Control of Directionality in Lambda Site Specific Recombination

Self Cite

From bacterial viruses to humans, site-specific recombination and transposition are the major pathways for rearranging genomes on both long-and short-time scales. The site-specific pathways can be divided into 2 groups based on whether they are stochastic or regulated. Recombinases Cre and Int are well-studied examples of each group, respectively. Both have been widely exploited as powerful and flexible tools for genetic engineering: Cre primarily in vivo and Int primarily in vitro. Although Cre and Int use the same mechanism of DNA strand exchange, their respective reaction pathways are very different. Cre-mediated recombination is bidirectional, unregulated, does not require accessory proteins, and has a minimal symmetric DNA target. We show that when Cre is fused to the small N-terminal domain of Int, the resulting chimeric Cre recombines complex higher-order DNA targets comprising >200 bp encoding 16 protein-binding sites. This recombination requires the IHF protein, is unidirectional, and is regulated by the relative levels of the 3 accessory proteins, IHF, Xis, and Fis. In one direction, recombination depends on the Xis protein, and in the other direction it is inhibited by Xis. It is striking that regulated directionality and complexity can be conferred in a simple chimeric construction. We suggest that the relative ease of constructing a chimeric Cre with these properties may simulate the evolutionary interconversions responsible for the large variety of site-specific recombinases observed in Archaea, Eubacteria, and Eukarya.bacteriophage ͉ recombinase ͉ site-specific recombination ͉ evolution

Section: Resultssupporting

confidence: 76%

A chimeric Cre recombinase with regulated directionality

Warren

Laxmikanthan²,

Landy³

2008

Self Cite

“…Consequently the P-arm would not be properly positioned to stabilize a synapsiscompetent attR. Because Xis also promotes cooperative binding of Int at P2 (37)(38)(39), it seems likely that only low-affinity Int-attR interactions would be possible in its absence.…”

Section: Discussionmentioning

confidence: 99%

Nucleoprotein architectures regulating the directionality of viral integration and excision

Seah

Warren

Tong

et al. 2014

Self Cite

The virally encoded site-specific recombinase Int collaborates with its accessory DNA bending proteins IHF, Xis, and Fis to assemble two distinct, very large, nucleoprotein complexes that carry out either integrative or excisive recombination along regulated and essentially unidirectional pathways. The core of each complex consists of a tetramer of Integrase protein (Int), which is a heterobivalent DNA binding protein that binds and bridges a core-type DNA site (where strand cleavage and ligation are executed), and a distal arm-type site, that is brought within range by one or more DNA bending proteins. The recent determination of the patterns of these Int bridges has made it possible to think realistically about the global architecture of the recombinogenic complexes. Here, we combined the previously determined Int bridging patterns with in-gel FRET experiments and in silico modeling to characterize and differentiate the two 400-kDa multiprotein Holiday junction recombination intermediates formed during λ integration and excision. The results lead to architectural models that explain how integration and excision are regulated in λ site-specific recombination. Our confidence in the basic features of these architectures is based on the redundancy and self-consistency of the underlying data from two very different experimental approaches to establish bridging interactions, a set of strategic intracomplex distances from FRET experiments, and the model's ability to explain key aspects of the integrative and excisive recombination pathways, such as topological changes, the mechanism of capturing attB, and the features of asymmetry and flexibility within the complexes.regulation of directionality | topology | recombinogenic architectures | molecular machines H igh-precision DNA transactions responsible for a variety of fundamental processes are typically promoted and modulated by large multiprotein machines that use cooperative interactions and involve DNA bending and/or wrapping. One well-studied example is the tightly regulated and highly directional site-specific recombination by which bacteriophage λ inserts and excises its DNA into and out of the Escherichia coli host chromosome, using the phage attP and the bacterial attB DNA sequences for integration and the resulting junction sequences, attL and attR, for excision. These reactions are catalyzed by the phage-encoded Integrase protein (Int), the founding member of the tyrosine recombinase family of site-specific recombinases (1). In addition to mediating integration and excision of viral genomes, members of this family function in a variety of other cellular processes including chromosome segregation, gene regulation, and conjugative transposition (2).Int has three well-characterized domains: an N-terminal DNA-binding domain (NTD), a central core-binding domain (CB), and a C-terminal catalytic domain (CAT). The CB and CAT domains (referred to here as the CTD) are together responsible for binding to the core-type DNA sequences where strand exchange and ligation t...

“…The Gateway technology is based on the bacteriophage lambda site-specific recombination system that facilitates the integration of lambda into the Escherichia coli chromosome (45). In the Gateway technology, the components of the lambda recombination system are modified to improve the specificity and efficiency of the system (46). Coding regions for viral (VARV-G1R, ECTV-002, MPXV-003, CPXV-006, and MYXV-M063) and human (NF-B1 and SKP1A) proteins were PCR amplified, using primers containing gene-specific sequences flanked by sequences representing modified components of the lambda recombination system (Table S3) and incorporated into pDNOR222 plasmid (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

Proteomic screening of variola virus reveals a unique NF-κB inhibitor that is highly conserved among pathogenic orthopoxviruses

Mohamed

Rahman

Lanchbury

et al. 2009

Identification of the binary interactions between viral and host proteins has become a valuable tool for investigating viral tropism and pathogenesis. Here, we present the first systematic protein interaction screening of the unique variola virus proteome by using yeast 2-hybrid screening against a variety of human cDNA libraries. Several protein-protein interactions were identified, including an interaction between variola G1R, an ankryin/F-box containing protein, and human nuclear factor kappa-B1 (NF-B1)/p105. This represents the first direct interaction between a pathogen-encoded protein and NF-B1/p105. Orthologs of G1R are present in a variety of pathogenic orthopoxviruses, but not in vaccinia virus, and expression of any one of these viral proteins blocks NF-B signaling in human cells. Thus, proteomic screening of variola virus has the potential to uncover modulators of the human innate antiviral responses.ankyrin repeats ͉ Skp1 ͉ yeast 2-hybrid