Genetic control of the error-prone repair of a chromosomal double-strand break with 5' overhangs in yeast.
Genetic control of the error-prone repair of a chromosomal double-strand break with 5' overhangs in yeast.
复制标题
酵母中 5 突出端染色体双链断裂的易错修复的遗传控制。
DOI：
10.1101/2023.05.04.539391
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Jinks-Robertson,Sue
中科院分区：
文献类型：
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作者：
Shaltz,Samantha;Jinks-Robertson,Sue
当同源重组不是一种选择时，引入到酵母基因组中的靶向双链断裂通过相对易错的非同源末端连接（NHEJ）途径修复。为了研究NHEJ基因的遗传调控，将一个锌指核酸酶切割位点插入到一株单倍体酵母LYS 2基因座的框外，研究了当末端含有5′端突出端时NHEJ基因的遗传调控。将破坏切割位点的修复事件鉴定为选择性培养基上的Lys+菌落或丰富培养基上的存活菌落。Lys+事件中的连接序列仅反映NHEJ，并受Mre 11的核酸酶活性以及NHEJ特异性聚合酶Pol 4和转座合成DNA聚合酶Pol η和Pol η的存在/不存在的影响。虽然大多数NHEJ事件依赖于Pol 4，但具有3-bp重复中的端点的29-bp缺失是例外。Pol 4非依赖性缺失需要跨损伤合成聚合酶以及复制型Pol δ DNA聚合酶的核酸外切酶活性。存活者在NHEJ事件和反映微同源介导的末端连接（MMEJ）的1.2或11.7kb缺失之间相等地分裂。MMEJ事件需要Exo 1/Sgs 1的进行性切除活性，但出乎意料的是，对于去除假定的3′尾，不依赖于Rad 1-Rad 10内切核酸酶。最后，NHEJ是更有效的nongrowing比在生长中的细胞，是最有效的G 0细胞。这些研究为酵母中易错DSB修复的灵活性和复杂性提供了新的见解。
A targeted double-strand break introduced into the genome ofSaccharomyces cerevisiaeis repaired by the relatively error-prone nonhomologous end joining (NHEJ) pathway when homologous recombination is not an option. A zinc finger nuclease cleavage site was inserted out-of-frame into theLYS2locus of a haploid yeast strain to study the genetic control of NHEJ when the ends contain 5′ overhangs. Repair events that destroyed the cleavage site were identified either as Lys+colonies on selective medium or as surviving colonies on rich medium. Junction sequences in Lys+events solely reflected NHEJ and were influenced by the nuclease activity of Mre11 as well as by the presence/absence of the NHEJ-specific polymerase Pol4 and the translesion-synthesis DNA polymerases Pol ζ and Pol η. Although most NHEJ events were dependent on Pol4, a 29-bp deletion with endpoints in 3-bp repeats was an exception. The Pol4-independent deletion required translesion synthesis polymerases as well as the exonuclease activity of the replicative Pol δ DNA polymerase. Survivors were equally split between NHEJ events and 1.2 or 11.7 kb deletions that reflected microhomology-mediated end joining (MMEJ). MMEJ events required the processive resection activity of Exo1/Sgs1, but there unexpectedly was no dependence on the Rad1–Rad10 endonuclease for the removal of presumptive 3′ tails. Finally, NHEJ was more efficient in nongrowing than in growing cells and was most efficient in G0 cells. These studies provide novel insights into the flexibility and complexity of error-prone DSB repair in yeast.