Repair of mismatched templates during Rad51-dependent Break-Induced Replication.
Repair of mismatched templates during Rad51-dependent Break-Induced Replication.
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DOI：
10.1371/journal.pgen.1010056
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发表时间：
2022-09
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影响因子：
4.5
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中科院分区：
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--
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利用芽殖酵母，我们研究了Rad 51依赖的断裂诱导复制（BIR），其中侵入的位点特异性双链断裂（DSB）的3'端和供体模板共享108 bp的同源性，可以容易地改变。当每6个碱基错配时，BIR仍然发生的频率为完全匹配供体的10%。在这里，我们探讨了更详细的错配的容忍度，通过检查供体模板，每个带有10个错配，每个具有不同的空间安排。虽然我们测试的6种排列中有2种几乎与均匀间隔的参考一样有效，但4种的效率明显较低。与错配在5'端成簇的排列相比，具有在DSB的3'侵入端成簇的所有10个错配的供体没有受损。我们的数据表明，链侵入的效率主要由热力学因素决定，即，通过可以形成的碱基对的总数;但是错配位置特异性效应也很重要。我们还解决了体外和体内链交换测定之间的明显差异，其中体外研究表明，在一个连续的8个碱基的单一延伸需要配对稳定的链配对，而在体内测定使用108-bp底物发现显着的重组，即使当每6个碱基错配。现在，使用90或108 nt的底物-后者是体内模板的大小-我们发现体外D-环结果与体内结果非常相似。然而，体内和体外试验之间仍存在显著差异，尤其是在不均匀分布的错配情况下。供体模板中的错配以强极性方式掺入BIR产物中，从3'端起多达约40个核苷酸。错配掺入依赖于DNA聚合酶δ的3 '→ 5'校正核酸外切酶活性，Msh 2/Mlh 1错配修复蛋白、Rad 1-Rad 10瓣状核酸酶或Mph 1解旋酶的贡献很小。令人惊讶的是，从3'端27 nt处的错配被供体序列取代的概率是相同的，无论前面的26个核苷酸是每第6个碱基错配还是完全同源。这些数据表明，DNA聚合酶δ“咬回”入侵链的3'末端，而没有来自链入侵结构的任何错配依赖性线索。然而，似乎存在在供体的3'端的第一个碱基处掺入错配的替代方式。DNA双链断裂（DSB）是最致命的DNA损伤形式，这些断裂的不准确修复对基因组完整性和细胞活力构成严重威胁。断裂诱导复制（BIR）是一种同源重组途径，导致染色体末端的非相互易位。我们使用芽殖酵母酿酒酵母来研究Rad 51介导的BIR，其中入侵的DSB的3'端和供体模板共享108 bp的同源性。我们研究了同源供体模板上不同分布的错配的耐受性。与错配在5'端聚集的排列相比，在DSB的3'侵入端每6个碱基聚集所有10个错配的供体没有受损。我们还比较了体内BIR与体外D-环形成的效率，发现对于相同长度的底物，对错配的耐受性是相当的。然而，体内和体外测定之间仍然存在显著差异，这在具有不均匀分布的错配的底物中尤其明显。错配以强极性方式掺入BIR产物中，距离3'端约40个核苷酸，这取决于DNA聚合酶δ的5'至3'校对活性。Pol δ可以“咬回”入侵链的3'端，即使被移除的序列在前26个核苷酸中没有错配。然而，第一个碱基的错配可以通过另一种未鉴定的机制从3'端去除。
Using budding yeast, we have studied Rad51-dependent break-induced replication (BIR), where the invading 3’ end of a site-specific double-strand break (DSB) and a donor template share 108 bp of homology that can be easily altered. BIR still occurs about 10% as often when every 6th base is mismatched as with a perfectly matched donor. Here we explore the tolerance of mismatches in more detail, by examining donor templates that each carry 10 mismatches, each with different spatial arrangements. Although 2 of the 6 arrangements we tested were nearly as efficient as the evenly-spaced reference, 4 were significantly less efficient. A donor with all 10 mismatches clustered at the 3’ invading end of the DSB was not impaired compared to arrangements where mismatches were clustered at the 5’ end. Our data suggest that the efficiency of strand invasion is principally dictated by thermodynamic considerations, i.e., by the total number of base pairs that can be formed; but mismatch position-specific effects are also important. We also addressed an apparent difference between in vitro and in vivo strand exchange assays, where in vitro studies had suggested that at a single contiguous stretch of 8 consecutive bases was needed to be paired for stable strand pairing, while in vivo assays using 108-bp substrates found significant recombination even when every 6th base was mismatched. Now, using substrates of either 90 or 108 nt–the latter being the size of the in vivo templates–we find that in vitro D-loop results are very similar to the in vivo results. However, there are still notable differences between in vivo and in vitro assays that are especially evident with unevenly-distributed mismatches. Mismatches in the donor template are incorporated into the BIR product in a strongly polar fashion up to ~40 nucleotides from the 3’ end. Mismatch incorporation depends on the 3’→ 5’ proofreading exonuclease activity of DNA polymerase δ, with little contribution from Msh2/Mlh1 mismatch repair proteins, or from Rad1-Rad10 flap nuclease or the Mph1 helicase. Surprisingly, the probability of a mismatch 27 nt from the 3’ end being replaced by donor sequence was the same whether the preceding 26 nucleotides were mismatched every 6th base or fully homologous. These data suggest that DNA polymerase δ “chews back” the 3’ end of the invading strand without any mismatch-dependent cues from the strand invasion structure. However, there appears to be an alternative way to incorporate a mismatch at the first base at the 3’ end of the donor. DNA double-strand breaks (DSBs) are the most lethal forms of DNA damage and inaccurate repair of these breaks presents a serious threat to genomic integrity and cell viability. Break-induced replication (BIR) is a homologous recombination pathway that results in a nonreciprocal translocation of chromosome ends. We used budding yeast Saccharomyces cerevisiae to investigate Rad51-mediated BIR, where the invading 3’ end of the DSB and a donor template share 108 bp of homology. We examined the tolerance of differently distributed mismatches on a homologous donor template. A donor with all 10 mismatches clustered every 6th base at the 3’ invading end of the DSB was not impaired compared to arrangements where mismatches were clustered at the 5’ end. We also compared the efficiency of in vivo BIR with in vitro D-loop formation and find that for substrates of the same length, the tolerance for mismatches is comparable. However, there are still notable differences between in vivo and in vitro assays that are especially evident in substrates with unevenly-distributed mismatches. Mismatches are incorporated into the BIR product in a strongly polar fashion as far as about 40 nucleotides from the 3’ end, dependent on the 5’ to 3’ proofreading activity of DNA polymerase δ. Pol δ can “chew back” the 3’ end of the invading strand even when the sequences removed have no mismatches for the first 26 nucleotides. However, a mismatch at the first base can be removed from the 3’ end by another, unidentified mechanism.
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