Double-strand break repair-associated intragenic deletions and tandem duplications suggest the architecture of the repair replication fork.
Double-strand break repair-associated intragenic deletions and tandem duplications suggest the architecture of the repair replication fork.
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双链断裂修复相关的基因内缺失和串联重复表明了修复复制叉的结构。
DOI：
10.1101/2023.10.09.561461
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Haber,JamesE
中科院分区：
文献类型：
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作者：
Dalin,Simona;Webster,Sophie;Sugawara,Neal;Zhang,Shu;Wu,Qiuqin;Cui,Tracy;Liang,Victoria;Beroukhim,Rameen;Haber,JamesE
与相同序列的正常复制相比，双链断裂（DSB）修复与突变增加1000倍相关。在芽殖酵母中，MATα基因座处HO内切酶诱导的DSB的修复可以通过使用含有转录沉默的URA 3基因的同源异染色质HMR：：Kl-URA 3供体来修复，从而产生MAT：：URA 3（Ura+）修复产物，其中URA 3表达。修复相关的ura 3 −突变可以通过对5-氟乳清酸（FOA）的抗性来选择。使用这个系统，我们发现一个主要的突变类型是-1缺失，几乎总是在同源序列中，但很少有+1插入。相比之下，同源序列中+1和-1插入在自发突变中几乎相等。大约10%的修复相关突变是染色体间模板开关（ICTS），尽管K。Iactis URA 3序列与S.酿酒酵母URA 3 -52序列在不同的染色体上。ICTS事件开始和结束于短微同源性区域，平均7 bp。长的微同源性是有利的，但一些ICTS连接短至2bp。修复相关的基因内缺失（ID）和串联重复（TD）的恢复，与路口共享短的延伸，平均6 bp的微同源性。基因内缺失的频率是TD的5倍以上。ID的平均长度为60 bp，但令人惊讶的是，几乎没有短于25 bp的缺失。相比之下，TD平均仅为12 bp。基因内缺失之间的微同源性的使用并不强烈的影响相邻同源性的程度。这些数据一起提供了修复复制分叉的结构图。我们认为ID和TD发生在迁移D环中，其中DNA聚合酶δ复制模板，其中部分复制的新DNA链的3'端可以解离并与位于3'端前面或后面的微同源单链区域退火，在迁移D环的开放结构中。我们的数据表明，聚合酶前面约100 bp是“开放的”，但修复复制装置的一部分仍然结合在新复制的DNA前面25 bp，防止退火。相比之下，聚合酶后面的模板区域似乎被快速再退火，将模板转换限制在非常短的区域。
Double-strand break (DSB) repair is associated with a 1000-fold increase in mutations compared to normal replication of the same sequences. In budding yeast, repair of an HO endonuclease-induced DSB at the MATα locus can be repaired by using a homologous, heterochromatic HMR::Kl-URA3 donor harboring a transcriptionally silenced URA3 gene, resulting in a MAT::URA3 (Ura+) repair product where URA3 is expressed. Repair-associated ura3− mutations can be selected by resistance to 5-fluoroorotic acid (FOA). Using this system, we find that a major class of mutations are −1 deletions, almost always in homonucleotide runs, but there are few +1 insertions. In contrast, +1 and −1 insertions in homonucleotide runs are nearly equal among spontaneous mutations. Approximately 10% of repair-associated mutations are interchromosomal template switches (ICTS), even though the K. lactis URA3 sequence embedded in HMR is only 72% identical with S. cerevisiae ura3-52 sequences on a different chromosome. ICTS events begin and end in regions of short microhomology, averaging 7 bp. Long microhomologies are favored, but some ICTS junctions are as short as 2 bp. Both repair-associated intragenic deletions (IDs) and tandem duplications (TDs) are recovered, with junctions sharing short stretches of, on average, 6 bp of microhomology. Intragenic deletions are more than 5 times more frequent than TDs. IDs have a mean length of 60 bp, but, surprisingly there are almost no deletions shorter than 25 bp. In contrast, TDs average only 12 bp. The usage of microhomologies among intragenic deletions is not strongly influenced by the degree of adjacent homeology. Together, these data provide a picture of the structure of the repair replication fork. We suggest that IDs and TDs occur within the migrating D-loop in which DNA polymerase δ copies the template, where the 3’ end of a partly copied new DNA strand can dissociate and anneal with a single-stranded region of microhomology that lies either in front or behind the 3’ end, within the open structure of a migrating D-loop. Our data suggest that ~100 bp ahead of the polymerase is “open,” but that part of the repair replication apparatus remains bound in the 25 bp ahead of the newly copied DNA, preventing annealing. In contrast, the template region behind the polymerase appears to be rapidly reannealed, limiting template switching to a very short region.