Slieman T A, Rebeil R, Nicholson W L
Department of Veterinary Science and Microbiology, University of Arizona, Tucson, Arizona 85721, USA.
J Bacteriol. 2000 Nov;182(22):6412-7. doi: 10.1128/JB.182.22.6412-6417.2000.
The predominant photolesion in the DNA of UV-irradiated dormant bacterial spores is the thymine dimer 5-thyminyl-5,6-dihydrothymine, commonly referred to as spore photoproduct (SP). A major determinant of SP repair during spore germination is its direct reversal by the enzyme SP lyase, encoded by the splB gene in Bacillus subtilis. SplB protein containing an N-terminal tag of six histidine residues [(6His)SplB] was purified from dormant B. subtilis spores and shown to efficiently cleave SP but not cyclobutane cis,syn thymine-thymine dimers in vitro. In contrast, SplB protein containing an N-terminal 10-histidine tag [(10His)SplB] purified from an Escherichia coli overexpression system was incompetent to cleave SP unless the 10-His tag was first removed by proteolysis at an engineered factor Xa site. To assay the parameters of binding of SplB protein to UV-damaged DNA, a 35-bp double-stranded oligonucleotide was constructed which carried a single pair of adjacent thymines on one strand. Irradiation of the oligonucleotide in aqueous solution or at 10% relative humidity resulted in formation of cyclobutane pyrimidine dimers (Py lozengePy) or SP, respectively. (10His)SplB was assayed for oligonucleotide binding using a DNase I protection assay. In the presence of (10His)SplB, the SP-containing oligonucleotide was selectively protected from DNase I digestion (half-life, >60 min), while the Py lozengePy-containing oligonucleotide and the unirradiated oligonucleotide were rapidly digested by DNase I (half-lives, 6 and 9 min, respectively). DNase I footprinting of (10His)SplB bound to the artificial substrate was carried out utilizing the (32)P end-labeled 35-bp oligonucleotide containing SP. DNase I footprinting showed that SplB protected at least a 9-bp region surrounding SP from digestion with DNase I with the exception of two DNase I-hypersensitive sites within the protected region. (10His)SplB also caused significant enhancement of DNase I digestion of the SP-containing oligonucleotide for at least a full helical turn 3' to the protected region. The data suggest that binding of SP lyase to SP causes significant bending or distortion of the DNA helix in the vicinity of the lesion.
紫外线照射的休眠细菌芽孢DNA中的主要光损伤是胸腺嘧啶二聚体5 - 胸腺嘧啶基 - 5,6 - 二氢胸腺嘧啶，通常称为芽孢光产物（SP）。芽孢萌发过程中SP修复的一个主要决定因素是由枯草芽孢杆菌中splB基因编码的SP裂解酶对其进行直接逆转。从休眠的枯草芽孢杆菌芽孢中纯化出含有六个组氨酸残基N端标签的SplB蛋白[（6His）SplB]，并证明其在体外能有效切割SP，但不能切割环丁烷顺式、反式胸腺嘧啶 - 胸腺嘧啶二聚体。相比之下，从大肠杆菌过表达系统中纯化出的含有N端10个组氨酸标签的SplB蛋白[（10His）SplB]无法切割SP，除非通过在工程化的因子Xa位点进行蛋白水解先去除10 - His标签。为了测定SplB蛋白与紫外线损伤DNA的结合参数，构建了一个35个碱基对的双链寡核苷酸，其一条链上带有一对相邻的胸腺嘧啶。在水溶液中或10%相对湿度下照射该寡核苷酸分别导致形成环丁烷嘧啶二聚体（Py菱形Py）或SP。使用DNase I保护试验测定（10His）SplB与寡核苷酸的结合。在（10His）SplB存在下，含SP的寡核苷酸被选择性保护免受DNase I消化（半衰期，>60分钟），而含Py菱形Py的寡核苷酸和未照射的寡核苷酸则被DNase I快速消化（半衰期分别为6分钟和9分钟）。利用含SP的（32）P末端标记的35个碱基对寡核苷酸对与人工底物结合的（10His）SplB进行DNase I足迹分析。DNase I足迹分析表明，除了受保护区域内的两个DNase I高敏位点外，SplB保护了围绕SP的至少9个碱基对区域不被DNase I消化。（10His）SplB还导致在受保护区域3'端至少一个完整螺旋圈的含SP寡核苷酸的DNase I消化显著增强。数据表明，SP裂解酶与SP的结合会导致损伤附近的DNA螺旋发生显著弯曲或扭曲。
