Sharma Suveena, Sathyanarayana Bangalore K, Bird Jeremy G, Hoskins Joel R, Lee Byungkook, Wickner Sue
Laboratory of Molecular Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Biol Chem. 2004 Feb 13;279(7):6027-34. doi: 10.1074/jbc.M310917200. Epub 2003 Nov 21.
DNA replication of plasmid P1 requires a plasmid-encoded origin DNA-binding protein, RepA. RepA is an inactive dimer and is converted by molecular chaperones into an active monomer that binds RepA binding sites. Although the sequence of RepA is not homologous to that of F plasmid RepE, we found by using fold-recognition programs that RepA shares structural homology with RepE and built a model based on the RepE crystal structure. We constructed mutants in the two predicted DNA binding domains to test the model. As expected, the mutants were defective in P1 DNA binding. The model predicted that RepA binds the first half of the binding site through interactions with the C-terminal DNA binding domain and the second half through interactions with the N-terminal domain. The experiments supported the prediction. The model was further supported by the observation that mutants defective in dimerization map to the predicted subunit interface region, based on the crystal structure of pPS10 RepA, a RepE family member. These results suggest P1 RepA is structurally homologous to plasmid initiators, including those of F, R6K, pSC101, pCU1, pPS10, pFA3, pGSH500, Rts1, RepHI1B, RepFIB, and RSF1010.
质粒P1的DNA复制需要一种由质粒编码的起始DNA结合蛋白RepA。RepA是一种无活性的二聚体，通过分子伴侣转化为有活性的单体，该单体可结合RepA结合位点。尽管RepA的序列与F质粒RepE的序列不同源，但我们通过使用折叠识别程序发现RepA与RepE具有结构同源性，并基于RepE晶体结构构建了一个模型。我们在两个预测的DNA结合结构域中构建了突变体以测试该模型。正如预期的那样，这些突变体在P1 DNA结合方面存在缺陷。该模型预测，RepA通过与C端DNA结合结构域的相互作用结合结合位点的前半部分，并通过与N端结构域的相互作用结合后半部分。实验支持了这一预测。基于RepE家族成员pPS10 RepA的晶体结构，二聚化有缺陷的突变体映射到预测的亚基界面区域这一观察结果进一步支持了该模型。这些结果表明，P1 RepA与质粒起始蛋白在结构上同源，包括F、R6K、pSC101、pCU1、pPS10、pFA3、pGSH500、Rts1、RepHI1B、RepFIB和RSF1010的起始蛋白。
