H Fukuda1, E Ohtsubo. 1. Institute of Molecular and Cellular Biosciences, the University of Tokyo, Japan.
Abstract
BACKGROUND: The plasmid R100 encodes the TraI protein, which is required for conjugal DNA transfer. TraI has the activity of site- and strand-specific nicking of the supercoiled plasmid DNA. The molecular mechanism of this specific nicking, which is supposed to be the initiation reaction of DNA transfer, is not understood. RESULTS: We have demonstrated that TraI has the ability to cleave the single-stranded DNA at the same site as the nicking site (nic) in a region, which we here refer to as sbi. The product contained the TraI protein which was covalently linked to the newly generated 5' end of the nicking reaction. Both the cleaving and nicking reactions took place under almost the same conditions and required the presence of the sbi region. DNase I-footprinting analysis revealed that the TraI bound to the single-stranded DNA of the sbi region. TraI did not cleave the double-stranded DNA fragment, but it did cleave the double-stranded DNA with a single-stranded DNA portion in the sbi region. KMnO4 mapping analysis revealed that TraI can melt the sbi region in the supercoiled DNA to generate a single-stranded portion. We have also demonstrated that TraI was able to rejoin the cleaved products. The rejoining reaction required the 5' end of one cleaved product with the TraI covalently attached and the 3' end of the other product containing the sbi region. CONCLUSIONS: Our results demonstrate that the nicking reaction-the initiation reaction of DNA transfer-is actually the cleaving reaction of the single-stranded DNA. TraI, which has both cleaving and rejoining activities, is thought to be involved in the termination of DNA transfer, to give a copy of the conjugative plasmid by joining the 5' end, which is generated by the initiation reaction, with the 3' end, which will be generated upon cleavage of the sbi region appearing after one round of the rolling circle replication of the plasmid.
BACKGROUND: The plasmid R100 encodes the TraI protein, which is required for conjugal DNA transfer. TraI has the activity of site- and strand-specific nicking of the supercoiled plasmid DNA. The molecular mechanism of this specific nicking, which is supposed to be the initiation reaction of DNA transfer, is not understood. RESULTS: We have demonstrated that TraI has the ability to cleave the single-stranded DNA at the same site as the nicking site (nic) in a region, which we here refer to as sbi. The product contained the TraI protein which was covalently linked to the newly generated 5' end of the nicking reaction. Both the cleaving and nicking reactions took place under almost the same conditions and required the presence of the sbi region. DNase I-footprinting analysis revealed that the TraI bound to the single-stranded DNA of the sbi region. TraI did not cleave the double-stranded DNA fragment, but it did cleave the double-stranded DNA with a single-stranded DNA portion in the sbi region. KMnO4 mapping analysis revealed that TraI can melt the sbi region in the supercoiled DNA to generate a single-stranded portion. We have also demonstrated that TraI was able to rejoin the cleaved products. The rejoining reaction required the 5' end of one cleaved product with the TraI covalently attached and the 3' end of the other product containing the sbi region. CONCLUSIONS: Our results demonstrate that the nicking reaction-the initiation reaction of DNA transfer-is actually the cleaving reaction of the single-stranded DNA. TraI, which has both cleaving and rejoining activities, is thought to be involved in the termination of DNA transfer, to give a copy of the conjugative plasmid by joining the 5' end, which is generated by the initiation reaction, with the 3' end, which will be generated upon cleavage of the sbi region appearing after one round of the rolling circle replication of the plasmid.
Authors: Nicholas J Clark; Madushi Raththagala; Nathan T Wright; Elizabeth A Buenger; Joel F Schildbach; Susan Krueger; Joseph E Curtis Journal: J Mol Model Date: 2014-06-06 Impact factor: 1.810