Nucleic Acids Research, 2005, Vol. 33, No. 19 6211 Figure 6. (A) Proposed pathway for transposition of a HEG via cleavage of its own genome. Occasional cleavage of the own genome by the homing endonuclease at the cleavage site gives it the opportunity to invade sites with sequence similarity at other positions in the genome, or in another genome during a mixed infection event. Single strand invasion followed by Join-cut- copy replication (38) and resolution of the Holiday junction will form a chimeric chromosome where the HEG has been joined to the new site. Short regions of initial pairing will be stabilized by DNA replication from the 30 end of the invading strand. In those cases where the homing endonuclease can cleave the alternative site the chimeric chromosome may subsequently be used as template for double strand break repair (DSBR) using the identical part (black lines) and any other short sequence similarity on the opposite side of the HEG for initiation of repair. This will result in the HEG being inserted at the new position in the genome. (B) Scheme of horizontal transfer of HEG to a cognate site within a phage population and invasion and spread to new sites within the same genome or an unrelated genome. The scheme is an adaptation of the cycle of intron gain and loss from reference (19). Heavy arrows indicate preferred transfer, and light arrows less frequent events. A novel gene was also found in phage U5 between nrdA and nrdB (in place of the mobE gene in T4). For in vitro cleavage assays the product of this gene generates a double strand cut in the downstream nrdB gene, and we name this gene hef