RESUMO
Efficient DNA inversion catalysed by the invertase Gin requires the cis-acting recombinational enhancer and the Escherichia coliFIS protein. Binding of FIS bends the enhancer DNA and, on a negatively supercoiled DNA inversion substrate, facilitates the formation of a synaptic complex with specific topology. Previous studies have indicated that FIS-independent Gin mutants can be isolated which have lost the topological constraints imposed on the inversion reaction yet remain sensitive to the stimulatory effect of FIS. Whether the effect of FIS is purely architectural, or whether in addition direct protein contacts between Gin and FIS are required for efficient catalysis has remained an unresolved question. Here we show that FIS mutants impaired in DNA binding are capable of either positively or negatively affecting the inversion reaction both in vivo and in vitro. We further demonstrate that the mutant protein FIS K25E/V66A/M67T dramatically enhances the cleavage of recombination sites by FIS-independent Gin in an enhancer-independent manner. Our observations suggest that FIS plays a dual role in the inversion reaction and stimulates both the assembly of the synaptic complex as well as DNA strand cleavage.
Assuntos
Proteínas de Transporte/metabolismo , DNA Nucleotidiltransferases/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Escherichia coli , Sequência de Bases , Sítios de Ligação/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , DNA Nucleotidiltransferases/química , DNA Nucleotidiltransferases/genética , Primers do DNA/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Elementos Facilitadores Genéticos , Escherichia coli/genética , Escherichia coli/metabolismo , Fator Proteico para Inversão de Estimulação , Fatores Hospedeiros de Integração , Mutagênese Sítio-Dirigida , Mutação , Recombinação Genética , TermodinâmicaRESUMO
BACKGROUND: Matching for the HLA class I loci A and B and for the HLA class II locus DRB is known to influence the survival rate of kidney transplants. It is unknown whether matching for the HLA class II locus DPB also exerts an influence on graft outcome. METHODS: The influence of matching for the HLA-DPB locus was analyzed based on DNA typing results obtained in more than 3600 first and 1300 repeat cadaver kidney transplants. RESULTS: HLA-DPB mismatches had no deleterious influence on the outcome of first cadaver transplants. However, the influence was statistically significant for retransplants. One-year graft survival rates were 83+/-2% with no mismatch (n=345), as compared with 76+/-2% with one mismatch (n=702, P=0.02), and 73+/-3% with two mismatches (n=258, P=0.003). The deleterious influence of HLA-DPB mismatches was particularly strong in retransplant recipients with >50% reactivity of preformed lymphocytotoxic antibodies, for which the 1-year graft survival rate was 70+/-4% with no mismatch, as compared with 69+/-3% with one mismatch (P=0.05) and 61+/-5% with two mismatches (P=0.003). CONCLUSIONS: These results indicate that HLA-DPB is a clinically relevant histocompatibility locus in cadaver kidney retransplantation. It is proposed that prospective typing and matching for HLA-DPB should be implemented for cadaver kidney retransplants.