In vivo and in vitro characterization of site-specific recombination of actinophage R4 integrase

The Journal of General and Applied Microbiology

Makoto Shirai²  Yang Yan-Zhuo²  Tomoyasu Nishizawa²  Munehiko Asayama²  Hideo Takahashi¹  Takamasa Miura²  Yayoi Hosaka² 

[1] Department of Applied Biological Science, College of Bioresource Sciences, Nihon University;Laboratory of Molecular Genetics, College of Agriculture

关键词: integrase;    R4 phage;    serine recombinase;    site-specific recombination;   

DOI  :  10.2323/jgam.57.45

学科分类：微生物学和免疫学

来源: Applied Microbiology, Molecular and Cellulrar Biosciences Research Foundation

【 摘 要 】

The site-specific integrase of actinophage R4 belongs to the serine recombinase family. During the lysogenization process, it catalyzes site-specific recombination between the phage genome and the chromosome of Streptomyces parvulus 2297. An in vivo assay using Escherichia coli cells revealed that the minimum lengths of the recombination sites attB and attP are 50-bp and 49-bp, respectively, for efficient intramolecular recombination. The in vitro assay using overproduced R4 integrases as a hexahistidine (His6)-glutathione-S-transferase (GST)-R4 integrase fusion protein, showed that the purified protein preparation retains the site-specific recombination activity which catalyzes the site-specific recombination between attP and attB in the intermolecular reaction. It also revealed that the inverted repeat within attP is essential for efficient in vitro intermolecular recombination. In addition, a gel shift assay showed that His6-GST-R4 integrase bound to the 50-bp attB and 49-bp attP specifically. Moreover, based on a detailed comparison analysis of amino acid sequences of serine integrases, we found the DNA binding region that is conserved in the serine recombinase containing the large C-terminal domain. Based on the results presented on this report, attachment sites needed in vitro and in vivo for site-specific recombination by the R4 integrase have been defined more precisely. This knowledge is useful for developing new genetic manipulation tools in the future.

【 授权许可】

Unknown