Nucleic Acids Research, 2005, Vol. 33, No. 19 6203–6213 doi:10.1093/nar/gki932 SegH and Hef: two novel homing endonucleases whose genes replace the mobC and mobE genes in several T4-related phages Linus Sandegren, David Nord and Britt-Marie Sjo¨ berg* Department of Molecular Biology and Functional Genomics, Stockholm University, Svante Arrhenius va¨ g 16, F3. SE-10691 Stockholm, Sweden Received August 16, 2005; Revised and Accepted October 11, 2005 ABSTRACT T4 contains two groups of genes with similarity to homing endonucleases, the seg-genes (similarity to endonucleases encoded by group I introns) contain- ing GIY-YIG motifs and the mob-genes (similarity to mobile endonucleases) containing H-N-H motifs. The four seg-genes characterized to date encode homing endonucleases with cleavage sites close to their respective gene loci while none of the mob- genes have been shown to cleave DNA. Of 18 phages screened, only T4 was found to have mobC while mobE genes were found in five additional phages. Interestingly, three phages encoded a seg-like gene (hereby called segH ) with a GIY-YIG motif in place of mobC. An additional phage has an unrelated gene called hef (homing endonuclease-like function) in place of the mobE gene. The gene products of both novel genes displayed homing endonuclease activity with cleavage site specificity close to their respective genes. In contrast to intron encoded homing endo- nucleases, both SegH and Hef can cleave their own DNA as well as DNA from phages without the genes. Both segH and mobE (and most likely hef ) can home between phages in mixed infections. We discuss why it might be a selective advantage for phage free- standing homing endonucleases to cleave both HEG-containing and HEG-less genomes. INTRODUCTION Homing endonucleases are mobile genetic elements that can promote their own horizontal transfer by a process called DDBJ/EMBL/GenBank accession nos+ homing (1). This occurs if the homing endonuclease encoun- ters a genome that does not contain the endonuclease gene (HEG) at the cognate site. The HEG-less genome is cleaved by the endonuclease and repaired by the cell machinery via dou- ble strand break repair using the HEG-containing allele as template thereby inserting the HEG at the cognate site in the cleaved genome [reviewed in (2)]. HEGs are found both intergenically (freestanding) and frequently inserted within intervening sequences (group I, or group II introns, or inteins) where they confer mobility both to themselves and the surrounding splicing element via homing (3–8). Inser- tion of a HEG into an intervening sequence is thought to be beneﬁcial for the HEG because it increases the number of potential homing targets since the splicing capability of the intron allows an intron-associated HEG to be inserted also into coding sequences without disrupting their function (5,6). Four families of homing endonucleases have been described based on conserved sequence motifs within them, LAGLIDADG, GIY-YIG, H-N-H and His-Cys box [reviewed in (9)]. Bacteriophage T4 contains three intron-associated HEGs and at least 12 freestanding genes with similarity to intron- encoded homing endonucleases (10,11). Only two of the intron-associated HEGs encode active homing endonucleases (I-TevI and I-TevII) while the third (I-TevIII in the nrdB intron) is rendered inactive due to a large internal deletion (12). Seven of the freestanding HEGs have similarities to the GIY-YIG family of intron-encoded homing endonucleases (10,13,14) and the remaining ﬁve have similarity to the H- N-H family of homing endonucleases (11). The GIY-YIG genes are called segA-G (for similarity to endonucleases encoded by group I introns) and four of them have been shown experimentally to encode freestanding homing endo- nucleases (segA (10,15), segE (16), segF (formerly gene 69) (13) and segG (formerly gene 32.1) (14). The H-N-H genes are called mobA-E (for similarity to mobile endonucleases), but none of the mob-genes have so far been shown to possess *To whom correspondence should be addressed. Tel: +46 8 164150; Fax: +46 8 166488; Email: britt-marie.sjoberg@molbio.su.se Present address: Linus Sandegren, Department of Medical Biochemistry and Microbiology, Uppsala, University, Sweden +AY262125–AY262139, AY262142, AY262144, AY262145 and DQ178119-DQ178121 The Author 2005. Published by Oxford University Press. 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