Interlocking activities of DNA polymerase β in the base excision repair pathway.
Interlocking activities of DNA polymerase β in the base excision repair pathway.
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DOI：
10.1073/pnas.2118940119
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发表时间：
2022-03-08
影响因子：
11.1
通讯作者：
Suo Z
中科院分区：
文献类型：
--
作者：
Kumar A;Reed AJ;Zahurancik WJ;Daskalova SM;Hecht SM;Suo Z
碱基切除修复（BER）是用于修复无数细胞DNA损伤的主要DNA修复途径之一。BER中涉及的酶，包括DNA聚合酶β（Polβ），已被鉴定和表征，但它们如何共同作用以有效地执行BER尚未完全了解。通过凝胶电泳、质谱和动力学分析，我们发现Polβ的两种酶活性在处理BER中形成的DNA中间体时可以互锁，而不是彼此独立地起作用。这一发现促使我们假设一个修改的BER途径。通过常规和时间分辨X射线晶体学方法，我们解析了11个交联Polβ复合物的高分辨率晶体结构，并提出了Polβ 5′-脱氧核糖-5-磷酸裂解酶活性的详细化学机制。碱基切除修复（BER）是DNA损伤修复的主要细胞途径。在BER过程中，DNA聚合酶β（Polβ）首先通过其聚合酶活性进行DNA合成，然后通过其dRP裂解酶活性切割5′-脱氧核糖-5-磷酸（dRP）部分。通过凝胶电泳和部分BER重建的动力学分析，我们证明聚合酶活性的缺口填充DNA合成可能发生在Schiff碱形成之后但在β-消除之前，这两个化学反应由dRP裂解酶活性催化。通过硼氢化钠还原捕获席夫碱形成和β-消除中间体，并通过质谱和X射线晶体学鉴定。稳态前动力学分析表明，交联Polβ（即，还原希夫碱）的聚合酶效率比未交联的Polβ高17倍。交联Polβ的常规和时间分辨X射线晶体学显示了其dRP裂解酶和聚合酶活性的重要中间体，导致dRP裂解酶活性的化学机制改变。观察到的Polβ的连锁酶活性使我们能够提出BER途径的改变机制，至少在所采用的条件下。可能的是，在两个Polβ活性位点的时间协调的活性很可能是Polβ具有嵌入在单个多肽链中的两个活性位点的原因。这一拟议的途径表明BER和DNA修复的正确方面，并可能使治疗干预的替代化学策略成为可能，因为Polβ功能障碍是几种疾病共同的关键因素。
Base excision repair (BER) is one of the major DNA repair pathways used to fix a myriad of cellular DNA lesions. The enzymes involved in BER, including DNA polymerase β (Polβ), have been identified and characterized, but how they act together to efficiently perform BER has not been fully understood. Through gel electrophoresis, mass spectrometry, and kinetic analysis, we discovered that the two enzymatic activities of Polβ can be interlocked, rather than functioning independently from each other, when processing DNA intermediates formed in BER. The finding prompted us to hypothesize a modified BER pathway. Through conventional and time-resolved X-ray crystallography, we solved 11 high-resolution crystal structures of cross-linked Polβ complexes and proposed a detailed chemical mechanism for Polβ’s 5′-deoxyribose-5-phosphate lyase activity. Base excision repair (BER) is a major cellular pathway for DNA damage repair. During BER, DNA polymerase β (Polβ) is hypothesized to first perform gap-filling DNA synthesis by its polymerase activity and then cleave a 5′-deoxyribose-5-phosphate (dRP) moiety via its dRP lyase activity. Through gel electrophoresis and kinetic analysis of partial BER reconstitution, we demonstrated that gap-filling DNA synthesis by the polymerase activity likely occurred after Schiff base formation but before β-elimination, the two chemical reactions catalyzed by the dRP lyase activity. The Schiff base formation and β-elimination intermediates were trapped by sodium borohydride reduction and identified by mass spectrometry and X-ray crystallography. Presteady-state kinetic analysis revealed that cross-linked Polβ (i.e., reduced Schiff base) exhibited a 17-fold higher polymerase efficiency than uncross-linked Polβ. Conventional and time-resolved X-ray crystallography of cross-linked Polβ visualized important intermediates for its dRP lyase and polymerase activities, leading to a modified chemical mechanism for the dRP lyase activity. The observed interlocking enzymatic activities of Polβ allow us to propose an altered mechanism for the BER pathway, at least under the conditions employed. Plausibly, the temporally coordinated activities at the two Polβ active sites may well be the reason why Polβ has both active sites embedded in a single polypeptide chain. This proposed pathway suggests a corrected facet of BER and DNA repair, and may enable alternative chemical strategies for therapeutic intervention, as Polβ dysfunction is a key element common to several disorders.
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发现聚合酶诱导的氧化核苷酸的细胞毒性。
影响因子：
64.8
作者：
Freudenthal, Bret D.;Beard, William A.;Perera, Lalith;Shock, David D.;Kim, Taejin;Schlick, Tamar;Wilson, Samuel H.
通讯作者：
Wilson, Samuel H.
DNA聚合酶β的结构和机制。
影响因子：
2.9
作者：
Beard WA;Wilson SH
通讯作者：
Wilson SH
DOI：
10.1038/nsb902
发表时间：
2003-03-01
期刊：
NATURE STRUCTURAL BIOLOGY
影响因子：
--
作者：
Fromme, JC;Bruner, SD;Verdine, GL
通讯作者：
Verdine, GL
影响因子：
5.6
作者：
Brown, Jessica A.;Duym, Wade W.;Suo, Zucai
通讯作者：
Suo, Zucai
影响因子：
14.9
作者：
Dianov GL;Hübscher U
通讯作者：
Hübscher U