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Regulation of site-specific recombination by the C-terminus of lambda integrase.

ABSTRACT: Site-specific recombination catalyzed by bacteriophage lambda integrase (Int) is essential for establishment and termination of the viral lysogenic life cycle. Int is the archetype of the tyrosine recombinase family whose members are responsible for DNA rearrangement in prokaryotes, eukaryotes and viruses. The mechanism regulating catalytic activity during recombination is incompletely understood. Studies of tyrosine recombinases bound to their target substrates suggest that the C-termini of the proteins are involved in protein-protein contacts that control the timing of DNA cleavage events during recombination. We investigated an Int truncation mutant (W350) that possesses enhanced topoisomerase activity but greater than 100-fold reduced recombination activity. Alanine scanning mutagenesis of the C-terminus indicates that two mutants, W350A and I353A, cannot perform site-specific recombination although their DNA binding, cleavage and ligation activities are at wild-type levels. Two other mutants, R346A and R348A, are deficient solely in the ability to cleave DNA. To explain these results, we have constructed a homology-threaded model of the Int structure using a Cre crystal structure. We propose that residues R346 and R348 are involved in orientation of the catalytic tyrosine that cleaves DNA, whereas W350 and I353 control and make intermolecular contacts with other Int proteins in the higher order recombination structures known as intasomes. These results suggest that Int and the other tyrosine recombinases have evolved regulatory contacts that coordinate site-specific recombination at the C-terminus.

SUBMITTER: Kazmierczak RA

PROVIDER: S-EPMC137966 | biostudies-literature | 2002 Dec

REPOSITORIES: biostudies-literature

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Regulation of site-specific recombination by the C-terminus of lambda integrase.

Kazmierczak Robert A RA Swalla Brian M BM Burgin Alex B AB Gumport Richard I RI Gardner Jeffrey F JF

Nucleic acids research 20021201 23

Site-specific recombination catalyzed by bacteriophage lambda integrase (Int) is essential for establishment and termination of the viral lysogenic life cycle. Int is the archetype of the tyrosine recombinase family whose members are responsible for DNA rearrangement in prokaryotes, eukaryotes and viruses. The mechanism regulating catalytic activity during recombination is incompletely understood. Studies of tyrosine recombinases bound to their target substrates suggest that the C-termini of the ...[more]

PMID: 12466544

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Project description:SSV-type integrases, encoded by fuselloviruses which infect the hyperthermophilic archaea of the Sulfolobales, are archaeal members of the tyrosine recombinase family. These integrases catalyze viral integration into and excision from a specific site on the host genome. In the present study, we have established an in vitro integration/excision assay for SSV2 integrase (Int(SSV2)). Int(SSV2) alone was able to catalyze both integration and excision reactions in vitro. A 27-bp specific DNA sequence is minimally required for the activity of the enzyme, and its flanking sequences influence the efficiency of integration by the enzyme in a sequence-nonspecific manner. The enzyme forms a tetramer through interactions in the N-terminal part (residues 1 to 80), interacts nonspecifically with DNA and performs chemical catalysis in the C-terminal part (residues 165 to 328), and appears to recognize and bind the specific site of recombination in the middle portion (residues 81 to 164). It is worth noting that an N-terminally truncated mutant of Int(SSV2) (residues 81 to 328), which corresponded to the putative product of the 3'-end sequence of the Int(SSV2) gene of the integrated SSV2 genome, was unable to form tetramers but possessed all the catalytic properties of full-length Int(SSV2) except for the slightly reduced recombination activity. Our results suggest that, unlike ? integrase, SSV-type integrases alone are capable of catalyzing viral DNA recombination with the host genome in a simple and reversible fashion.Archaea are host to a variety of viruses. A number of archaeal viruses are able to integrate their genome into the host genome. Many known archaeal viral integrases belong to a unique type, or the SSV type, of tyrosine recombinases. SSV-type integrases catalyze viral integration into and excision from a specific site on the host genome. However, the molecular details of the recombination process have yet to be fully understood because of the lack of an established in vitro recombination assay system. Here we report an in vitro assay for integration and excision by SSV2 integrase, a member of the SSV-type integrases. We show that SSV2 integrase alone is able to catalyze both integration and excision and reveal how different parts of the target DNA and the enzyme serve their roles in these processes. Therefore, our results provide mechanistic insights into a simple recombination process catalyzed by an archaeal integrase.

Dataset Information

Regulation of site-specific recombination by the C-terminus of lambda integrase.

Publications

Regulation of site-specific recombination by the C-terminus of lambda integrase.

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