homing endonuclease genes (23). The toxicity to E.coli, its scattered distribution in phages closely related to T4 and the strong exclusion of T2 markers around mobE in mixed infec- tions with T4 (14,19) suggest that MobE is an endonuclease with homing properties. SegH and Hef: two novel sequence specific endonucleases To test if any of these putative homing endonucleases are functional we in vitro expressed the gene products of three of the mobE genes (from T4, T6 and RB3, respectively), T4 mobC, LZ2 segH and U5 hef from PCR fragments with a T7 promoter sequence inserted before the start codon of the respective gene. All in vitro reactions yielded proteins of the expected molecular masses (Figure 2A). After degradation of RNA the in vitro translated proteins were used directly for endonuclease assays. As can be seen in the left part of Figure 2B incubation of LZ2 SegH translation extract with a PCR fragment ﬂuorescently labelled at one end and spanning the nrdD-nrdG region from T2 generated a speciﬁc double strand cleavage product while no such activity was detected with a T4 MobC translation or with a mock translation reaction where no T7 translation template was added. SegH could also cleave DNA of the same genetic region from T4 and LZ2, and from the lengths of the cleavage products generated with DNA targets ampliﬁed with different primer combinations we were able to locate the SegH cleavage site to the end of the nrdD gene for all phages (data not shown). Double strand cleavage was also shown to occur when in vitro translated U5 Hef was incubated with a ﬂuorescently labelled PCR fragment spanning the nrdA-nrdB region from T2 (Figure 2B, right part). However, in vitro translated T4 MobE or a mock translation reaction without added T7 tem- plate had no endonuclease activity. Further analysis showed that Hef could also cleave T4, T6 and RB3 DNA at the same position around 250 bp into the nrdB gene (data not shown). We further tested the T4, T6 and RB3 variants of MobE for cleavage of T2, T6 and RB3 DNA ranging from the inter- genic region upstream of nrdA to the end of the nrdB gene ( 2800 bp for T2, 3800 bp for T6 and 4000 bp for RB3) with seven different buffer conditions. No cleavage products were detected with any templates or cleavage conditions tested. The same negative results were obtained with T4 MobC on T2, T4 and LZ2 DNA spanning 1500–2000 bp including the whole nrdG gene and half of the nrdD gene (data not shown). Since several proteins with the H-N-H motif recently have been shown to nick only one strand of the DNA instead of intro- ducing double strand breaks (24,25) we also tested T4, T6 and RB3 MobE and T4 MobC for cleavage with PCR fragments that were isotopically labelled on one strand or the other but Nucleic Acids Research, 2005, Vol. 33, No. 19 6209 still without detecting any cleavage (data not shown). We conclude that MobE and MobC do not display any endonuc- lease activity in vitro with the templates and under conditions tested. Mapping of SegH and Hef cleavage sites and competition between different target sites The freestanding T4 homing endonucleases characterized so far differ from intron-encoded homing endonucleases in that their cleavage sites are located several 100 bp away from the HEG insertion site (10,13–16). As mentioned above we roughly mapped the SegH cleavage site to the end of nrdD and the Hef site to within the ﬁrst 300 bp of nrdB. By isotopic labelling of only one DNA strand at a time we were able to determine the exact cleavage sites on each strand (Figure 3A and B). Both SegH and Hef introduce double strand breaks with 2 nt 30 extensions like many characterized phage homing endonucleases (6,13,14,26). Cleavage by SegH occurs 82 and 84 nt from the end of the nrdD gene, on the coding and template strand respectively (Figure 3C). Interestingly, the SegH cleavage site coincides exactly with the position of a major shift in sequence similarity between the segH containing phages T6, RB3 and LZ2 and the T-even-like phages that do not contain segH (Figure 3C). Upstream of the cleavage site there is >95% sequence identity throughout the nrdD gene among all phages sequenced (19) while the last part of the nrdD gene, downstream of the cleav- age site in the segH containing phages, shows only about 60% identity to segH-less phages. This shift in sequence similarity ﬁts well with the co-conversion of ﬂanking markers between the endonuclease cleavage site and the inserted HEG that is generated upon homing (27–29) and suggests that segH originates from a phage whose sequence has diverged sub- stantially from the rest of the T-even-like phages. Cleavage by U5 Hef occurs 229 and 231 nt downstream of the start of nrdB, on the template and coding strands respect- ively (Figure 3D). This region of nrdB is quite well conserved at the DNA level among the T-even-like phages. Therefore it is not surprising that Hef cleaves DNA from T2, T4, T6 and RB3 as well as from U5. The position of the cleavage site in the middle of the ﬁrst U5 nrdB exon ﬁts well with our previous prediction that it is in fact the homing activity of Hef that mobilizes the U5 nrdB intron (that contains a non-functional I-TevIII HEG, cf. Figure 5A) (19). For most intron-encoded homing endonucleases, insertion of the intron disrupts the recognition site. However, for freest- anding homing endonucleases the separation of the cleavage site and the insertion site frequently results in both HEG- containing as well as HEG-less alleles being cut (13–16). Extensive sequence variation which limits the frequency of Figure 5. (A) Distribution of optional genes and gene order of flanking genes for the nrdD-nrdG, and the td-nrdA-nrdB regions in phages used as donors and recipients in the homing experiments. Genes are indicated by large boxes, introns with thin boxes and intergenic regions with black lines. Optional genes are dark grey with white text. Note that frame-shifted HEGs are indicated by short/interrupted ORF boxes. The cleavage positions of SegH and Hef are shown. (B) Frequency of screened introns and HEGs in progeny of mixed infections. Error bars show the standard error of the mean. Homing proficient genes are generally over represented in progeny from mixed infections (i.e. U5 td IVS) while non-homing genes are expected to have around 50% occurrence (i.e. U5 nrdD IVS). General exclusion by the recipient phage can reduce the effect of homing and therefore some homing proficient genes are present in less than 50% of the progeny. Such progeny were screened for recombination points between donor and recipient sequences close to the gene of interest, indicative of homing. (C) Screen for co-conversion of flanking markers around the segH gene in RB3/T2 and LZ2/T2 crosses. (D) Screen for co-conversion of flanking markers around the mobE in T6/T2 crosses. Gene order and position of restriction sites used to determine sequence specific markers are shown at the top. White boxes denote recipient alleles, black boxes denote donor alleles and dashed lines denote regions where recombination has occurred. Frequencies of the different chimerical sequences in the progeny are indicated to the left.