DNA polymerase theta suppresses mitotic crossing over.
DNA polymerase theta suppresses mitotic crossing over.
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DNA聚合酶theta抑制有丝分裂交换。
DOI：
10.1371/journal.pgen.1009267
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发表时间：
2021-03
期刊：
影响因子：
4.5
通讯作者：
Sekelsky J
中科院分区：
文献类型：
--
作者：
Carvajal-Garcia J;Crown KN;Ramsden DA;Sekelsky J
聚合酶- β介导的末端连接（TMEJ）是一种染色体断裂修复途径，能够挽救与同源重组早期步骤（例如BRCA1/2）中涉及的蛋白质丢失相关的致病性。这是由于聚合酶θ (Pol θ)能够使用切除的3 '单链DNA尾部来修复染色体断裂。这些被切除的DNA尾部也是同源重组的起始底物。然而，在同源重组过程中，TMEJ是否可以弥补下游步骤中蛋白质的损失尚不清楚。本研究表明，在没有Pol θ的情况下，黑腹果蝇的存活需要Holliday连接分解酶SLX4和GEN1，缺乏这三种蛋白会导致高水平的凋亡。缺乏Pol θ和SLX4的果蝇对DNA损伤剂极其敏感，而哺乳动物细胞需要Pol θ或SLX4才能存活。我们的研究结果表明，TMEJ和Holliday结的形成/分解具有共同的DNA底物，可能是同源重组中间体，如果不进行修复，将导致细胞死亡。SLX4和GEN1的Holliday结分解的一个主要后果是由于有丝分裂交叉导致的杂合性的致癌损失。我们测量了cas9诱导的染色体断裂后果蝇的有丝分裂交叉，并观察到在没有Pol θ的情况下，这种致突变的修复形式会增加。这表明TMEJ可以在Holiday连接分解酶的上游发挥作用，以保护细胞免受杂合性的丧失。我们的研究表明，Pol θ可以通过使用同源重组中间体来补偿Holliday连接分解酶的损失，从而抑制有丝分裂交叉并保持细胞的基因组稳定性。染色体断裂是对DNA稳定性的常见威胁。参与同源重组（BRCA1和BRCA2）早期步骤的基因突变导致遗传性乳腺癌，这是一种基本无错误的染色体断裂修复途径。缺乏BRCA1和BRCA2的细胞依靠DNA聚合酶来生存，这是一种更容易出错的途径的关键蛋白质。利用果蝇和哺乳动物细胞，我们发现参与同源重组后期步骤的基因突变（SLX4和GEN1）也使细胞依赖于聚合酶。此外，我们已经证明聚合酶作用于一种同源重组的上游，这种重组容易出错，依赖于SLX4和GEN1。这种形式的同源重组，被称为霍利迪结分解，产生有丝分裂交叉，这可能导致杂合性的丧失和癌症。我们的研究结果扩展了细胞依赖聚合酶生存的细胞环境，以及这种蛋白质可以用来修复染色体断裂的底物。
Polymerase theta-mediated end joining (TMEJ) is a chromosome break repair pathway that is able to rescue the lethality associated with the loss of proteins involved in early steps in homologous recombination (e.g., BRCA1/2). This is due to the ability of polymerase theta (Pol θ) to use resected, 3’ single stranded DNA tails to repair chromosome breaks. These resected DNA tails are also the starting substrate for homologous recombination. However, it remains unknown if TMEJ can compensate for the loss of proteins involved in more downstream steps during homologous recombination. Here we show that the Holliday junction resolvases SLX4 and GEN1 are required for viability in the absence of Pol θ in Drosophila melanogaster, and lack of all three proteins results in high levels of apoptosis. Flies deficient in Pol θ and SLX4 are extremely sensitive to DNA damaging agents, and mammalian cells require either Pol θ or SLX4 to survive. Our results suggest that TMEJ and Holliday junction formation/resolution share a common DNA substrate, likely a homologous recombination intermediate, that when left unrepaired leads to cell death. One major consequence of Holliday junction resolution by SLX4 and GEN1 is cancer-causing loss of heterozygosity due to mitotic crossing over. We measured mitotic crossovers in flies after a Cas9-induced chromosome break, and observed that this mutagenic form of repair is increased in the absence of Pol θ. This demonstrates that TMEJ can function upstream of the Holiday junction resolvases to protect cells from loss of heterozygosity. Our work argues that Pol θ can thus compensate for the loss of the Holliday junction resolvases by using homologous recombination intermediates, suppressing mitotic crossing over and preserving the genomic stability of cells. Chromosome breaks are a common threat to the stability of DNA. Mutations in genes involved in the early steps of homologous recombination (BRCA1 and BRCA2), a mostly error-free chromosome break repair pathway, lead to hereditary breast cancer. Cells lacking BRCA1 and BRCA2 rely on DNA polymerase theta, a key protein for a more error-prone pathway, for survival. Using fruit flies and mammalian cells, we have shown that mutations in genes involved in later steps of homologous recombination (SLX4 and GEN1) also make cells reliant on polymerase theta. Moreover, we have shown that polymerase theta acts upstream of a type of homologous recombination that is error-prone and depends on SLX4 and GEN1. This form of homologous recombination, termed Holliday junction resolution, creates mitotic crossovers, which can lead to loss of heterozygosity and cancer. Our results expand the cellular contexts that make cells depend on polymerase theta for survival, and the substrates that this protein can use to repair chromosome breaks.
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