Institute of Chemical Biology and Fundamental Medicine Siberian Division of the Russian Academy of Sciences, Prospect Lavrentieva 8, Novosibirsk 630090, Russia.
DNA Repair (Amst). 2011 Aug 15;10(8):815-25. doi: 10.1016/j.dnarep.2011.04.031. Epub 2011 May 23.
To characterize proteins that interact with base excision/single-strand interruption repair DNA intermediates in cell free extracts of Saccharomyces cerevisiae, we used a combination of photoaffinity labeling with the protein identification by MALDI-TOF-MS peptide mapping. Photoreactive analogue of dCTP, namely exo-N-[4-(4-azido-2,3,5,6,-tetrafluorobenzylidenehydrazinocarbonyl)-butylcarbamoyl]-2'-deoxycytidine-5'-triphosphate, and [(32)P]-labeled DNA duplex containing one nucleotide gap were used to generate nick-containing DNA with a photoreactive dCMP residue at the 3'-margin of the nick. This photoreactive DNA derivative was incubated with the yeast cell extract and after UV irradiation a number of proteins were labeled. Two of the crosslinked proteins were identified as the catalytic subunit of DNA polymerase ɛ and Ddc1 checkpoint protein. Labeling of DNA polymerase ɛ catalytic subunit with the nick-containing DNA repair intermediate indicates that the DNA polymerase is involved in the DNA repair synthesis in yeast, at least at DNA single-strand interruptions. Crosslinking of Ddc1 to DNA nicks took place independently of the other components of checkpoint clamp, Mec3 and Rad17, suggesting that the protein alone is able to recognize DNA single-strand breaks. Indeed, purified GST-tagged Ddc1 protein was efficiently crosslinked to nick-containing DNA. The interaction of Ddc1 with DNA nicks may provide a link between the DNA damage checkpoint and DNA base excision/single-strand breaks repair pathways in yeast. In addition, we found that absence of Ddc1 protein greatly influences the overall pattern of other proteins crosslinked to DNA nick. We suggested that this last effect of Ddc1 is at least partially due to its capacity to prevent proteolytic degradation of the DNA-protein adducts.
为了鉴定与酿酒酵母无细胞提取物中碱基切除/单链中断修复 DNA 中间产物相互作用的蛋白质，我们使用光亲和标记与 MALDI-TOF-MS 肽图谱的蛋白质鉴定相结合的方法。使用外消旋-N-[4-(4-叠氮-2,3,5,6-四氟苯甲亚肼羰基)-丁基氨甲酰基]-2'-脱氧胞苷-5'-三磷酸，即 dCTP 的光反应类似物，和含有一个核苷酸缺口的 [(32)P]-标记 DNA 双链体，在缺口的 3'-边缘生成含有光反应性 dCMP 残基的带缺口 DNA。将该光反应性 DNA 衍生物与酵母细胞提取物孵育，然后用 UV 照射，标记了许多蛋白质。交联的两种蛋白质被鉴定为 DNA 聚合酶ɛ的催化亚基和 Ddc1 检查点蛋白。用含有缺口的 DNA 修复中间体标记 DNA 聚合酶ɛ催化亚基表明，该 DNA 聚合酶至少在酵母的 DNA 单链中断处参与 DNA 修复合成。Ddc1 与 DNA 缺口的交联发生在检查点钳的其他成分（Mec3 和 Rad17）之外，表明该蛋白本身能够识别 DNA 单链断裂。事实上，纯化的 GST 标记的 Ddc1 蛋白可有效地与含有缺口的 DNA 交联。Ddc1 与 DNA 缺口的相互作用可能为酵母中的 DNA 损伤检查点与 DNA 碱基切除/单链断裂修复途径之间提供了联系。此外，我们发现 Ddc1 蛋白的缺失极大地影响了与 DNA 缺口交联的其他蛋白质的整体模式。我们认为，Ddc1 的最后一个影响至少部分是由于其防止 DNA-蛋白加合物的蛋白水解降解的能力。
