点突变降低了双端突触的形成，并强烈降低了 Polμ 和聚合酶 λ 在体外进行非同源末端连接反应的能力。
