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Nucleic Acids Res. 2011 Apr;39(7):2658-70. doi: 10.1093/nar/gkq1137. Epub 2010 Nov 24.

Tracking F plasmid TraI relaxase processing reactions provides insight into F plasmid transfer.

Author information

1
Department of Biology, Johns Hopkins University, 3400 N. Charles St, Baltimore, MD 21218, USA.

Abstract

Early in F plasmid conjugative transfer, the F relaxase, TraI, cleaves one plasmid strand at a site within the origin of transfer called nic. The reaction covalently links TraI Tyr16 to the 5'-ssDNA phosphate. Ultimately, TraI reverses the cleavage reaction to circularize the plasmid strand. The joining reaction requires a ssDNA 3'-hydroxyl; a second cleavage reaction at nic, regenerated by extension from the plasmid cleavage site, may generate this hydroxyl. Here we confirm that TraI is transported to the recipient during transfer. We track the secondary cleavage reaction and provide evidence it occurs in the donor and F ssDNA is transferred to the recipient with a free 3'-hydroxyl. Phe substitutions for four Tyr within the TraI active site implicate only Tyr16 in the two cleavage reactions required for transfer. Therefore, two TraI molecules are required for F plasmid transfer. Analysis of TraI translocation on various linear and circular ssDNA substrates supports the assertion that TraI slowly dissociates from the 3'-end of cleaved F plasmid, likely a characteristic essential for plasmid re-circularization.

PMID:
21109533
PMCID:
PMC3074121
DOI:
10.1093/nar/gkq1137
[Indexed for MEDLINE]
Free PMC Article

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