Characterization of the 3′ exonuclease subunit DP1 of Methanococcus jannaschii replicative DNA polymerase D
Characterization of the 3′ exonuclease subunit DP1 of Methanococcus jannaschii replicative DNA polymerase D
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DOI：
10.1093/nar/gkh558
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发表时间：
2004-04-01
影响因子：
14.9
通讯作者：
Syväoja, JE
中科院分区：
文献类型：
--
作者：
Jokela, M;Eskelinen, A;Syväoja, JE
与复制型DNA聚合酶相关的B亚基从古生物到人类都是保守的，而相应的催化亚基则不相关。后者分别属于真核生物和古细菌中的B和D DNA聚合酶家族。序列分析将B亚基置于钙调神经磷酸酶样磷酸酯酶超家族内。由于涉及金属结合和催化的残基在古细菌D家族DNA聚合酶中很好地保守，因此已经假设B亚基可能负责这些酶的3 '-5'校正外切核酸酶活性。为了验证这一假设，我们在大肠杆菌中表达了詹氏甲烷球菌DP 1（MjaDP 1），DNA聚合酶D的B亚基，并证明MjaDP 1单独作为一种中等活性、热稳定、Mn 2+依赖性3 '-5'核酸外切酶发挥作用。不需要假定的聚合酶亚基DP 2。核酸酶活性被磷酸酯酶结构域中的单个氨基酸突变强烈降低，表明该结构域对活性的要求。MjaDP 1作为一种单向的、非进行性的外切核酸酶，偏好错配的核苷酸和单链DNA，这表明MjaDP 1作为古细菌家族D DNA聚合酶的校正外切核酸酶发挥作用。
The B-subunits associated with the replicative DNA polymerases are conserved from Archaea to humans, whereas the corresponding catalytic subunits are not related. The latter belong to the B and D DNA polymerase families in eukaryotes and archaea, respectively. Sequence analysis places the B-subunits within the calcineurin-like phosphoesterase superfamily. Since residues implicated in metal binding and catalysis are well conserved in archaeal family D DNA polymerases, it has been hypothesized that the B-subunit could be responsible for the 3'-5' proofreading exonuclease activity of these enzymes. To test this hypothesis we expressed Methanococcus jannaschii DP1 (MjaDP1), the B-subunit of DNA polymerase D, in Escherichia coli, and demonstrate that MjaDP1 functions alone as a moderately active, thermostable, Mn2+-dependent 3'-5' exonuclease. The putative polymerase subunit DP2 is not required. The nuclease activity is strongly reduced by single amino acid mutations in the phosphoesterase domain indicating the requirement of this domain for the activity. MjaDP1 acts as a unidirectional, non-processive exonuclease preferring mispaired nucleotides and single-stranded DNA, suggesting that MjaDP1 functions as the proofreading exonuclease of archaeal family D DNA polymerase.