In vitro cleavage of double- and single-stranded DNA by plasmid RSF1010-encoded mobilization proteins.

AUTOR(ES)

Scherzinger, E

RESUMO

We have used purified RSF1010 mobilization proteins to reproduce in vitro a strand-specific nicking at the plasmid origin of transfer, oriT. In the presence of Mg2+, the proteins MobA (78-kDa form of RSF1010 DNA primase), MobB, and MobC and supercoiled or linear duplex oriT DNA form large amounts of a cleavage complex, which is characterized by its sensitivity to protein-denaturant treatment. Upon addition of SDS to such a complex, a single strand break is generated in the DNA, and MobA is found linked to the 5' nick terminus, presumably covalently. The double-strand nicking activity of MobA requires, in addition to Mg2+, the presence of MobC and is stimulated by the presence of MobB. The nick site has been shown by DNA sequencing to lie at the position cleaved in vivo during transfer, between nucleotides 3138/3139 in the r strand of RSF1010. We have found that MobA will also cleave DNA at sites other than oriT if the DNA is present in single-stranded form. Breakage in this case occurs in the absence of denaturing conditions, and after prolonged incubation, reclosure can be demonstrated.

Documentos Relacionados

• A silencer element for the lipoprotein lipase gene promoter and cognate double- and single-stranded DNA-binding proteins.
• Cleavage of single-stranded DNA by plasmid pT181-encoded RepC protein.
• Common double- and single-stranded DNA binding factor for a sterol regulatory element.
• Mechanism of origin unwinding: sequential binding of DnaA to double- and single-stranded DNA
• Plasmid RSF1010 DNA replication in vitro promoted by purified RSF1010 RepA, RepB and RepC proteins.