title: Site-Specific Recombination by SSV2 Integrase: Substrate Requirement and Domain Functions.
abstract: 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 λ integrase, SSV-type integrases alone are capable of catalyzing viral DNA recombination with the host genome in a simple and reversible fashion.
  
  
  OBJECTIVE
  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.
authors: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, People's Republic of China.
authors: Zhengyan
authors: Z
authors: Zhan
authors: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, People's Republic of China.
authors: Ju
authors: J
authors: Zhou
authors: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, People's Republic of China huangl@sun.im.ac.cn.
authors: Li
authors: http://orcid.org/0000-0003-2421-8162
authors: L
authors: Huang
mesh_terms: D001483:Base Sequence; D002850:Chromatography, Gel; D016000:Cluster Analysis; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D019241:Fuselloviridae / Q000201:enzymology*; D066298:In Vitro Techniques; D019426:Integrases / Q000502:physiology*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000235:genetics; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D016942:Sulfolobales / Q000821:virology*; D016662:Virus Integration / Q000235:genetics*
chemical_list: D009841:Oligonucleotides; D004254:DNA Nucleotidyltransferases; D019426:Integrases; C077570:Site-specific recombinase