| 论文题目: | Site-specific recombination by SSV2 integrase: substrate requirement and domain functions |
|---|---|
| 作者: | Zhan Zhengyan, Zhou Ju, and Huang Li*. |
| 联系作者: | Huang, Li* |
| 刊物名称: | J Virol |
| 期: | |
| 卷: | |
| 页: | |
| 年份: | |
| 影响因子: | 4.609 |
| 论文下载: | 下载地址 |
| 摘要: | 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 (IntSSV2). IntSSV2 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 1-80), interacts nonspecifically with DNA and performs chemical catalysis in the C-terminal part (residues 165-328), and appears to recognize and bind the specific site of recombination in the middle portion (residues 81-164). It is worth noting that an N-terminally truncated mutant of IntSSV2 (residues 81-328), which corresponded to the putative product of the 3' -end sequence of the IntSSV2 gene of the integrated SSV2 genome, was unable to form tetramers but possessed all the catalytic properties of full-length IntSSV2 except for the slightly reduced recombination activity. Our results suggest that, unlike lambda integrase, SSV-type integrases alone are capable of catalyzing viral DNA recombination with the host genome in a simple and reversible fashion. IMPORTANCE: 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 the processes. Therefore, our results provide mechanistic insights into a simple recombination process catalyzed by an archaeal integrase. |
京公网安备 11010502044263号