DOD domain
	<----> 53-106 AA			<--> 4-18 AA			<-----> 0-23 AA
EN1		EN2			EN3			EN4

The first and third EN motifs (C and E) are the DOD motifs found in the DOD homing endonucleases . The two motifs are similar to each other and probably have similar roles. Protein structure of the yeast Sce VMA intein and of a DOD type endonuclease showed the motifs to be alpha helices holding together the two halves of the protein and also forming the endonuclease's active site. The Sce VMA structure also showed the conserved basic residue in the second position of the second EN motif (motif D Perler et al. 97') to be another part of the active site. Mutating DOD motifs in the Tli pol2 (Hodges et al. 92') and Sce VMA (Gimble and Stephens 95') inteins abolished their endonuclease activity. However, the protein splicing activity of Tli pol2 was not affected by the mutation. Genetically engineered Sce VMA and Mtu recA inteins lacking the EN domain were both shown to protein splice. The EN domain is also naturally missing from various inteins. All this clearly shows that the endonuclease domain is optional and not crucial for intein splicing.

the positions of the motifs are conserved in different inteins and relative to each other. This can be seen in the inteins motif map. Intein structures show that the motifs have important functional and structural roles, forming the protein splicing and endonuclease active sites.

Pietrokovski S. (2001). Inteins- Protein Introns. http://bioinfo.weizmann.ac.il/~pietro/inteins

HO endonuclease initiates a mating-type switch in the yeast Saccharomyces cerevisiae by making a double-strand break (DSB) in a 24-bp cognate sequence in the mating-type gene MAT. Repair of the DSB is by gene conversion using one of the silent HM cassettes as a template and results in substitution of the resident MAT allele with a sequence of the opposite mating type. Ho has homology to LAGLIDADG homing endonucleases, rare-cutting enzymes that cleave long ( 14-40 bp) asymmetrical target sequences in the minor groove, leaving 4-nucleo-tide (nt) 3 cohesive ends. Ho (F-SceII) is encoded by a freestanding nuclear gene; however, other homing endonucleases are encoded by open reading frames (ORF) embedded in genetically mobile introns or within self-splicing inteins [protein introns (PI); Belfort and Roberts 1997; Jurica and Stoddard 1999; Kowalski and Derbyshire 2002]. Repair of the DSB made by the endonuclease promotes mobility of its host intron/intein into its cognate site. The survival of Ho has been attributed to its ability to promote a mating-type switch, thus allowing progeny of a single haploidspore to mate and sporulate (Gimble 2000). Eight conserved sequence motifs are found in intein-encoded LAGLIDADG endonucleases, and the primary sequence of Ho displays all the intein motifs, including thoseinvolved in protein splicing (Pietrokovski 1994; Perler 2000).

Structures have been determined using X-ray crystal lography for a number of LAGLIDADG endonucleases (Chevalier and Stoddard 2001). The Chlamydomonasreinhardtii 23S rRNA intron-encoded I-CreI has a singl ecopy of the LAGLIDADG motif and acts as a homodimer, cleaving an almost palindromic homing site (Heath et al. 1997; Jurica et al. 1998; Chevalier et al. 2001). The alpha beta beta alpha beta beta alpha fold of the I-CreI subunit is found in two copies in the monomeric endonucleases, yeast PI-SceI (Duan et al. 1997; Hu et al. 2000), I-DmoI (Silva et al. 1999), and PI-PfuI (Ichiyanagi et al. 2000). These monomeric forms are thought to have arisen by gene duplication (Lykke-Andersen et al. 1996; Silva et al. In all the structures the two LAGLIDADG alpha-helices pack against each other with a pair of catalytic aspartic acid residues at the C-terminal ends of each helix. The LAGLIDADG alpha-helix is followed by two anti-parallel beta-strands with a loop of varying length between them. The length of the beta-strands and size of the intervening loop are correlated with cognate site length, whereas individual residues in this region dictate site specificity. All LAGLIDADG enzyme structures solved to date show the central two-helix bundle followed by to date show the central two-helix bundle followed by represent the domain topology for thr family. (Anya Bakhrat, Melissa S. Jurica,1 Barry L. Stoddard† and Dina Raveh: Homology Modeling and Mutational Analysis of Ho Endonuclease of Yeast: publiced October 31, 2003)