Defective resection at DNA double-strand breaks leads to de novo telomere formation and enhances gene targeting.
Defective resection at DNA double-strand breaks leads to de novo telomere formation and enhances gene targeting.
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DOI：
10.1371/journal.pgen.1000948
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发表时间：
2010-05-13
期刊：
影响因子：
4.5
通讯作者：
Ira G
中科院分区：
文献类型：
--
作者：
Chung WH;Zhu Z;Papusha A;Malkova A;Ira G
在双链断裂（DSB）末端形成单链DNA（ssDNA）是通过同源重组进行修复所必需的，并且由DNA解旋酶和核酸酶介导。在这里，我们估计了DSB修复过程中产生的ssDNA的长度，并分析了消除Sgs 1解旋酶和Exo 1核酸酶介导的进行性切除途径对DSB修复保真度的影响。在野生型细胞中，在等位基因转换期间，平均2- 4kb的ssDNA在断裂的每一侧积累。更长的ssDNA在异位重组或断裂诱导复制（BIR）过程中形成，反映了慢得多的修复动力学。这种相对广泛的切除可能有助于确定参与同源性搜索的序列，并防止断裂附近的短DNA重复序列内的重组。在仅形成非常短的ssDNA的sgs 1 Δ exo 1 Δ突变体中，等位基因转换降低5倍，并且DSB通过BIR或从头端粒形成修复，导致杂合性丢失。端粒酶抑制剂PIF 1的缺乏使从头端粒途径的使用增加到约50%。Cdc 13（一种募集端粒酶的蛋白质）在sgs 1 Δ exo 1 Δ断裂位点的积累增加，Ku复合物对新端粒形成的要求被部分绕过。与这种减少的和替代的DSB修复相反，在sgs 1 Δ exo 1 Δ细胞中基因靶向的效率和准确性显著增加，表明转化的DNA在这些突变体中非常稳定。总之，这些数据建立了一个新的作用，进行性切除的保真度DSB修复。染色体断裂是自发发生的，或由电离辐射和许多化疗药物诱导。DNA双链断裂由酵母和人类中的核酸酶和解旋酶处理以产生单链DNA，然后通过与同源染色体重组用于修复。染色体断裂处的单链DNA也构成细胞阻止细胞周期进展的信号，直到DNA损伤被修复。本研究考察了消除处理染色体断裂为单链DNA的酶对模式生物酵母中修复保真度和基因组稳定性的影响。缺乏这些酶的突变体通常不能通过同源重组修复断裂，而是在断裂处添加新的端粒。新端粒的形成与断裂远端染色体臂的部分丢失有关。这种染色体畸变经常在肿瘤细胞中观察到，并且导致约10%的由染色体异常引起的人类基因组疾病。我们还观察到，消除将染色体断裂成单链DNA的酶极大地刺激了通过基因靶向的基因组操作，这表明转化的DNA也是这些酶降解的底物。我们讨论了在哺乳动物细胞中使用类似方法的可能性，与酵母相比，基因靶向不准确且效率较低。
The formation of single-stranded DNA (ssDNA) at double-strand break (DSB) ends is essential in repair by homologous recombination and is mediated by DNA helicases and nucleases. Here we estimated the length of ssDNA generated during DSB repair and analyzed the consequences of elimination of processive resection pathways mediated by Sgs1 helicase and Exo1 nuclease on DSB repair fidelity. In wild-type cells during allelic gene conversion, an average of 2–4 kb of ssDNA accumulates at each side of the break. Longer ssDNA is formed during ectopic recombination or break-induced replication (BIR), reflecting much slower repair kinetics. This relatively extensive resection may help determine sequences involved in homology search and prevent recombination within short DNA repeats next to the break. In sgs1Δ exo1Δ mutants that form only very short ssDNA, allelic gene conversion decreases 5-fold and DSBs are repaired by BIR or de novo telomere formation resulting in loss of heterozygosity. The absence of the telomerase inhibitor, PIF1, increases de novo telomere pathway usage to about 50%. Accumulation of Cdc13, a protein recruiting telomerase, at the break site increases in sgs1Δ exo1Δ, and the requirement of the Ku complex for new telomere formation is partially bypassed. In contrast to this decreased and alternative DSB repair, the efficiency and accuracy of gene targeting increases dramatically in sgs1Δ exo1Δ cells, suggesting that transformed DNA is very stable in these mutants. Altogether these data establish a new role for processive resection in the fidelity of DSB repair. Chromosomal breaks occur spontaneously or are induced by ionizing radiation and many chemotherapeutic drugs. DNA double-strand breaks are processed by nucleases and helicases in yeast and human to generate single-stranded DNA that is then used for repair by recombination with homologous chromosome. Single-stranded DNA at chromosomal breaks also constitutes a signal for cells to arrest cell cycle progression until the DNA damage is repaired. This study examines the consequences of elimination of enzymes that process chromosomal breaks to single-stranded DNA on the fidelity of repair and genome stability in the model organism yeast. Mutants deficient in these enzymes often fail to repair the breaks by homologous recombination and instead add new telomeres at the breaks. Formation of new telomeres is associated with partial loss of the chromosome arm distal from the break. Such chromosomal aberrations were frequently observed in tumor cells and are responsible for about 10% of human genomic disorders resulting from chromosomal abnormalities. We also observed that elimination of enzymes that process chromosomal breaks into single-stranded DNA greatly stimulates genome manipulation by gene targeting, suggesting that transformed DNA is also a substrate for degradation by these enzymes. We discuss the possibility of using a similar approach in mammalian cells where gene targeting is inaccurate and less efficient when compared to yeast.
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