6212 Nucleic Acids Research, 2005, Vol. 33, No. 19 (for homing endonuclease-like function). Even though hef encodes a domain related to a diverse group of endonucleases it does not show any sequence similarity to the other known HEG families. In line with the nomenclature proposed for homing endonucleases (9,32) we suggest that the endo- nuclease products of segH and hef are called F-TevVIII and F-TevIX, respectively. Our results show that segH from LZ2 and RB3 can induce homing in mixed infections with T2 as recipient phage and that T6 mobE in mixed infections with T2 also promotes its own propagation via recombination although we have still not been able to demonstrate mobE cleavage in vitro. Since T-even phages use hydroxymethylcytosine instead of cytosine and also glucosylate the HMC residues in vivo, with the glucosyla- tion varying in pattern and amount between phages (33), it is not unlikely that our in vitro experiments with unmodiﬁed DNA do not produce the conditions needed for recognition and cleavage by these enzymes. Both SegH and Hef produce double strand cuts generating 2 nt 30 extensions and, at least in vitro, they cleave both HEG- containing and HEG-less alleles as seen in previously tested T4 Seg-endonucleases (13–16). This is in sharp contrast to the majority of intron-encoded homing endonucleases that do not cleave their own intron-containing alleles. Cleavage of the HEG-containing allele is likely to reduce the efﬁciency of homing. Why then do so many of the freestanding homing endonucleases cleave their own DNA? We propose that modest cleavage of the own genome does not reduce phage viability and that it may increase the frequency of trans- position of the HEG to new sites within the same genome or between genomes (Figure 6). Frequent transposition to new sites followed by new spread between phages via homing is vital for the survival of a HEG in a population (19,34,35). T4 utilizes its very efﬁcient recombination machinery to ensure replication of the ends of its linear chromosome and at the same time increases the number of replication forks on the phage DNA (36,37). Single stranded 30 ends of T4 chromosomes are highly recombinogenic and invade cognate sites on sister chromosomes followed by replication with the invaded DNA as template [for mechanism see (38)]. We pro- pose that it is beneﬁcial for the spreading of a homing endo- nuclease that the DNA is cut near the HEG region since this will generate recombinogenic DNA ends close to the HEG that can invade a second genome-copy. Invasion will in the major- ity of cases, due to sequence identity, occur at a cognate site in an uncleaved genome-copy resulting in increased genome replication from that position and will therefore not have a negative effect on phage viability. However, in the event of invasion at a secondary site within the same genome or the genome of a second infecting phage, strand invasion will lead to relocation of sequences from the cleavage site and onwards including the HEG (Figure 6A). This can result in genome shufﬂing within a single genome or in mixing of genomes between phages infecting the same bacterial cell (39,40). Due to the requirement of sequence similarity for strand invasion, such sites may be secondary low-level cleavage sites for the homing endonuclease. After transposition to the new site, HEGs that can cleave HEG-less alleles at the new site will be able to initiate homing to that site and will spread in the population (Figure 6B). However, extensive cleavage of the HEG-containing genome is likely to compromise the efﬁciency of homing and therefore mutations (either in the recognition site of the HEG-containing genome or in the HEG itself) that lower self-cleavage will produce HEGs with a higher homing frequency that will promote their own super- Mendelian inheritance in the phage population. In agreement with this, many of the characterized T4 seg-genes have relaxed cleavage site speciﬁcity with several alternative cleavage sites in other parts of the genome and most of them also cleave their own DNA although they have a preference for cleaving HEG-less alleles (13–16). In addition variations in in vivo DNA modiﬁcation patterns between phages may alter the cleavage speciﬁcity further. Our results show that SegH from LZ2 has a preference for site-speciﬁc cleavage of T2 and T4 over LZ2 in vitro. T2 and T4 both lack the segH but T4 has the mobC gene instead at the equivalent site. The sequence variation surrounding the SegH cleavage site is less between T2 and T4 but quite high downstream of the cleavage site between LZ2 on the one hand and T2 and T4 on the other. This high variation downstream of the site, differentiating the segH-containing phages LZ2, RB3 and T6 from T2 and T4 may contribute to the difference in target preference. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. ACKNOWLEDGEMENTS We thank Marcus Boman for help with the mobC screen, Kajsa Eriksson for help with the purification of SegH, and Patrick Young for helpful discussion and constructive criticism on the manuscript. This work was supported by the Swedish Science Research Council. Funding to pay the Open Access publication charges for this article was provided by the Swedish Science Research Council. Conflict of interest statement. None declared. REFERENCES 1. Dujon,B. (1989) Group I introns as mobile genetic elements: facts and mechanistic speculations—a review. 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