文章基本信息

标题：Lagging strand replication of rolling-circle plasmids: Specific recognition of the ssoA-type origins in different gram-positive bacteria
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作者：M. Gabriela Kramer ; Manuel Espinosa ; Tapan K. Misra 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1998
卷号：95
期号：18
页码：10505-10510
DOI：10.1073/pnas.95.18.10505
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Many bacterial plasmids replicate by a rolling-circle mechanism that involves the generation of single-stranded DNA (ssDNA) intermediates. Replication of the lagging strand of such plasmids initiates from their single strand origin (sso). Many different types of ssos have been identified. One group of ssos, termed ssoA, which have conserved sequence and structural features, function efficiently only in their natural hosts in vivo. To study the host specificity of sso sequences, we have analyzed the functions of two closely related ssoAs belonging to the staphylococcal plasmid pE194 and the streptococcal plasmid pLS1 in Staphylococcus aureus. The pLS1 ssoA functioned poorly in vivo in S. aureus as evidenced by accumulation of high levels of ssDNA but supported efficient replication in vitro in staphylococcal extracts. These results suggest that one or more host factors that are present in sufficient quantities in S. aureus cell-free extracts may be limiting in vivo. Mapping of the initiation points of lagging strand synthesis in vivo and in vitro showed that DNA synthesis initiates from specific sites within the pLS1 ssoA. These results demonstrate that specific initiation of replication can occur from the pLS1 ssoA in S. aureus although it plays a minimal role in lagging strand synthesis in vivo. Therefore, the poor functionality of the pLS1 in vivo in a nonnative host is caused by the low efficiency rather than a lack of specificity of the initiation process. We also have identified ssDNA promoters and mapped the primer RNAs synthesized by the S. aureus and Bacillus subtilis RNA polymerases from the pE194 and pLS1 ssoAs. The S. aureus RNA polymerase bound more efficiently to the native pE194 ssoA as compared with the pLS1 ssoA, suggesting that the strength of RNA polymerase-ssoA interaction may play a major role in the functionality of the ssoA sequences in Gram-positive bacteria.
关键词：single strand origin/single-stranded DNA promoter/RNA polymerase/ in vitro replication