PURIFICATION AND CHARACTERIZATION OF THE SEGA PROTEIN OF BACTERIOPHAGE-T4, AN ENDONUCLEASE RELATED TO PROTEINS ENCODED BY GROUP-I INTRONS

Although not encoded by an intron, the bacteriophage T4 SegA protein s hares common amino acid motifs with a family of proteins found within mobile group I introns present in fungi and phage. Each of these intro n encoded proteins is thought to initiate the homing of its own intron by cleaving the intronless DNA at or near the site of insertion. Prev iously, we have found that SegA also cleaves DNA. In this report, we h ave purified the SegA protein and characterized this endonuclease acti vity extensively. SegA protein cleaved circular and linear plasmids, D NA containing unmodified cytosines, and wild-type T4 DNA containing hy droxymethylated, glucosylated cytosines. In all cases, certain sites o n the DNA were highly preferred for cleavage, but with increasing prot ein concentration or time of incubation, cleavage occurred at many sit es. SegA cleaving activity was stimulated by the presence of ATP or AT P gamma S. Sequence analysis of three highly preferred cleavage sites did not reveal a simple consensus sequence, suggesting that even among highly preferred sites, SegA tolerates many different sequences. A T4 segA amber mutant that we constructed had no phenotype, and PCR analy ses indicated that several T-even-related phages lack the segA gene. T aken together, our results show that SegA is an endonuclease with a hi erarchy of site specificity, and these results are consistent with the insertion of segA DNA into the T4 genome some time after the divergen ce of the closely related T-even phages.