PMID:18281701

Citation	Brok-Volchanskaya, VS, Kadyrov, FA, Sivogrivov, DE, Kolosov, PM, Sokolov, AS, Shlyapnikov, MG, Kryukov, VM and Granovsky, IE (2008) Phage T4 SegB protein is a homing endonuclease required for the preferred inheritance of T4 tRNA gene region occurring in co-infection with a related phage. Nucleic Acids Res. 36:2094-105
Abstract	Homing endonucleases initiate nonreciprocal transfer of DNA segments containing their own genes and the flanking sequences by cleaving the recipient DNA. Bacteriophage T4 segB gene, which is located in a cluster of tRNA genes, encodes a protein of unknown function, homologous to homing endonucleases of the GIY-YIG family. We demonstrate that SegB protein is a site-specific endonuclease, which produces mostly 3' 2-nt protruding ends at its DNA cleavage site. Analysis of SegB cleavage sites suggests that SegB recognizes a 27-bp sequence. It contains 11-bp conserved sequence, which corresponds to a conserved motif of tRNA TpsiC stem-loop, whereas the remainder of the recognition site is rather degenerate. T4-related phages T2L, RB1 and RB3 contain tRNA gene regions that are homologous to that of phage T4 but lack segB gene and several tRNA genes. In co-infections of phages T4 and T2L, segB gene is inherited with nearly 100% of efficiency. The preferred inheritance depends absolutely on the segB gene integrity and is accompanied by the loss of the T2L tRNA gene region markers. We suggest that SegB is a homing endonuclease that functions to ensure spreading of its own gene and the surrounding tRNA genes among T4-related phages.
Links	PubMed PMC2330249 Online version:10.1093/nar/gkn053
Keywords	Bacteriophage T4/enzymology; Bacteriophage T4/genetics; Base Sequence; Conserved Sequence; DNA, Viral/chemistry; DNA, Viral/metabolism; Endodeoxyribonucleases/genetics; Endodeoxyribonucleases/metabolism; Gene Conversion; Inheritance Patterns; Myoviridae/genetics; Open Reading Frames; RNA, Transfer/genetics; Substrate Specificity; T-Phages/genetics; Viral Proteins/genetics; Viral Proteins/metabolism
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
BPT4:SEGB		GO:0004520: endodeoxyribonuclease activity	ECO:0000314:		F		The T4 SegB protein is a site-specific endonuclease that recognizes a 27-bp sequence. 666 bp segB ORF was placed under the control of T7 promoter in pET-15b vector. SegB protein was overproduced in E. coli T7 lac promoter system and purified. The purified protein was incubated with a linear plasmid DNA and products were analyzed of the reaction through gel electrophoresis (Figure 3). SegB cleaved DNA by introducing double-strand breaks in reactions requiring Mg2+, Mn2+, or Ca2+ cations. With Mg2+ and Ca2+ cation, SegB cleaved the DNA at 1 point, creating 2.2 kb and 1.6 kb fragments. In the presence of Mn2+, multiple cleavage sites were present.	complete CACAO 13238
BPT4:A0A023ZV65		GO:0004519: endonuclease activity	ECO:0000247:		F		Bacteriophage T4 segB gene encodes for a protein of unknown function, but is homologous to homing endonucleases of the GIY-YIG family. By using sequence alignment with PCR, the study demonstrates that the SegB protein is a site-specific endonuclease, by analyszing the DNA cleavage site. The cleavage site analysis suggests SegB is a homing endonuclease that functions to spread its own gene among T4-related phages. In Figure 3, it is shown that Phage T4 SegB protein demonstrates endonuclease activity by cleaving DNA in the presence of Mg2+, Mn2+ or Ca2+ cations. Although the study uses a wet lab, the focus is on sequencing; the evidence is sequence alignment, instead of direct assay.	Missing: with/from CACAO 13299

References

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