PubAg

Main content area

Biochemical and mutagenic analysis of I-CreII reveals distinct but important roles for both the H-N-H and GIY-YIG motifs

Author:
Corina, Laura E., Qiu, Weihua, Desai, Ami, Herrin, David L.
Source:
Nucleic acids research 2009 v.37 no.17 pp. 5810-5821
ISSN:
0305-1048
Subject:
DNA damage, alanine, asparagine, colicins, enzymes, histidine, introns, leaves, mutagens, supercoiled DNA
Abstract:
Homing endonucleases typically contain one of four conserved catalytic motifs, and other elements that confer tight DNA binding. I-CreII, which catalyzes homing of the Cr.psbA4 intron, is unusual in containing two potential catalytic motifs, H-N-H and GIY-YIG. Previously, we showed that cleavage by I-CreII leaves ends (2-nt 3' overhangs) that are characteristic of GIY-YIG endonucleases, yet it has a relaxed metal requirement like H-N-H enzymes. Here we show that I-CreII can bind DNA without an added metal ion, and that it binds as a monomer, akin to GIY-YIG enzymes. Moreover, cleavage of supercoiled DNA, and estimates of strand-specific cleavage rates, suggest that I-CreII uses a sequential cleavage mechanism. Alanine substitution of a number of residues in the GIY-YIG motif, however, did not block cleavage activity, although DNA binding was substantially reduced in several variants. Substitution of conserved histidines in the H-N-H motif resulted in variants that did not promote DNA cleavage, but retained high-affinity DNA binding--thus identifying it as the catalytic motif. Unlike the non-specific H-N-H colicins, however; substitution of the conserved asparagine substantially reduced DNA binding (though not the ability to promote cleavage). These results indicate that, in I-CreII, two catalytic motifs have evolved to play important roles in specific DNA binding. The data also indicate that only the H-N-H motif has retained catalytic ability.
Agid:
2205478