Mechanism of suppression of chromosomal instability by DNA polymerase POLQ.
Mechanism of suppression of chromosomal instability by DNA polymerase POLQ.
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DNA 聚合酶 POLQ 抑制染色体不稳定性的机制。
DOI：
10.1371/journal.pgen.1004654
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发表时间：
2014-10
期刊：
影响因子：
4.5
通讯作者：
Wood RD
中科院分区：
文献类型：
--
作者：
Yousefzadeh MJ;Wyatt DW;Takata K;Mu Y;Hensley SC;Tomida J;Bylund GO;Doublié S;Johansson E;Ramsden DA;McBride KM;Wood RD
虽然DNA聚合酶POLQ的缺陷导致哺乳动物细胞对电离辐射敏感，但相关的酶途径尚未确定。在这里，我们定义了POLQ限制有害DNA不稳定性的具体机制。我们的实验表明，POLQ -null小鼠细胞对DNA链断裂剂选择性超敏感，并且这种损伤抗性需要POLQ的DNA聚合酶活性。利用细胞中的DNA断裂端连接试验，我们监测了具有长3 '单链悬垂的DNA末端的修复。End joining事件保留了大部分的悬垂，依赖于POLQ，独立于Ku70。为了更详细地分析修复功能，我们检测了抗体基因中DNA片段之间的免疫球蛋白类开关连接。在免疫球蛋白类转换过程中，POLQ参与DNA断裂的末端连接，在与IgH开关区序列同源的连接处产生碱基对插入。用纯化的人POLQ蛋白进行的生化实验揭示了在DNA末端连接过程中产生插入的机制，该机制依赖于POLQ从最小配对引物延伸DNA的独特能力。DNA在IgH位点的断裂有时会与Myc的断裂结合，造成染色体易位。我们发现在POLQ缺陷小鼠中Myc/IgH易位显著增加，表明POLQ抑制基因组不稳定性和起源于DNA双链断裂的基因组重排。这项工作清楚地定义了哺乳动物POLQ在抑制染色体易位形成的另一末端连接途径中的作用和机制。我们的研究结果背离了主流观点，即替代末端连接过程通常容易易位。polq缺陷哺乳动物细胞对电离辐射过敏的原因一直难以捉摸。在这里，我们表明polq缺陷的哺乳动物细胞选择性地易受DNA双链断裂的影响。我们在哺乳动物细胞中进行的实验表明，特定的双链断裂修复途径依赖于polq。为了更详细地分析修复功能，我们检查了抗体基因中DNA片段之间的类开关连接。DNA碱基的插入有时在这些片段之间的连接处被发现，但这些插入的起源一直是神秘的。我们表明，在免疫球蛋白类转换过程中，这类插入连接完全依赖于polq。在纯化的人POLQ蛋白实验中，我们发现了一种新的解释插入形成的生化机制。POLQ具有独特的生化能力，以最小的碱基配对扩展DNA。最后，我们研究了染色体稳定性的生物学后果。出乎意料的是，在没有POLQ的情况下，伯基特淋巴瘤易位（一种主要的与癌症相关的基因组不稳定性）会增强。这改变了目前关于哺乳动物细胞中DNA末端连接作用的观点，揭示了依赖于polq的DNA修复途径可以对抗潜在的破坏性染色体易位。
Although a defect in the DNA polymerase POLQ leads to ionizing radiation sensitivity in mammalian cells, the relevant enzymatic pathway has not been identified. Here we define the specific mechanism by which POLQ restricts harmful DNA instability. Our experiments show that Polq-null murine cells are selectively hypersensitive to DNA strand breaking agents, and that damage resistance requires the DNA polymerase activity of POLQ. Using a DNA break end joining assay in cells, we monitored repair of DNA ends with long 3′ single-stranded overhangs. End joining events retaining much of the overhang were dependent on POLQ, and independent of Ku70. To analyze the repair function in more detail, we examined immunoglobulin class switch joining between DNA segments in antibody genes. POLQ participates in end joining of a DNA break during immunoglobulin class-switching, producing insertions of base pairs at the joins with homology to IgH switch-region sequences. Biochemical experiments with purified human POLQ protein revealed the mechanism generating the insertions during DNA end joining, relying on the unique ability of POLQ to extend DNA from minimally paired primers. DNA breaks at the IgH locus can sometimes join with breaks in Myc, creating a chromosome translocation. We found a marked increase in Myc/IgH translocations in Polq-defective mice, showing that POLQ suppresses genomic instability and genome rearrangements originating at DNA double-strand breaks. This work clearly defines a role and mechanism for mammalian POLQ in an alternative end joining pathway that suppresses the formation of chromosomal translocations. Our findings depart from the prevailing view that alternative end joining processes are generically translocation-prone. The reason for the hypersensitivity of POLQ-defective mammalian cells to ionizing radiation has been elusive. Here we show that POLQ-defective mammalian cells are selectively susceptible to double-strand breaks in DNA. We present experiments in mammalian cells showing that a specific double-strand break repair pathway is POLQ-dependent. To analyze the repair function in more detail, we examined class switch joining between DNA segments in antibody genes. Insertions of DNA bases are sometimes found at the joins between such segments, but the origin of these insertions has been mysterious. We show that this class of insertion joins during immunoglobulin class-switching is entirely POLQ-dependent. In experiments with purified human POLQ protein, we found a novel biochemical mechanism explaining the formation of the insertions. POLQ has a unique biochemical ability to extend DNA with minimal base pairing. Finally, we examined the biological consequences for chromosome stability. Unexpectedly, the Burkitt lymphoma translocation (a major cancer-associated genome instability) is enhanced in the absence of POLQ. This alters the current view about the action of DNA end joining in mammalian cells, revealing that a POLQ-dependent DNA repair pathway combats potentially damaging chromosome translocations.
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影响因子：
16
作者：
Callen, Elsa;Jankovic, Mila;Wong, Nancy;Zha, Shan;Chen, Hua-Tang;Difilippantonio, Simone;Di Virgilio, Michela;Heidkamp, Gordon;Alt, Frederick W.;Nussenzweig, Andre;Nussenzweig, Michel
通讯作者：
Nussenzweig, Michel
影响因子：
3.8
作者：
Frit, Philippe;Barboule, Nadia;Calsou, Patrick
通讯作者：
Calsou, Patrick
影响因子：
4.5
作者：
Chan SH;Yu AM;McVey M
通讯作者：
McVey M
影响因子：
14.9
作者：
Arana, Mercedes E.;Seki, Mineaki;Wood, Richard D.;Rogozin, Igor B.;Kunkel, Thomas A.
通讯作者：
Kunkel, Thomas A.
DOI：
10.1083/jcb.201011083
发表时间：
2011-04-04
期刊：
The Journal of cell biology
影响因子：
--
作者：
Harrigan JA;Belotserkovskaya R;Coates J;Dimitrova DS;Polo SE;Bradshaw CR;Fraser P;Jackson SP
通讯作者：
Jackson SP