Liu Yuan, Prasad Rajendra, Beard William A, Kedar Padmini S, Hou Esther W, Shock David D, Wilson Samuel H
Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
J Biol Chem. 2007 May 4;282(18):13532-41. doi: 10.1074/jbc.M611295200. Epub 2007 Mar 12.
The individual steps in single-nucleotide base excision repair (SN-BER) are coordinated to enable efficient repair without accumulation of cytotoxic DNA intermediates. The DNA transactions and various proteins involved in SN-BER of abasic sites are well known in mammalian systems. Yet, despite a wealth of information on SN-BER, the mechanism of step-by-step coordination is poorly understood. In this study we conducted experiments toward understanding step-by-step coordination during BER by comparing DNA binding specificities of two major human SN-BER enzymes, apurinic/aprymidinic endonuclease 1 (APE) and DNA polymerase beta (Pol beta). It is known that these enzymes do not form a stable complex in solution. For each enzyme, we found that DNA binding specificity appeared sufficient to explain the sequential processing of BER intermediates. In addition, however, we identified at higher enzyme concentrations a ternary complex of APE.Pol beta.DNA that formed specifically at BER intermediates containing a 5'-deoxyribose phosphate group. Formation of this ternary complex was associated with slightly stronger Pol beta gap-filling and much stronger 5'-deoxyribose phosphate lyase activities than was observed with the Pol beta.DNA binary complex. These results indicate that step-by-step coordination in SN-BER can rely on DNA binding specificity inherent in APE and Pol beta, although coordination also may be facilitated by APE.Pol beta.DNA ternary complex formation with appropriate enzyme expression levels or enzyme recruitment to sites of repair.
单核苷酸碱基切除修复（SN-BER）中的各个步骤相互协调，以实现高效修复，同时避免细胞毒性DNA中间体的积累。在哺乳动物系统中，与无碱基位点SN-BER相关的DNA交易和各种蛋白质已为人熟知。然而，尽管有大量关于SN-BER的信息，但对逐步协调机制的了解仍很有限。在本研究中，我们通过比较两种主要的人类SN-BER酶——脱嘌呤/脱嘧啶内切核酸酶1（APE）和DNA聚合酶β（Polβ）的DNA结合特异性，进行了旨在了解BER过程中逐步协调的实验。已知这些酶在溶液中不会形成稳定的复合物。对于每种酶，我们发现DNA结合特异性似乎足以解释BER中间体的顺序加工。然而，此外，我们在较高酶浓度下鉴定出一种APE.Polβ.DNA三元复合物，该复合物在含有5'-脱氧核糖磷酸基团的BER中间体处特异性形成。与Polβ.DNA二元复合物相比，这种三元复合物的形成与略强的Polβ缺口填充活性以及更强得多的5'-脱氧核糖磷酸裂解酶活性相关。这些结果表明，SN-BER中的逐步协调可以依赖于APE和Polβ固有的DNA结合特异性，尽管通过以适当的酶表达水平形成APE.Polβ.DNA三元复合物或使酶募集到修复位点也可能促进协调。
