RESUMO
The integrase (Int) protein of coliphage HK022 can catalyze in Escherichia coli as well as in in vitro integrative and excisive recombinase-mediated cassette exchange reactions between plasmids as substrates. Atomic force microscopy images have revealed that in the protein-DNA complexes that are formed, the plasmid substrates are connected via one and not two pairs of attachment sites. This observation, together with the elucidation of intermediate co-integrates between the two circular plasmids, suggest that a sequential mechanism of the RMCE reaction is possible.
Assuntos
Proteínas de Bactérias/genética , Bacteriófago HK022/enzimologia , DNA Nucleotidiltransferases , Escherichia coli K12/virologia , Integrases/metabolismo , Plasmídeos/genética , Antibacterianos/farmacologia , Sítios de Ligação Microbiológicos , Proteínas de Bactérias/metabolismo , Bacteriófago HK022/genética , Bacteriófago HK022/fisiologia , Biocatálise , Cloranfenicol/farmacologia , DNA Nucleotidiltransferases/metabolismo , Farmacorresistência Bacteriana/genética , Escherichia coli K12/efeitos dos fármacos , Escherichia coli K12/genética , Técnicas Genéticas , Microscopia de Força Atômica , Recombinação Genética , Integração ViralRESUMO
The integrase (Int) proteins of coliphages HK022 and lambda, are phosphorylated in one or more of their tyrosine residues. In Int of HK022 the phosphorylated residue(s) belong to its core-binding/catalytic domains. Wzc, a protein tyrosine kinase of Escherichia coli, is not required for Int phosphorylation in vivo, however, it can transphosphorylate the conserved Tyr(342) catalytic residue of Int in vitro. Int purified from cells that overexpress Wzc has a reduced activity in vitro. In vivo, the lysogenization of wild type HK022 as well as of lambda is not affected by the overexpression of Wzc. However, the nin5 mutant of lambda, which lacks a protein-tyrosine phosphatase gene, shows a significantly reduced lysogenization. It is suggested that phosphorylation of Int by Wzc down regulates the activity of Int.
