Homology modeling and mutational analysis of Ho endonuclease of yeast.
Ho endonuclease is a LAGLIDADG homing endonuclease that initiates mating-type interconversion in yeast. Ho is encoded by a free-standing gene but shows 50% primary sequence similarity to the intein (protein-intron encoded) PI-SceI. Ho is unique among LAGLIDADG endonucleases in having a 120-residue C-terminal putative zinc finger domain. The crystal structure of PI-SceI revealed a bipartite enzyme with a protein-splicing domain (Hint) and intervening endonuclease domain. We made a homology model for Ho on the basis of the PI-SceI structure and performed mutational analysis of putative critical residues, using a mating-type switch as a bioassay for activity and GFP-fusion proteins to detect nuclear localization. We found that residues of the N-terminal sequence of the Hint domain are important for Ho activity, in particular the DNA recognition region. C-terminal residues of the Hint domain are dispensable for Ho activity; however, the C-terminal putative zinc finger domain is essential. Mutational analysis indicated that residues in Ho that are conserved relative to catalytic, active-site residues in PI-SceI and other related homing endonucleases are essential for Ho activity. Our results indicate that in addition to the conserved catalytic residues, Hint domain residues and the zinc finger domain have evolved a critical role in Ho activity.
Bakhrat
Anya
A
Department of Life Sciences, Ben Gurion University of the Negev, Beersheva, 84105 Israel.
Jurica
Melissa S
MS
Stoddard
Barry L
BL
Raveh
Dina
D
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D004252:DNA Mutational Analysis; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics*; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D006180:Proton-Translocating ATPases / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D016415:Sequence Alignment; D017385:Sequence Homology; D016335:Zinc Fingers
D029701:Saccharomyces cerevisiae Proteins; C530845:HO protein, S cerevisiae; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific; D006180:Proton-Translocating ATPases; C081687:VMA1 protein, S cerevisiae