Connor D A, Falick A M, Young M C, Shetlar M D
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143-0446, USA.
Photochem Photobiol. 1998 Sep;68(3):299-308.
In recent years, there has been a significant number of studies in which UV light has been used as a reagent to induce cross-links in nucleic acid-protein complexes. An area of considerable interest among those interested in structural biology is the garnering of information about the sites of cross-linking within the protein and nucleic acid members of photolinked conjugates, under the assumption that such knowledge should lead to identification of contact regions or sites within the native complexes. In this paper, we present our results from a photocross-linking study of the complex of the single-stranded DNA-binding domain of rat DNA polymerase beta (pol beta-ss) with the oligonucleotide d(ATATATA). In this study, we have used single nanosecond laser pulses as the cross-linking reagent and matrix-assisted laser desorption/ionization-time of flight mass spectrometry as an analytical tool to identify cross-linked peptides purified from proteolytic digests of the cross-linked complex. Six cross-linked peptides have been identified in tryptic digests of the protein-oligonucleotide conjugates that result from irradiation of the pol beta-ss-d(ATATATA) complex with a single laser pulse. Comparisons with NMR data in the literature for the same complex show that each of the cross-linked peptides contains amino acids that are in contact with the nucleic acid component of the complex.
近年来，有大量研究将紫外光用作试剂来诱导核酸 - 蛋白质复合物中的交联。在结构生物学领域，一个备受关注的方面是获取有关光交联共轭物中蛋白质和核酸成员内交联位点的信息，前提是这些信息应有助于识别天然复合物中的接触区域或位点。在本文中，我们展示了对大鼠DNA聚合酶β的单链DNA结合结构域（pol β - ss）与寡核苷酸d(ATATATA)形成的复合物进行光交联研究的结果。在这项研究中，我们使用单纳秒激光脉冲作为交联试剂，并采用基质辅助激光解吸/电离飞行时间质谱作为分析工具，以鉴定从交联复合物的蛋白水解消化物中纯化出的交联肽段。在用单激光脉冲照射pol β - ss - d(ATATATA)复合物后得到的蛋白质 - 寡核苷酸共轭物的胰蛋白酶消化物中，已鉴定出六个交联肽段。与文献中关于同一复合物的核磁共振数据比较表明，每个交联肽段都包含与复合物核酸成分接触的氨基酸。
