Laverty Daniel J, Greenberg Marc M
Department of Chemistry , Johns Hopkins University , 3400 North Charles Street , Baltimore , Maryland 21218 , United States.
Biochemistry. 2018 Oct 23;57(42):6119-6127. doi: 10.1021/acs.biochem.8b00911. Epub 2018 Oct 9.
DNA polymerase θ (Pol θ) is a multifunctional enzyme with double-strand break (DSB) repair, translesion synthesis, and lyase activities. Pol θ lyase activity on ternary substrates containing a 5'-dRP that are produced during base excision repair of abasic sites (AP) is weak compared to that of DNA polymerase β (Pol β), a polymerase integrally involved in base excision repair. This led us to explore whether Pol θ utilizes its lyase activity to remove 5'-dRP and incise abasic sites from alternative substrates that might be produced during DNA damage and repair. We found that Pol θ exhibited lyase activity on abasic lesions near DSB termini and on clustered lesions. To calibrate the Pol θ activity, Pol β reactivity was examined with the same substrates. Pol β excised 5'-dRP from within a 5'-overhang 80 times faster than did Pol θ. Pol θ and Pol β also incised AP within clustered lesions but showed opposite preferences with respect to the polarity of the lesions. AP lesions in 5'-overhangs were typically excised by Pol β 35-50 times faster than those in a duplex substrate but 15-20-fold more slowly than 5'-dRP in a ternary complex. This is the first report of Pol θ exhibiting lyase activity within an unincised strand. These results suggest that bifunctional polymerases may exhibit lyase activity on a greater variety of substrates than previously recognized. A role in DSB repair could potentially be beneficial, while the aberrant activity exhibited on clustered lesions may be deleterious because of their conversion to DSBs.
DNA聚合酶θ（Pol θ）是一种多功能酶，具有双链断裂（DSB）修复、跨损伤合成和裂解酶活性。与DNA聚合酶β（Pol β）相比，Pol θ对在无碱基位点（AP）碱基切除修复过程中产生的含有5'-脱氧核糖磷酸（5'-dRP）的三元底物的裂解酶活性较弱，Pol β是一种全面参与碱基切除修复的聚合酶。这促使我们探究Pol θ是否利用其裂解酶活性从DNA损伤和修复过程中可能产生的其他底物上去除5'-dRP并切割无碱基位点。我们发现Pol θ在DSB末端附近的无碱基损伤和簇状损伤上表现出裂解酶活性。为了校准Pol θ活性，我们用相同的底物检测了Pol β的反应性。Pol β从5'-突出端内切除5'-dRP的速度比Pol θ快80倍。Pol θ和Pol β也能切割簇状损伤中的AP，但在损伤极性方面表现出相反的偏好。5'-突出端中的AP损伤通常被Pol β切除的速度比双链底物中的快35 - 50倍，但比三元复合物中的5'-dRP慢15 - 20倍。这是关于Pol θ在未切割链内表现出裂解酶活性的首次报道。这些结果表明，双功能聚合酶可能在比以前认识到的更多种类的底物上表现出裂解酶活性。在DSB修复中的作用可能是有益的，而在簇状损伤上表现出的异常活性可能是有害的，因为它们会转化为DSB。
