Daley James M, Laan Renee L Vander, Suresh Aswathi, Wilson Thomas E
Graduate Program in Cellular and Molecular Biology and Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109-0602, USA.
J Biol Chem. 2005 Aug 12;280(32):29030-7. doi: 10.1074/jbc.M505277200. Epub 2005 Jun 17.
DNA double strand breaks (DSBs) can be rejoined directly by the nonhomologous end-joining (NHEJ) pathway of repair. Nucleases and polymerases are required to promote accurate NHEJ when the terminal bases of the DSB are damaged. The same enzymes also participate in imprecise rejoining and joining of incompatible ends, important mutagenic events. Previous work has shown that the Pol X family polymerase Pol4 is required for some but not all NHEJ events that require gap filling in Saccharomyces cerevisiae. Here, we systematically analyzed DSB end configurations and found that gaps on both strands and overhang polarity are the principal factors that determine whether a joint requires Pol4. DSBs with 3'-overhangs and a gap on each strand strongly depended on Pol4 for repair, DSBs with 5'-overhangs of the same sequence did not. Pol4 was not required when 3'-overhangs contained a gap on only one strand, however. Pol4 was equally required at 3'-overhangs of all lengths within the NHEJ-dependent range but was dispensable outside of this range, indicating that Pol4 is specific to NHEJ. Loss of Pol4 did not affect the rejoining of DSBs that utilized a recessed microhomology or DSBs bearing 5'-hydroxyls but no gap. Finally, mammalian Pol X polymerases were able to differentially complement a pol4 mutation depending on the joint structure, demonstrating that these polymerases can participate in yeast NHEJ but with distinct properties.
DNA双链断裂（DSBs）可通过非同源末端连接（NHEJ）修复途径直接重新连接。当DSB的末端碱基受损时，需要核酸酶和聚合酶来促进准确的NHEJ。相同的酶也参与不精确的重新连接以及不相容末端的连接，这些都是重要的诱变事件。先前的研究表明，在酿酒酵母中，Pol X家族聚合酶Pol4对于某些但并非所有需要填补缺口的NHEJ事件是必需的。在这里，我们系统地分析了DSB末端构型，发现两条链上的缺口和突出端极性是决定连接是否需要Pol4的主要因素。具有3'突出端且每条链上都有一个缺口的DSB在修复时强烈依赖于Pol4，而具有相同序列5'突出端的DSB则不需要。然而，当3'突出端仅在一条链上有缺口时，不需要Pol4。在NHEJ依赖范围内，所有长度的3'突出端都同样需要Pol4，但在此范围之外则不需要，这表明Pol4对NHEJ具有特异性。Pol4的缺失并不影响利用隐性微同源性的DSB或带有5'羟基但无缺口的DSB的重新连接。最后，哺乳动物Pol X聚合酶能够根据连接结构差异互补pol4突变，表明这些聚合酶可以参与酵母NHEJ，但具有不同的特性。
