Li Y, Kurokawa K, Matsuo M, Fukuhara N, Murakami K, Sekimizu K
Laboratory of Developmental Biochemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 3-1, 7-Chome, Hongo, Bunkyo-ku, 113-0033 Tokyo, Japan.
Mol Genet Genomics. 2004 May;271(4):447-57. doi: 10.1007/s00438-004-0996-6. Epub 2004 Mar 24.
The DnaD protein in Gram-positive bacteria is thought to be essential for the initiation step in DNA replication. In the present study, we characterized two Staphylococcus aureus mutants whose temperature-sensitive growth phenotype could be complemented by a plasmid carrying the dnaD gene. These mutants each had a single amino acid substitution in the DnaD protein and showed decreased DNA synthesis at restrictive temperature. Analyses of the origin to terminus ratio by Southern blotting, and of origin numbers per cell by flow cytometry, revealed that, at the restrictive temperature, one mutant continued ongoing DNA replication but failed to initiate DNA replication. The other mutant, in contrast, could not complete ongoing DNA replication and proceeded to degrade the chromosome. However, if protein synthesis was inhibited, the second mutant could complete DNA replication. These results suggest that DnaD protein is necessary not only for the initiation step, but also to avoid replication fork blockage. Moreover, both mutants were sensitive to mitomycin C, a drug that induces DNA damage, suggesting that the DnaD protein is also involved in DNA repair.
革兰氏阳性菌中的DnaD蛋白被认为对DNA复制的起始步骤至关重要。在本研究中，我们鉴定了两个金黄色葡萄球菌突变体，其温度敏感型生长表型可被携带dnaD基因的质粒互补。这些突变体在DnaD蛋白中各有一个单氨基酸替换，并且在限制温度下DNA合成减少。通过Southern印迹分析起始到终止的比例，以及通过流式细胞术分析每个细胞中的起始数，结果显示，在限制温度下，一个突变体能够继续进行正在进行的DNA复制，但无法起始DNA复制。相比之下，另一个突变体无法完成正在进行的DNA复制，并进而降解染色体。然而，如果蛋白质合成受到抑制，第二个突变体能够完成DNA复制。这些结果表明，DnaD蛋白不仅对起始步骤是必需的，而且对于避免复制叉阻塞也是必需的。此外，这两个突变体对诱导DNA损伤的药物丝裂霉素C敏感，表明DnaD蛋白也参与DNA修复。
