POLQ (Pol θ), a DNA polymerase and DNA-dependent ATPase in human cells
POLQ (Pol θ), a DNA polymerase and DNA-dependent ATPase in human cells
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DOI：
10.1093/nar/gkg814
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发表时间：
2003-11-01
影响因子：
14.9
通讯作者：
Wood, RD
中科院分区：
文献类型：
--
作者：
Seki, M;Marini, F;Wood, RD
真核细胞的基因组预测了多种DNA聚合酶的存在，这些聚合酶被认为在DNA复制和修复中起着特殊的作用。我们在这里报告的全长人类DNA POLQ基因的分离，其基因产物，DNA聚合酶θ的初步表征。POLQ是特别令人感兴趣的，因为它是果蝇Mus 308的直向同源基因，该基因涉及对链间DNA交联剂的细胞抗性。POLQ cDNA编码2592个氨基酸的多肽，在蛋白质的N-末端部分具有ATP酶解旋酶结构域，在C-末端部分具有中央间隔区结构域和DNA聚合酶结构域。这种排列方式在Mus 308中得到保留。在人细胞系中检测到类似于8.5kb的mRNA的表达。在对人类和小鼠组织的调查中，睾丸中的表达最高。用POLQ抗体的免疫印迹检测到HeLa细胞提取物中>250 kDa的蛋白质。类似于100 kDa的突出片段表明POLQ易于蛋白水解。从杆状病毒系统表达全长人POLQ。纯化的POLQ显示出DNA聚合酶活性的切口双链DNA和单引物的DNA模板。该酶的活性是耐阿非迪霉素，符合其成员的A家族的DNA聚合酶，并抑制双脱氧核苷酸。POLQ还表现出单链DNA依赖的ATP酶活性。
The genomes of eukaryotic cells predict the existence of multiple DNA polymerases, which are proposed to serve specialized roles in DNA replication and repair. We report here the isolation of the full-length human DNA POLQ gene, and an initial characterization of its gene product, DNA polymerase theta. POLQ is of particular interest as it is orthologous to Drosophila Mus308, a gene implicated in cellular resistance to interstrand DNA cross-linking agents. The POLQ cDNA encodes a polypeptide of 2592 amino acids with an ATPase-helicase domain in the N-terminal part of the protein, a central spacer domain, and a DNA polymerase domain in the C-terminal portion. This arrangement is conserved with Mus308. Expression of an mRNA of similar to8.5 kb was detected in human cell lines. In a survey of human and mouse tissues, expression was highest in testis. Immunoblotting with POLQ antibodies detected a protein of >250 kDa in extracts from HeLa cells. Prominent fragments of similar to100 kDa suggest that POLQ is readily proteolyzed. Full-length human POLQ was expressed from a baculovirus system. Purified POLQ showed DNA polymerase activity on nicked double-stranded DNA and on a singly primed DNA template. The enzyme activity was resistant to aphidicolin, consistent with its membership of the A family of DNA polymerases, and inhibited by dideoxynucleotides. POLQ further exhibited a single-stranded DNA-dependent ATPase activity.