The His Cys Box Family of Homing Endonucleases

Similar to members of other homing endonuclease families, the His-Cys box homing endonucleases are encoded by group I introns that exhibit mobility into intron-less alleles of their host genes. For all His-Cys box family members identified thus far, the host genes are nuclear rDNA loci from several species of protists including slime molds, fungi, algae, and amoebae. This endonuclease family is characterized by a series of conserved histidine and cysteine residues over -100 amino acid stretch of the protein (Johansen et al. 1993). The family now includes 6 enzymes that have demonstrated activity and 17 putative members from full-length open-reading frames (ORFs; Table 1). The enzymes with demonstrated activity are I-Ppol from Physarum polycephalum (Muscarella et al. 1990; Wittmayer et al. 1998), I-Dirl from Didymium iridis (Johansen et al. 1997), and a set of four highly related proteins that act as isoschizomers (I-Njal, I-NanI, I-NitI, and I-NgrI) from several Naegleria species (Elde et al. 1999,2000; Decatur et al. 2000). These enzymes recognize several variants of their respective homing sites which are -18 nucleotide (nt) pseudopalindromic sequences. In general, the His-Cys box proteins are small, on the order of 160-290 amino acids in length. It has been suggested that their size may be limited by length restrictions of their encoding mobile introns.

In addition to the full-length His-Cys box ORFs, 20 pseudogenes have also been identified in group I introns found at the same position of rDNA sequences as those encoding active endonucleases. These pseudogene sequences contain nonsense codons that interrupt the putative ORF and therefore are not likely to encode an active endonuclease. Due to their high sequence divergence, many of the His-Cys box ORFs and pseudogenes were found by locating their host group I intron in nuclear ribosomal genes and correlating the observation of a larger than normal intron with the presence of an ORF-like sequence. The large number of pseudogenes is taken as evidence that homing endonuclease genes may be unstable and therefore lost during evolution (Cho et al. 1998; Goddard and Burt 1999; Haugen et al. 1999; Foley et al. 2000; Muller et al. 2001; Bhattacharya et al. 2002; Nozaki et al. 2002)

It is interesting to note that in addition to the mobility of the group I introns encoding the homing endonucleases, there is accumulating evidence that the enzyme ORFs are also mobile (Mota and Collins 1988; Lambowitz and Belfort 1993; Loizos et al. 1994; Ogawa et al. 1997; Pellenz et al. 2002; Haugen et al. 2004). This prospect complicates the matter of the inheritance of homing introns, which center primarily on questions of vertical versus lateral transfer of the sequences. Bhattacharya and coworkers performed phylo-genetic analysis of the His-Cys box enzymes, examining their relationship to the encoding group I introns, as well as those introns' relationships to the host gene in terms of position of the ORF within the intron and position of the in-

Table 1. Members of the His-Cys box homing endonuclease family. ACT Enzyme with demonstrated activity; FL predicted full-length ORF, those marked with * contain predicted spliceosomal introns; PG pseudogene

insertion site

Intron insertion site3

No. aa

Type

Reference

I-Ppol

Physarum polycephalum

L1925

s-Pl

163

ACT

Muscarella et al. (1990)

I-NgrI

Naegleria gruberi

S516

s-P6

245

ACT

Einvik et al. (1997); Decatur et al. (2000)

I-Njal

Naegleria jamiesoni

S516

s-P6

245

ACT

Elde et al. (1999)

I-NanI

Naegleria andersoni

S516

s-P6

245

ACT

Elde et al. (2000)

I-NitI

Naegleria italica

S516

s-P6

245

ACT

Elde et al. (2000)

I-Dirl

Didymium iridis

S956

s-P2

261""

ACT

Decatur et al. (1995)

I-NaeIIP

Naegleria sp. NG874

L1926

s-Pl

148

FL

Haugen et al. (2002)

I-NmoIP

Naegleria morganensis (NG236)

L2563

s-Pl

175

FL

de Jonckheere and Brown (1998)

I-NcaIP

Naegleria carteri

S516

s-P6

245

FL

de Jonckheere and Brown (1998)

I-NclIP

Naegleria clarki

S516

s-P6

245

FL

de Jonckheere (1994)

I-NaeIP

Naegleria sp. NG872

S516

s-P6

244

FL

de Jonckheere and Brown (1998)

Naegleria sp. NG597

S516

s-P6

245

FL

de Jonckheere (1994)

Naegleria sp. NG434

S516

s-P6

245

FL

de Jonckheere (1994)

Naegleria sp. NG560

S516

s-P6

245

FL

de Jonckheere (1994)

I-PteIP

Porphyra tenera

S516

s-P2

162

FL

Haugen et al. (1999)

I-PabIP

Porphyra abbottae

S516

s-P2

FL

Muller et al. (2001)

I-EmyIP

Ericoid mycorrhizal

S943

s-P8

294""

FL

Perotto et al. (2000)

I-MteIP

Monoraphidium terrestre

S943

a-P8

276

FL

Haugen et al. (2002)

I-CpiIP

Capronia pilosella

S943

s-P8

307

FL

Haugen et al. (2004)

I-CmoIP

Coemansia mojavensis

S943

a-P8

171

FL

Tanabe et al. (2002)

I-PchIP

Pleopsidium chlorophanum

S943

a-P8

256

FL

Haugen et al. (2004)

I-SdiIP

Scytalidium dimidiatum

S943

?

290

FL

P. Haugen (pers. comm.)

I-DirIIP

Didymium iridis

S952

a-P8

192""

FL

Vader(1998)

-

Candida albicans

L1923

a-P2.1

PG

Haugen et al. (2004)

Table 1. (Continue)

insertion site

Intron insertion site3

No. aa Type

Reference

-

Candida dublineinsis

L1923

a-P2.1

PG

Haugen et al. (2004)

-

Cercomonas sp.

SI 190

P9

PG

P. Haugen (pers. comm.)

-

Ericoid mycorrhizal

SI 199

a-P9

PG

Haugen et al. (2004)

-

Nectria galligena

SI 199

a-P9

PG

Johansen and Haugen (1999)

-

Protomyces pachydermus

S1506

s-P9

PG

Haugen et al. (2004)

-

Arthrobotrys superba

S1506

s-P9

PG

Haugen et al. (2004)

-

Tilletiopsis oryzicola

S1506

a-P9

PG

Haugen et al. (2004)

-

Porphyra spiralis

S1506

a-Pl

PG

Haugen et al. (1999)

-

Bangia atropupurea

S1506

a-Pl

PG

Haugen et al. (1999)

-

Bangia fuscopurpurea

S1506

a-Pl

PG

Muller et al. (2001)

-

Porphyra tenera

S1506

a-Pl

PG

Haugen et al. (1999)

-

Porphyra sp. 2

S1506

a-Pl

PG

Haugen et al. (1999)

-

Porphyra umbilicus

S1506

a-Pl

PG

Muller et al. (2001)

-

Bangia fuscopurpurea

S516

s-P2

PG

Haugen et al. (1999)

-

Acantliamoeba sp. KA/E4

S516

s-P2

PG

Haugen et al. (2004)

Porphyra kanakaensise

S516

s-P2

PG

Muller et al. (2001)

-

Pseudohalonectria lignicola

S943

s-P8

PG

Haugen et al. (2004)

-

Cordyceps pseudomilitaris

S943

a-P8

PG

Haugen et al. (2004)

-

Beauveria bassiana

S943

s-P8

PG

Yokoyama et al. (2002)

as and a refer to ORF insertion into the sense or antisense orientation relative to the rDNA transcript.

as and a refer to ORF insertion into the sense or antisense orientation relative to the rDNA transcript.

tron in the rDNA gene (Haugen et al. 2004). They found that the Naeglaria family of enzymes, with the exceptions of I-Nmol and I-NaeII, appear to be examples of vertical inheritance. Here, highly related endonuclease ORFs situated in the same position (the P6 arm) of related group I introns are found in the same rDNA site (S516). However, examples of likely lateral transfer of endonuclease sequences independent of the encoding intron also exist. In one case, four endonuclease ORFs that group together phylogenetically (I-Ppol, I-Naell, and two Candida pseudogenes) are found at different sites in two different subclasses of group I introns. The ORFs of I-Ppol and I-NaeII are found in the sense orientation in the PI element of the IC1 subclass of group I introns, whereas the Candida pseudogenes are in the antisense orientation in the P2.1 element of the IE subclass. Interestingly, both of these introns are located in similar locations of the LSU gene (L1923 and L1926).

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