Mechanism of inhibition of site-specific recombination by the Holliday junction-trapping peptide WKHYNY: insights into phage lambda integrase-mediated strand exchange.
Holliday junctions are central intermediates in site-specific recombination reactions mediated by tyrosine recombinases. Because these intermediates are extremely transient, only artificially assembled Holliday junctions have been available for study. We have recently identified hexapeptides that cause the accumulation of natural Holliday junctions of bacteriophage lambda Integrase (Int)-mediated reactions. We now show that one of these peptides acts after the first DNA cleavage event to stabilize protein-bound junctions and to prevent their resolution. The peptide acts before the step affected by site affinity (saf) mutations in the core region, in agreement with a model that the peptide stabilizes the products of strand exchange (i.e. Holliday junctions) while saf mutations reduce ligation of exchanged strands.Strand exchange events leading to Holliday junctions in phage lambda integration and excision are asymmetric, presumably because interactions between Int and some of its core-binding sites determine the order of strand cleavage. We have compared the structure of Holliday junctions in one unidirectional and in two bidirectional Int-mediated pathways and show that the strand cleavage steps are much more symmetric in the bidirectional pathways. Thus Int-DNA interactions which determine the order of top and bottom strand cleavage and exchange are unique in each recombination pathway.
Cassell
Geoffrey D
GD
Department of Biology and Center for Microbial Sciences, San Diego State University, 5500 Campanile Drive, CA 92182-4614, USA.
Segall
Anca M
AM
D001287:Attachment Sites, Microbiological / Q000235:genetics; D010582:Bacteriophage lambda / Q000201:enzymology / Q000235:genetics*; D001665:Binding Sites; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008957:Models, Genetic; D009690:Nucleic Acid Conformation; D019000:Phosphotyrosine / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D045522:Recombinases; D011995:Recombination, Genetic / Q000235:genetics*; D014443:Tyrosine / Q000378:metabolism
D004269:DNA, Bacterial; D045522:Recombinases; D019000:Phosphotyrosine; D014443:Tyrosine; D004254:DNA Nucleotidyltransferases; D019426:Integrases