Functional interactions of a homolog of proliferating cell nuclear antigen with DNA polymerases in Archaea
Functional interactions of a homolog of proliferating cell nuclear antigen with DNA polymerases in Archaea
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DOI：
10.1128/jb.181.21.6591-6599.1999
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发表时间：
1999-11-01
影响因子：
3.2
通讯作者：
Ishino, Y
中科院分区：
文献类型：
--
作者：
Cann, IKO;Ishino, S;Ishino, Y
增殖细胞核抗原（PCNA）是真核生物DNA复制和修复机制的重要组成部分。我们克隆的基因编码的PCNA同源物（PfuPCNA）从euryarchaeote，激烈的火球菌，在大肠杆菌中表达，其特征在于基因产物的生化特性。PfuPCNA蛋白刺激聚合酶（Pol）I和Pol II的体外引物延伸能力，这是迄今为止在该生物体中鉴定的两种DNA聚合酶。免疫学实验表明PfuPCNA与Pol I和Pol II两者相互作用。Pol I是具有与家族B（α样）DNA聚合酶相似的序列的单一多肽，而Pol II是异二聚体。PfuPCNA与异二聚体复合物的催化亚基DP2相互作用。这些结果有力地支持了这样的想法，即PCNA同源物作为一个滑动夹的DNA聚合酶在P. furiosus，和进行性DNA合成的基本机制是保守的域细菌，真核生物，和真核生物。PfuPCNA对DNA合成的刺激作用通过在Pol I和Pol II中使用不具有钳加载器（复制因子C [RFC]）的环状DNA模板来观察，与真核生物的情况相反，真核生物的反应已知需要RFC在加载到环状DNA上之前打开PCNA的环结构。由于RFC同源物已被发现在古细菌基因组中，它们可能允许更有效地刺激DNA合成的古细菌DNA聚合酶在PCNA的存在下。这是阐明古细菌DNA复制机制的第一阶段。
Proliferating cell nuclear antigen (PCNA) is an essential component of the DNA replication and repair machinery in the domain Eucarya. We cloned the gene encoding a PCNA homolog (PfuPCNA) from an euryarchaeote, Pyrococcus furiosus, expressed it in Escherichia coli, and characterized the biochemical properties of the gene product. The protein PfuPCNA stimulated the in vitro primer extension abilities of polymerase (Pol) I and Pol II, which are the two DNA polymerases identified in this organism to date. An immunological experiment showed that PfuPCNA interacts with both Pol I and Pol II. Pol I is a single polypeptide with a sequence similar to that of family B (alpha-like) DNA polymerases, while Pol II is a heterodimer. PfuPCNA interacted with DP2, the catalytic subunit of the heterodimeric complex. These results strongly support the idea that the PCNA homolog works as a sliding clamp of DNA polymerases in P. furiosus, and the basic mechanism for the processive DNA synthesis is conserved in the domains Bacteria, Eucarya, and Archaea. The stimulatory effect of PfuPCNA on the DNA synthesis was observed by using a circular DNA template without the clamp loader (replication factor C [RFC]) in both Pol I and Pol II, reactions in contrast to the case of eukaryotic organisms, which are known to require the RFC to open the ring structure of PCNA prior to loading onto a circular DNA. Because RFC homologs have been found in the archaeal genomes, they may permit more efficient stimulation of DNA synthesis by archaeal DNA polymerases in the presence of PCNA. This is the first stage in elucidating the archaeal DNA replication mechanism.