Institute of Veterinary Biochemistry and Molecular Biology, University of Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland.
Nucleic Acids Res. 2014 Jan;42(1):553-66. doi: 10.1093/nar/gkt889. Epub 2013 Oct 4.
Alkylating agents often generate 3-methylcytosine (3meC) lesions that are efficiently repaired by AlkB homologues. If AlkB homologue proteins are not functional, or the number of 3meC lesions exceeds the cellular repair capacity, the damage will persist in the genome and become substrate of DNA polymerases (Pols). Though alkylating agents are present in our environment and used in the clinics, currently nothing is known about the impact of 3meC on the accuracy and efficiency of human Pols. Here we compared the 3meC bypass properties of six human Pols belonging to the three families: B (Pol δ), X (Pols β and λ) and Y (Pols κ, ι and η). We show that under replicative conditions 3meC impairs B-family, blocks X-family, but not Y-family Pols, in particular Pols η and ι. These Pols successfully synthesize opposite 3meC; Pol ι preferentially misincorporates dTTP and Pol η dATP. The most efficient extenders from 3meC base-paired primers are Pols κ and η. Finally, using xeroderma pigmentosum variant patient cell extracts, we provide evidence that the presence of functional Pol η is mandatory to efficiently overcome 3meC by mediating complete bypass or extension. Our data suggest that Pol η is crucial for efficient 3meC bypass.
烷基化试剂通常会产生 3-甲基胞嘧啶（3meC）损伤，这些损伤可以被 AlkB 同源物有效修复。如果 AlkB 同源蛋白没有功能，或者 3meC 损伤的数量超过了细胞的修复能力，那么损伤将在基因组中持续存在，并成为 DNA 聚合酶（Pols）的底物。虽然烷基化试剂存在于我们的环境中，并在临床上使用，但目前还不知道 3meC 对人类 Pols 的准确性和效率的影响。在这里，我们比较了属于三个家族的六种人类 Pol 的 3meC 旁路特性：B（Pol δ）、X（Pols β 和 λ）和 Y（Pols κ、ι 和 η）。我们表明，在复制条件下，3meC 会损害 B 家族，阻止 X 家族，但不会阻止 Y 家族的 Pols，特别是 Pols η 和 ι。这些 Pols 成功地合成了相反的 3meC；Poli 优先掺入 dTTP，而 Pol η 掺入 dATP。从 3meC 碱基配对引物中最有效的延伸物是 Pol κ 和 η。最后，使用着色性干皮病变异型患者细胞提取物，我们提供了证据表明，功能性 Pol η 的存在对于通过介导完全旁路或延伸来有效地克服 3meC 是必需的。我们的数据表明，Poli 对于有效绕过 3meC 至关重要。
