Saccharomyces cerevisiae DNA ligase IV supports imprecise end joining independently of its catalytic activity.
Saccharomyces cerevisiae DNA ligase IV supports imprecise end joining independently of its catalytic activity.
复制标题
DOI：
10.1371/journal.pgen.1003599
复制
发表时间：
2013-06
期刊：
影响因子：
4.5
通讯作者：
Wilson TE
中科院分区：
文献类型：
--
作者：
Chiruvella KK;Liang Z;Birkeland SR;Basrur V;Wilson TE
DNA连接酶IV（在出芽酵母中称为dn14）是一种用于DNA双链断裂（DSBs）非同源末端连接（NHEJ）的特殊连接酶。虽然人类连接酶IV综合征中出现点突变和截短突变，但dn14在DSB修复中的作用主要是通过基因缺失来研究的。在这里，产生了Dnl4催化点突变体，这些突变体在体外的自腺苷化和体内的NHEJ活性方面存在严重缺陷，尽管它们被高度招募到dsb中，并支持野生型的Lif1相互作用和dsb中含有Ku-和Lif1复合物的组装。有趣的是，与基因缺失菌株相比，在Dnl4催化突变体的缺失检测中，尤其不精确的NHEJ残留水平明显更高，这表明dsb结合的Dnl4在支持由不同连接酶催化的NHEJ模式中发挥了作用。同样，在一项独特的单dsb试验中，下一代修复关节测序显示，Dnl4 - k466a突变与野生型Dnl4相比，具有明显不同的不精确连接谱，并且在缺乏Dnl4的情况下很少观察到这种修复。在野生型和dnl4点突变菌株中，dsb上的DNA连接酶I（酵母中的Cdc9）富集，dnl4和Cdc9在5 '切除后从dsb上消失，这没有受到催化活性dnl4存在的阻碍。这些发现表明，dn14可以独立于其催化活性促进致突变末端连接，可能通过一种涉及Cdc9的机制。染色体重排是人类遗传疾病和癌症中常见的驱动突变。在重排中观察到的连接通常只显示伴侣之间的几个碱基对，这表明它们是通过端到端连接过程形成的，即非同源端连接（NHEJ）。然而，实际上产生突变连接的机制还不确定。DNA连接酶IV在典型的NHEJ通路中催化恢复性双链断裂（DSB）连接，但越来越多的证据表明，使用DNA连接酶I和/或III的不同的NHEJ通路可能对突变更重要。我们使用酵母来研究具有催化活性的DNA连接酶IV在dsb中正常积累的体内后果。我们在一些实验中检测到突变的连接需要DNA连接酶IV蛋白而不需要其催化活性。这种模式表明，当DNA连接酶IV存在时，DNA连接酶I会产生许多突变连接，当DNA连接酶IV活性低下时，这可能成为主要的修复模式。我们的酵母连接酶IV突变具有类似于在人类连接酶IV综合征中观察到的特性，强调了这些观察结果的相关性。
DNA ligase IV (Dnl4 in budding yeast) is a specialized ligase used in non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs). Although point and truncation mutations arise in the human ligase IV syndrome, the roles of Dnl4 in DSB repair have mainly been examined using gene deletions. Here, Dnl4 catalytic point mutants were generated that were severely defective in auto-adenylation in vitro and NHEJ activity in vivo, despite being hyper-recruited to DSBs and supporting wild-type levels of Lif1 interaction and assembly of a Ku- and Lif1-containing complex at DSBs. Interestingly, residual levels of especially imprecise NHEJ were markedly higher in a deletion-based assay with Dnl4 catalytic mutants than with a gene deletion strain, suggesting a role of DSB-bound Dnl4 in supporting a mode of NHEJ catalyzed by a different ligase. Similarly, next generation sequencing of repair joints in a distinct single-DSB assay showed that dnl4-K466A mutation conferred a significantly different imprecise joining profile than wild-type Dnl4 and that such repair was rarely observed in the absence of Dnl4. Enrichment of DNA ligase I (Cdc9 in yeast) at DSBs was observed in wild-type as well as dnl4 point mutant strains, with both Dnl4 and Cdc9 disappearing from DSBs upon 5′ resection that was unimpeded by the presence of catalytically inactive Dnl4. These findings indicate that Dnl4 can promote mutagenic end joining independently of its catalytic activity, likely by a mechanism that involves Cdc9. Chromosomal rearrangements are common driver mutations in human genetic disease and cancer. The junctions observed at rearrangements typically show only a few base pairs in common between the partners, suggesting that they were formed by the end-to-end joining process, nonhomologous end joining (NHEJ). However, there is uncertainty about the mechanisms that actually create mutated junctions. DNA ligase IV catalyzes restorative double-strand break (DSB) joining in the canonical NHEJ pathway, but increasing evidence suggests that distinct NHEJ pathways that use DNA ligases I and/or III might be more important for mutations. We used yeast to study the in vivo consequence of having DNA ligase IV that was catalytically inactive but that nonetheless accumulated at DSBs normally. We detected mutated junctions in some assays that required DNA ligase IV protein but not its catalytic activity. This pattern suggests that DNA ligase I creates many mutated junctions when DNA ligase IV is present and that this can become a predominant mode of repair when DNA ligase IV activity is inefficient. Our yeast ligase IV mutations have properties similar to those observed in the human ligase IV syndrome, underscoring the relevance of these observations.
登录
查看更多内容
影响因子：
4.8
作者：
Della-Maria, Julie;Zhou, Yi;Tomkinson, Alan E.
通讯作者：
Tomkinson, Alan E.
影响因子：
3.5
作者：
Girard, PM;Kysela, B;Jeggo, PA
通讯作者：
Jeggo, PA
影响因子：
5.3
作者：
Moore, JK;Haber, JE
通讯作者：
Haber, JE
影响因子：
16
作者：
Ma, YM;Lu, HH;Lieber, MR
通讯作者：
Lieber, MR
影响因子：
12.3
作者：
Langmead B;Trapnell C;Pop M;Salzberg SL
通讯作者：
Salzberg SL