Sgs1 and exo1 redundantly inhibit break-induced replication and de novo telomere addition at broken chromosome ends.
Sgs1 and exo1 redundantly inhibit break-induced replication and de novo telomere addition at broken chromosome ends.
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DOI：
10.1371/journal.pgen.1000973
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发表时间：
2010-05-27
期刊：
影响因子：
4.5
通讯作者：
Haber JE
中科院分区：
文献类型：
--
作者：
Lydeard JR;Lipkin-Moore Z;Jain S;Eapen VV;Haber JE
在芽殖酵母中，HO内切酶诱导的双链断裂(DSB)被几条同源重组(HR)途径有效地修复。与基因转换(GC)不同的是，DSB的两端可以与相同的模板重组，而只有DSB的着丝粒近端才能定位同源序列时，才会发生断裂诱导复制(BIR)。GC导致一小块新的DNA合成，而BIR导致非互惠易位。完成BIR的要求与GC的要求有很大不同，但这两个过程都需要5‘到3’切除DSB末端以创建单链DNA，从而形成启动HR所需的RAD51细丝。切除通过两条途径进行，依赖于外显子1或BLM同源基因SGS1。我们报道，Exo1和SGS1各自抑制BIR，但对GC几乎没有影响，而过表达这两种蛋白都严重抑制BIR。相反，RAD51的过表达显著增加了BIR的效率，同样对GC几乎没有影响。在SGS1ΔExo1DNA株中，很少有5‘到3’的切除，BIR的水平与任何一个突变体都没有不同；令人惊讶的是，在HO诱导后，细胞活力增加了两倍，40%的细胞通过在Δ裂解部位几kb内形成新的端粒而存活。在野生型、SGS1Δ或Exo1Δ细胞中，端粒的从头增加是罕见的。在SGS1ΔExo1Δ中，GC的修复受到严重抑制，但由于新的端粒的形成，细胞的存活率仍然很高。这些数据表明，在BIR中新的DNA合成开始之前发生的广泛的5‘到3’切除可能阻碍了DSB末端附近RAD51丝的有效维持。对5‘到3’切除的严格限制，也取消了依赖于Mec1的DNA损伤检查点的激活，允许前所未有的新端粒增加。染色体双链断裂(DSB)对基因组完整性构成严重威胁，萌芽酵母细胞使用几种同源重组机制来修复这种断裂。在基因转换(GC)中，DSB的两端与一个完整的供体基因座有同源性，通过复制供体基因来修复断裂，创造一个新的DNA合成的小片段。在断裂诱导复制(BIR)中，只有DSB的一侧与供体有同源性，修复涉及重组依赖的复制叉的组装，该复制叉将序列复制到模板染色体的末端，从而产生非互惠易位。这两个过程都需要DSB末端被5‘到3’核酸外切酶切除，涉及几种蛋白质或蛋白质复合体，包括Exo1和SGS1-Rmi1-top3-DNA2。我们报道了异位BIR独立地被SGS1和Exo1抑制，RAD51重组酶的过表达进一步改善了BIR，而GC基本上没有受到影响。令人惊讶的是，当SGS1和Exo1同时缺失，切除受到严重损害时，一半的细胞获得新的端粒，而不是完成BIR或GC。新的端粒增加似乎是由于缺乏切除本身以及这样一个事实，即在没有切除的情况下，Mec1(ATR)DNA损伤检查点无法失活阻止新端粒形成的Pif1解旋酶。
In budding yeast, an HO endonuclease-inducible double-strand break (DSB) is efficiently repaired by several homologous recombination (HR) pathways. In contrast to gene conversion (GC), where both ends of the DSB can recombine with the same template, break-induced replication (BIR) occurs when only the centromere-proximal end of the DSB can locate homologous sequences. Whereas GC results in a small patch of new DNA synthesis, BIR leads to a nonreciprocal translocation. The requirements for completing BIR are significantly different from those of GC, but both processes require 5′ to 3′ resection of DSB ends to create single-stranded DNA that leads to formation of a Rad51 filament required to initiate HR. Resection proceeds by two pathways dependent on Exo1 or the BLM homolog, Sgs1. We report that Exo1 and Sgs1 each inhibit BIR but have little effect on GC, while overexpression of either protein severely inhibits BIR. In contrast, overexpression of Rad51 markedly increases the efficiency of BIR, again with little effect on GC. In sgs1Δ exo1Δ strains, where there is little 5′ to 3′ resection, the level of BIR is not different from either single mutant; surprisingly, there is a two-fold increase in cell viability after HO induction whereby 40% of all cells survive by formation of a new telomere within a few kb of the site of DNA cleavage. De novo telomere addition is rare in wild-type, sgs1Δ, or exo1Δ cells. In sgs1Δ exo1Δ, repair by GC is severely inhibited, but cell viaiblity remains high because of new telomere formation. These data suggest that the extensive 5′ to 3′ resection that occurs before the initiation of new DNA synthesis in BIR may prevent efficient maintenance of a Rad51 filament near the DSB end. The severe constraint on 5′ to 3′ resection, which also abrogates activation of the Mec1-dependent DNA damage checkpoint, permits an unprecedented level of new telomere addition. A chromosomal double-strand break (DSB) poses a severe threat to genome integrity, and budding yeast cells use several homologous recombination mechanisms to repair the break. In gene conversion (GC), both ends of the DSB share homology to an intact donor locus, and the break is repaired by copying the donor to create a small patch of new DNA synthesis. In break-induced replication (BIR), only one side of the DSB shares homology to a donor, and repair involves assembly of a recombination-dependent replication fork that copies sequences to the end of the template chromosome, yielding a nonreciprocal translocation. Both processes require that the DSB ends be resected by 5′ to 3′ exonucleases, involving several proteins or protein complexes, including Exo1 and Sgs1-Rmi1-Top3-Dna2. We report that ectopic BIR is inhibited independently by Sgs1 and Exo1 and that overexpression of Rad51 recombinase further improves BIR, while GC is largely unaffected. Surprisingly, when both Sgs1 and Exo1 are deleted, and resection is severely impaired, half of the cells acquire new telomeres rather than completing BIR or GC. New telomere addition appears to result from the lack of resection itself and from the fact that, without resection, the Mec1 (ATR) DNA damage checkpoint fails to inactivate the Pif1 helicase that discourages new telomere formation.
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