Human DNA polymerase theta possesses 5'-dRP lyase activity and functions in single-nucleotide base excision repair in vitro.
Human DNA polymerase theta possesses 5'-dRP lyase activity and functions in single-nucleotide base excision repair in vitro.
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DOI：
10.1093/nar/gkp035
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发表时间：
2009-04
影响因子：
14.9
通讯作者：
Wilson SH
中科院分区：
文献类型：
--
作者：
Prasad R;Longley MJ;Sharief FS;Hou EW;Copeland WC;Wilson SH
DNA聚合酶θ（Pol θ）是一种低保真DNA聚合酶，属于A聚合酶家族，被认为在体细胞高突变中起作用。Pol θ具有跨损伤DNA合成的能力，与AP位点或胸腺嘧啶乙二醇相对，最近提出它参与DNA损伤的碱基切除修复（BER）。在这里，我们发现Pol θ具有内在的5′-脱氧核糖磷酸（5′-dRP）裂解酶活性，该活性参与单核苷酸碱基切除DNA修复（SN-BER）。全长人Pol θ是一种分子量约为300 kDa的多肽，但我们在此发现，Pol θ的98 kDa C-末端区域同时具有DNA聚合酶活性和dRP裂解酶活性，足以在体外进行碱基切除修复。5′-dRP裂解酶活性与聚合酶活性无关，因为聚合酶失活突变体保留了完整的5′-dRP裂解酶活性。通过限制性蛋白水解和NaBH 4与BER中间体的交联对98-kDa酶进行结构域作图，发现dRP裂解酶活性位点位于Pol θ的24-kDa结构域。这些结果与Pol θ在BER中的作用一致。
DNA polymerase θ (Pol θ) is a low-fidelity DNA polymerase that belongs to the family A polymerases and has been proposed to play a role in somatic hypermutation. Pol θ has the ability to conduct translesion DNA synthesis opposite an AP site or thymine glycol, and it was recently proposed to be involved in base excision repair (BER) of DNA damage. Here, we show that Pol θ has intrinsic 5′-deoxyribose phosphate (5′-dRP) lyase activity that is involved in single-nucleotide base excision DNA repair (SN-BER). Full-length human Pol θ is a ∼300-kDa polypeptide, but we show here that the 98-kDa C-terminal region of Pol θ possesses both DNA polymerase activity and dRP lyase activity and is sufficient to carry out base excision repair in vitro. The 5′-dRP lyase activity is independent of the polymerase activity, in that a polymerase inactive mutant retained full 5′-dRP lyase activity. Domain mapping of the 98-kDa enzyme by limited proteolysis and NaBH4 cross-linking with a BER intermediate revealed that the dRP lyase active site resides in a 24-kDa domain of Pol θ. These results are consistent with a role of Pol θ in BER.
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