Mapping the λ Integrase bridges in the nucleoprotein Holliday junction intermediates of viral integrative and excisive recombination

Abstract

The site-specific recombinase encoded by bacteriophage λ [λ Integrase (Int)] is responsible for integrating and excising the viral chromosome into and out of the chromosome of its Escherichia coli host. In contrast to the other well-studied and highly exploited tyrosine recombinase family members, such as Cre and Flp, Int carries out a reaction that is highly directional, tightly regulated, and depends on an ensemble of accessory DNA bending proteins acting on 240 bp of DNA encoding 16 protein binding sites. This additional complexity enables two pathways, integrative and excisive recombination, whose opposite, and effectively irreversible, directions are dictated by different physiological and environmental signals. Int recombinase is a heterobivalent DNA binding protein that binds via its small amino-terminal domain to high affinity arm-type DNA sites and via its large, compound carboxyl-terminal domain to core-type DNA sites, where DNA cleavage and ligation are executed. Each of the four Int protomers, within a multiprotein 400-kDa recombinogenic complex, is thought to bind and, with the aid of DNA bending proteins, bridge one arm-and one core-type DNA site. Despite a wealth of genetic, biochemical, and functional information generated by many laboratories over the last 50 y, it has not been possible to decipher the patterns of Int bridges, an essential step in understanding the architectures responsible for regulated directionality of recombination. We used site-directed chemical cross-linking of Int in trapped Holliday junction recombination intermediates and recombination reactions with chimeric recombinases, to identify the unique and monogamous patterns of Int bridges for integrative and excisive recombination.site-specific recombination | regulation of directionality | recombinogenic architectures | molecular machines

Nucleoprotein architectures regulating the directionality of viral integration and excision

“…1). The resulting HJ protein-DNA complexes have been well characterized and shown to be stable both in gels and in solution for more than 24 h at room temperature (16), in agreement with the coherent FRET results described below. To form the heteroduplex bubble substrates for excisive recombination, we annealed and ligated appropriate combinations of synthetic oligonucleotides (∼40-50 nucleotides long) to generate the 95-to 107-bp attL and the 135-to 145-bp attR substrates.…”

“…We suggest that this flexibility is functionally important because it allows for the dynamic binding required to engage the bacterial chromosome and ultimately lock onto the attB sequence. This model is also consistent with, and explains, a difference between the two kinds of Int bridging experiments reported in the companion paper (16). Whereas chemical cross-linking of the P1-B Int bridge was the most robust of all of the Int bridges, in the genetic analyses, the P1-B Int bridge was the weakest, precisely the difference expected for a flexible arm.…”

“…We efficiently trapped stable HJ complexes as described previously (17,18) and as implemented in the companion paper (16). Briefly, the HJs were formed from recombination between attL and attR substrates containing heteroduplex bubbles such that the resulting DNA was fully base paired, but resolution of the junction in either direction was energetically disfavored by heterologies that would result during strand exchange (Fig.…”

“…Proteins (Int, IHF, Xis, and Fis) were purified, oligonucleotides were prepared, and recombination reactions were carried out as described in SI Text and ref. 16.…”

Bacteriophage lambda: Early pioneer and still relevant

“…The host integration host factor (IHF; named for this reaction but now known to have many pleiotropic functions in E. coli and Fis protein were found to be involved (Ball and Johnson, 1991). Genetic studies by Ethan Signer, Robert Weisberg and others combined with biochemical studies from the Art Landy lab have resulted in a marvelously detailed picture of the integration protein DNA complex bound to its target (the attachment site, att ) and its actions that result in att site-specific recombination and lambda prophage insertion (Seah et al , 2014; Tong et al , 2014 and references therein).…”