Relationship of HO to VDE

The HO protein has strong sequence similarity to inteins, which are unusual selfish genetic elements found primarily in bacteria (see Perler, this Vol.). Only two nuclear genes of eukaryotes are known to contain inteins (Perler 2002), one of which is the vacuolar H+-ATPase gene VMA1 of S. cerevisiae which contains the VDE intein (Gimble and Thorner 1992). HO has higher sequence similarity to VDE than to any other intein and clusters with it in a phyloge-netic tree (Dalgaard et al. 1997; Gogarten et al. 2002). This fact, together with their co-occurrence in S. cerevisiae despite the rarity of inteins in eukaryotes, suggests that HO shares a relatively recent common ancestor with VDE and is a sort of renegade intein. Although the VMA1 ATPase gene is a highly conserved gene with homologues in all eukaryotes, eubacteria and archaea, the distribution of the VDE intein is limited to a few hemiascomycete species closely related to S. cerevisiae. Among these species, VDE has a patchy phyl-ogenetic distribution (e.g., it is present in Candida tropicalis but not C. albicans) and there is evidence that it has been horizontally transferred among yeasts (Koufopanou et al. 2002; Okuda et al. 2003).

Now that the sequences of several HO and VDE proteins are known from different species it is possible to examine patterns of domain conservation, within HO and VDE separately, using the crystal structure of the S. cerevisiae VDE protein (also called Pl-Scel) to help interpretation (Moure et al. 2002; Bakhrat et al. 2004). Koufopanou et al. (2002) pointed out that mobile inteins are under continual selection for efficient protein splicing to maintain expression of the host gene, but their homing endonuclease activity is only "tested" by natural selection if there are inteinless alleles available for colonization. Thus, once an intein-containing allele has reached a 100% frequency in a population its endonuclease activity can decay. It is still necessary to retain an open reading frame in the endonuclease region of the gene so that the downstream parts of the protein-splicing domain and the host gene are translated, but there will be no selection to preserve amino acid residues needed for the nuclease activity. Indeed, only three of 13 VDE proteins from different yeast species tested by Posey et al. (2004) were active endonucleases. When HO and VDE proteins are compared among a uniform set of four yeast species it is apparent that the endonuclease domain of HO is much better conserved than that of VDE (Fig. 3). However, it should be noted that because VDE can be horizontally transferred among species, it is possible that the amount of evolutionary time involved in these comparisons is not equal for the two proteins. Of the VDE proteins in this alignment, only the S. cerevisiae one has been shown to have an active endonuclease; the Z. rouxii VDE has no nuclease activity (Posey et al. 2004), and the other two have not been tested.

HO also shows strong conservation of the N-terminal part of the protein-splicing domain, even though HO is a free-standing gene, and there is no host gene whose product needs to be spliced. Furthermore, deletion of the N-ter-minal 112 residues of HO, which correspond to the protein-splicing domain, results in loss of endonuclease activity (Bakhrat et al. 2004). One possible explanation for this conservation is that the protein-splicing domain (properly called domain I) of HO may contain some residues that bind to DNA, similar to the DRR (DNA recognition region) residues in domain I of VDE (Moure et al. 2002). However, the DRR residues are poorly conserved among VDE sequences, let alone HO sequences, so it is impossible to say whether DNA recognition is the sole reason for the conservation of the protein-splicing domain in HO. It is striking that this domain of VDE, which supports protein splicing, has tolerated more insertions and deletions than the equivalent region of HO, which does not (Fig. 3). The C-terminus of HO contains a putative zinc finger domain (Russell et al. 1986), which is an extension relative to VDE. Analysis of the S. cerevisiae sequence alone suggested that the domain has three fingers, each with two Cys/Cys or Cys/His pairs (Russell et al. 1986; Bakhrat et al. 2004). However, the last pair (H-X2-C at positions 574-577 of the S. cerevisiae protein) is not conserved in other species, and the conserved Cys residues in the first pair have a C-X3-C rather than the usual C-X2-C spacing, making it unlikely that this pair is actually part of a zinc finger (Fig. 3). Thus the original proposals for the structure of the zinc finger in HO may not be correct.

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in the late mitotic G1 phase of haploids. The complex way in which HO activity is regulated is reasonably well understood (Cosma 2004), whereas how VDE activity is restricted to meiosis - even though the protein is expressed at high levels in mitotically grown haploid or diploid cells - is unknown (Gim-ble and Thorner 1992).

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