Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
J Biol Chem. 2011 Sep 9;286(36):31180-93. doi: 10.1074/jbc.M111.258038. Epub 2011 Jul 22.
The hyperthermophilic crenarchaeon Sulfolobus solfataricus P2 encodes three B-family DNA polymerase genes, B1 (Dpo1), B2 (Dpo2), and B3 (Dpo3), and one Y-family DNA polymerase gene, Dpo4, which are related to eukaryotic counterparts. Both mRNAs and proteins of all four DNA polymerases were constitutively expressed in all growth phases. Dpo2 and Dpo3 possessed very low DNA polymerase and 3' to 5' exonuclease activities in vitro. Steady-state kinetic efficiencies (k(cat)/K(m)) for correct nucleotide insertion by Dpo2 and Dpo3 were several orders of magnitude less than Dpo1 and Dpo4. Both the accessory proteins proliferating cell nuclear antigen and the clamp loader replication factor C facilitated DNA synthesis with Dpo3, as with Dpo1 and Dpo4, but very weakly with Dpo2. DNA synthesis by Dpo2 and Dpo3 was remarkably decreased by single-stranded binding protein, in contrast to Dpo1 and Dpo4. DNA synthesis in the presence of proliferating cell nuclear antigen, replication factor C, and single-stranded binding protein was most processive with Dpo1, whereas DNA lesion bypass was most effective with Dpo4. Both Dpo2 and Dpo3, but not Dpo1, bypassed hypoxanthine and 8-oxoguanine. Dpo2 and Dpo3 bypassed uracil and cis-syn cyclobutane thymine dimer, respectively. High concentrations of Dpo2 or Dpo3 did not attenuate DNA synthesis by Dpo1 or Dpo4. We conclude that Dpo2 and Dpo3 are much less functional and more thermolabile than Dpo1 and Dpo4 in vitro but have bypass activities across hypoxanthine, 8-oxoguanine, and either uracil or cis-syn cyclobutane thymine dimer, suggesting their catalytically limited roles in translesion DNA synthesis past deaminated, oxidized base lesions and/or UV-induced damage.
嗜热古菌 Sulfolobus solfataricus P2 编码三种 B 族 DNA 聚合酶基因，B1（Dpo1）、B2（Dpo2）和 B3（Dpo3），以及一种 Y 族 DNA 聚合酶基因 Dpo4，它们与真核生物的对应物有关。所有四种 DNA 聚合酶的 mRNA 和蛋白质在所有生长阶段都持续表达。Dpo2 和 Dpo3 在体外具有非常低的 DNA 聚合酶和 3' 到 5' 外切核酸酶活性。Dpo2 和 Dpo3 正确核苷酸插入的稳态动力学效率（k(cat)/K(m)）比 Dpo1 和 Dpo4 低几个数量级。增殖细胞核抗原和夹取器复制因子 C 这两种辅助蛋白都促进了 Dpo3 的 DNA 合成，就像 Dpo1 和 Dpo4 一样，但与 Dpo2 非常弱。与 Dpo1 和 Dpo4 相反，单链结合蛋白显著降低了 Dpo2 和 Dpo3 的 DNA 合成。在增殖细胞核抗原、复制因子 C 和单链结合蛋白存在的情况下，Dpo1 的 DNA 合成最具连续性，而 Dpo4 的 DNA 损伤绕过最有效。Dpo2 和 Dpo3 都能绕过次黄嘌呤和 8-氧鸟嘌呤，但 Dpo1 不能。Dpo2 和 Dpo3 分别绕过尿嘧啶和顺式-顺式环丁烷胸腺嘧啶二聚体。高浓度的 Dpo2 或 Dpo3 不会减弱 Dpo1 或 Dpo4 的 DNA 合成。我们的结论是，Dpo2 和 Dpo3 在体外的功能比 Dpo1 和 Dpo4 差得多，也更不稳定，但具有绕过次黄嘌呤、8-氧鸟嘌呤以及尿嘧啶或顺式-顺式环丁烷胸腺嘧啶二聚体的活性，表明它们在跨损伤 DNA 合成中催化有限的作用，以修复脱氨基、氧化碱基损伤和/或 UV 诱导的损伤。
