IntroductionIn the lysogenic phase, a temperate phage can integrate its genomic DNA into a host chromosome in a site-specifi c manner. The phage genome-encoded integrase is a site-specifi c recombinase that catalyzes the recombination between two attachment sites on the phage genome (attP) and bacterial chromosome (attB), resulting in the complete integration of the phage genome (Champbell, 1962(Champbell, , 1992. Consequently, the integrated phage DNA is sandwiched between hybrid attachment sites, attL and attR. This prophage genome can be released from the bacterial chromosome by the action of an excisionase, which is an accessory phage-encoded protein that stimulates site-specifi c recombination between attL and attR sites.There are two major families of phage-encoded sitespecifi c recombinases, based on their conserved amino acid sequences. One of them is the tyrosine recom-J. Gen. Appl. Microbiol., 57, 45 57 (2011) The site-specifi c integrase of actinophage R4 belongs to the serine recombinase family. During the lysogenization process, it catalyzes site-specifi c 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 effi cient intramolecular recombination. The in vitro assay using overproduced R4 integrases as a hexahistidine (His 6 )-glutathione-S-transferase (GST)-R4 integrase fusion protein, showed that the purifi ed protein preparation retains the site-specifi c recombination activity which catalyzes the site-specifi c recombination between attP and attB in the intermolecular reaction. It also revealed that the inverted repeat within attP is essential for effi cient in vitro intermolecular recombination. In addition, a gel shift assay showed that His 6 -GST-R4 integrase bound to the 50-bp attB and 49-bp attP specifi cally. 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-specifi c recombination by the R4 integrase have been defi ned more precisely. This knowledge is useful for developing new genetic manipulation tools in the future.