PubMed · 9343669
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S H Lee, J B Clark. 1997. High-yield method for isolation of lambda DNA.. https://doi.org/10.2144/97234bm07
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The integrase protein of bacteriophage lambda mediates recombination via four distinct pathways. The recent in vitro reconstitution of the efficient bidirectional reaction between two attLtenP'1 target sites now allows comparisons of this pathway, known as the bent-L pathway, with the inefficient bidirectional straight-L pathway and with the efficient but unidirectional pathways of integration and excision. To this end, a series of higher order intermediates of the bent-L pathway was characterized using gel mobility shift assays, two-dimensional gel analysis, and footprinting. The analysis spans the initial binding of proteins to individual DNA target sites, synapsis of two partner DNA targets, and strand exchange. This study identifies a presynaptic "checkpoint" of recombination. It shows that synapsis is a slow step in the recombination reaction, while subsequent strand exchange is comparatively fast. Synaptic complexes contain a preponderance of recombinant products, suggesting that an energetically favorable but somewhat subtle conformational change drives strand exchange. In addition, comparison of wild-type integrase with a catalytically defective mutant of integrase, IntF, showed that, in addition to the catalysis defect, this mutant has different DNA-binding properties than the wild-type protein.
The oriC transducing phage lambda poriCc is a pseudovirulent phage capable of forming plaques on a lambda lysogen. This phenotype is dependent upon the presence of the oriC insert. The ability of lambda poriCc to form plaques on a lambda lysogen represents a potential phage assay system for studying aspects of oriC function. In the present study we establish that lambda poriCc infection of a lambda lysogen is a legitimate assay for oriC function. We use this assay to confirm the previously reported observation that initiation of DNA replication from oriC is transiently inhibited in a ultra violet (UV) irradiated cell at doses greater than 60 J/m2. We further demonstrate using this assay that the UV induced inhibition of initiation of DNA replication from oriC is not a SOS function nor a heat shock function. In the course of these studies, we found that lambda poriCc infection of a non-lysogenic cell is extremely sensitive to pre-irradiation of the Escherichia coli host. We postulate that the sensitivity of lambda poriCc replication to host cell pre-irradiation reflects in some way the transient inhibition of initiation of DNA replication from oriC following UV irradiation.
Termination of packaging of the lambda chromosome involves completion of translocation of the DNA into the head shell, and conversion of the translocation complex into a cleavage complex. The cleavage reaction introduces staggered nicks into the downstream cosN to generate the right cohesive end of the chromosome. cosQ, a site adjacent to cosN, was found to be required for nicking the bottom strand of cosN; bottom strand nicking was also sequence-specific for bps at the nick site. Nicking of the top strand of cosN (cosNL) was stimulated by cosQ, but fidelity and efficiency of cosNL nicking were largely dictated by other cos subsites (i.e. cosB and I2). Aberrant top-strand cleavage within cosQ was observed in the absence of I2, and nicking at a site 8 nt 5' to the normal cosNL nick site occurred in the absence of cosB. The presence of cosQ was found to be insufficient to arrest DNA translocation in vivo, indicating that cosQ, per se, is not a packaging stop signal. A model is presented in which the role of cosQ is to depolarize the asymmetric arrangement of terminase protomers in the translocation complex so that protomers are configured to match the 2-fold rotational symmetry of cosN.