NORMAL GENOMIC

• Edgell DR, Shub DA
• Related homing endonucleases I-BmoI and I-TevI use different strategies to cleave homologous recognition sites.
• Proc Natl Acad Sci U S A. 2001; 98: 7898-903
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• A typical homing endonuclease initiates mobility of its group I intron by recognizing DNA both upstream and downstream of the intron insertion site of intronless alleles, preventing the endonuclease from binding and cleaving its own intron-containing allele. Here, we describe a GIY-YIG family homing endonuclease, I-BmoI, that possesses an unusual recognition sequence, encompassing 1 base pair upstream but 38 base pairs downstream of the intron insertion site. I-BmoI binds intron-containing and intronless substrates with equal affinity but can nevertheless discriminate between the two for cleavage. I-BmoI is encoded by a group I intron that interrupts the thymidylate synthase (TS) gene (thyA) of Bacillus mojavensis s87-18. This intron resembles one inserted 21 nucleotides further downstream in a homologous TS gene (td) of Escherichia coli phage T4. I-TevI, the T4 td intron-encoded GIY-YIG endonuclease, is very similar to I-BmoI, but each endonuclease gene is inserted within a different position of its respective intron. Remarkably, I-TevI and I-BmoI bind a homologous stretch of TS-encoding DNA and cleave their intronless substrates in very similar positions. Our results suggest that each endonuclease has independently evolved the ability to distinguish intron-containing from intronless alleles while maintaining the same conserved recognition sequence centered on DNA-encoding active site residues of TS.

• Lucas P, Otis C, Mercier JP, Turmel M, Lemieux C
• Rapid evolution of the DNA-binding site in LAGLIDADG homing endonucleases.
• Nucleic Acids Res. 2001; 29: 960-9
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• Sequence analysis of chloroplast and mitochondrial large subunit rRNA genes from over 75 green algae disclosed 28 new group I intron-encoded proteins carrying a single LAGLIDADG motif. These putative homing endonucleases form four subfamilies of homologous enzymes, with the members of each subfamily being encoded by introns sharing the same insertion site. We showed that four divergent endonucleases from the I-CreI subfamily cleave the same DNA substrates. Mapping of the 66 amino acids that are conserved among the members of this subfamily on the 3-dimensional structure of I-CreI bound to its recognition sequence revealed that these residues participate in protein folding, homodimerization, DNA recognition and catalysis. Surprisingly, only seven of the 21 I-CreI amino acids interacting with DNA are conserved, suggesting that I-CreI and its homologs use different subsets of residues to recognize the same DNA sequence. Our sequence comparison of all 45 single-LAGLIDADG proteins identified so far suggests that these proteins share related structures and that there is a weak pressure in each subfamily to maintain identical protein-DNA contacts. The high sequence variability we observed in the DNA-binding site of homologous LAGLIDADG endonucleases provides insight into how these proteins evolve new DNA specificity.

• Lazarevic V
• Ribonucleotide reductase genes of Bacillus prophages: a refuge to introns and intein coding sequences.
• Nucleic Acids Res. 2001; 29: 3212-8
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• The ribonucleotide reductase gene tandem bnrdE/bnrdF in SPbeta-related prophages of different Bacillus spp. isolates presents different configurations of intervening sequences, comprising one to three of six non-homologous splicing elements. Insertion sites of group I introns and intein DNA are clustered in three relatively short segments encoding functionally important domains of the ribonucleotide reductase. Comparison of the bnrdE homologs reveals mutual exclusion of a group I intron and an intein coding sequence flanking the codon that specifies a conserved cysteine. In vivo splicing was demonstrated for all introns. However, for two of them a part of the mRNA precursor molecules remains unspliced. Intergenic bnrdE-bnrdF regions are unexpectedly long, comprising between 238 and 541 nt. The longest encodes a putative polypeptide related to HNH homing endonucleases.

• Bujnicki JM, Rychlewski L
• Unusual evolutionary history of the tRNA splicing endonuclease EndA: relationship to the LAGLIDADG and PD-(D/E)XK deoxyribonucleases.
• Protein Sci. 2001; 10: 656-60
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• The tRNA splicing endoribonuclease EndA from Methanococcus jannaschii is a homotetramer formed via heterologous interaction between the two pairs of homodimers. Each monomer consists of two alpha/beta domains, the N-terminal domain (NTD) and the C-terminal domain (CTD) containing the RNase A-like active site. Comparison of the EndA coordinates with the publicly available protein structure database revealed the similarity of both domains to site-specific deoxyribonucleases: the NTD to the LAGLIDADG family and the CTD to the PD-(D/E)XK family. Superposition of the NTD on the catalytic domain of LAGLIDADG homing endonucleases allowed a suggestion to be made about which amino acid residues of the tRNA splicing nuclease might participate in formation of a presumptive cryptic deoxyribonuclease active site. On the other hand, the CTD and PD-(D/E)XK endonucleases, represented by restriction enzymes and a phage lambda exonuclease, were shown to share extensive similarities of the structural framework, to which entirely different active sites might be attached in two alternative locations. These findings suggest that EndA evolved from a fusion protein with at least two distinct endonuclease activities: the ribonuclease, which made it an essential "antitoxin" for the cells whose RNA genes were interrupted by introns, and the deoxyribonuclease, which provided the means for homing-like mobility. The residues of the noncatalytic CTDs from the positions corresponding to the catalytic side chains in PD-(D/E)XK deoxyribonucleases map to the surface at the opposite side to the tRNA binding site, for which no function has been implicated. Many restriction enzymes from the PD-(D/E)XK superfamily might have the potential to maintain an additional active or binding site at the face opposite the deoxyribonuclease active site, a property that can be utilized in protein engineering.

• Kozlov NN
• Analysis of a set of overlapping genes.
• Dokl Biochem. 2000; 373: 119-22

• Elde M, Haugen P, Willassen NP, Johansen S
• I-NjaI, a nuclear intron-encoded homing endonuclease from Naegleria, generates a pentanucleotide 3' cleavage-overhang within a 19 base-pair partially symmetric DNA recognition site.
• Eur J Biochem. 1999; 259: 281-8
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• Different species of the amoebo-flagellate Naegleria harbor optional group I introns in the nuclear ribosomal DNA that contain open reading frames. Intron proteins from Naegleria jamiesoni, Naegleria andersoni, and Naegleria italica (named I-NjaI, I-NanI and I-NitI, respectively) were expressed in Escherichia coli and found to be isoschizomeric homing endonucleases that specifically recognize and cleave intron-lacking homologous alleles of ribosomal DNA. The I-NjaI endonuclease was affinity purified, characterized in more detail, and found to generate five-nucleotide 3' staggered ends at the intron insertion site which differs from the ends generated by all other known homing endonucleases. The recognition site was delimited and found to cover an approximately 19 base-pair partially symmetric sequence spanning both the cleavage site and the intron insertion site. The palindromic feature was supported by mutational analysis of the target DNA. All single-site substitutions within the recognition sequence were cleaved by the purified I-NjaI endonuclease, but at different efficiencies. The center of symmetry and cleavage was found to be completely degenerate in specificity, which resembles that of the subclass IIW bacterial restriction enzymes.

• Mackay JP, Crossley M
• Zinc fingers are sticking together.
• Trends Biochem Sci. 1998; 23: 1-4

• Pietrokovski S
• Modular organization of inteins and C-terminal autocatalytic domains.
• Protein Sci. 1998; 7: 64-71
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• Analysis of the conserved sequence features of inteins (protein "introns") reveals that they are composed of three distinct modular domains. The N-terminal (N) and C-terminal (C) domains are predicted to perform different parts of the autocatalytic protein splicing reaction. An optional endonuclease domain (EN) is shown to correspond to different types of homing endonucleases in different inteins. The N domain contains motifs predicted to catalyze the first steps of protein splicing, leading to the cleavage of the intein N terminus from its protein host. Intein N domain motifs are also found in C-terminal autocatalytic domains (CADs) present in hedgehog and other protein families. Specific residues in the N domain of intein and CADs are proposed to form a charge relay system involved in cleaving their N-termini. The intein C domain is apparently unique to inteins and contains motifs that catalyze the final protein splicing steps: ligation of the intein flanks and cleavage of its C terminus to release the free intein and spliced host protein. All intein EN domains known thus far have dodecapeptide (DOD, LAGLI-DADG) type homing endonuclease motifs. This work identifies an EN domain with an HNH homing-endonuclease motif and two new small inteins with no EN domains. One of these small inteins might be inactive or a "pseudo intein." The results suggest a modular architecture for inteins, clarify their origin and relationship to other protein families, and extend recent experimental findings on the functional roles of intein N, C, and EN motifs.

• Van Die I, Bakker H, Van den Eijnden DH
• Identification of conserved amino acid motifs in members of the beta1-->4-galactosyltransferase gene family.
• Glycobiology. 1997; 7: 0-0

• Dalgaard JZ, Moser MJ, Hughey R, Mian IS
• Statistical modeling, phylogenetic analysis and structure prediction of a protein splicing domain common to inteins and hedgehog proteins.
• J Comput Biol. 1997; 4: 193-214
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• Inteins, introns spliced at the protein level, and the hedgehog family of proteins involved in eucaryotic development both undergo autocatalytic proteolysis. Here, a specific and sensitive hidden Markov model (HMM) of protein splicing domain shared by inteins and the hedgehog proteins has been trained and employed for further analysis. The HMM characterizes the common features of this domain including the position where a site-specific DNA endonuclease domain is inserted in the majority of the inteins. The HMM was used to identify several new putative inteins, such as that in the Methanococcus jannaschii klbA protein, and to generate a multiple sequence alignment of sequences possessing this domain. Phylogenetic analysis suggests that hedgehog proteins evolved from inteins. Secondary and tertiary structure predictions suggest that the domain has a structure similar to a beta-sandwich. Similarities between the serine protease cleavage mechanism and the protein splicing reaction mechanism are discussed. Examination of the locations of inteins indicates that they are not inserted randomly in an extein, but are often inserted at functionally important positions in the host proteins. A specific and sensitive HMM for a domain present in klbA proteins identified several additional bacterial and archaeal family members, and analysis of the site of insertion of the intein suggests residues that may be functionally important. This domain may play a role in formation of surface-associated protein complexes.

• Garinot-Schneider C, Pommer AJ, Moore GR, Kleanthous C, James R
• Identification of putative active-site residues in the DNase domain of colicin E9 by random mutagenesis.
• J Mol Biol. 1996; 260: 731-42
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• We have used random mutagenesis to identify putative active-site residues in the C-terminal cytotoxic endonuclease domain of the bacterial toxin colicin E9. Six single-site mutations in the DNase domain were isolated which destroyed the toxic action of the colicin. DNA sequencing identified the mutations as Gly460Asp, Arg544Gly, Glu548Gly, Thr571Ile, His575Tyr and His579Tyr. All six wild-type residues are highly conserved in the DNase domains of both the E group colicins and the closely related pyocins. Site-directed mutagenesis was then used to substitute the wild-type amino acid residue at each of these positions for an alanine residue in order to distinguish important from unimportant sites. Two of the six alanine-mutant colicins (Gly460Ala and His579Ala) exhibited significant in vivo activity, unlike the original mutation of these residues, and were therefore not characterised further. The Thr571Ala mutant colicin, although not inactive, was significantly less active than the control. The other three alanine mutants (Arg544Ala, Glu548Ala and His575Ala remained completely inactive in the in vivo tests. Each 15 kDa alanine-mutant DNase domain was overexpressed and purified using a tandem-expression strategy which relies on the enzyme being able to bind to the natural inhibitor, Im9. Tryptophan emission spectra of the alanine mutants showed significant alterations in the emission maxima of all but the His575Ala mutant, suggesting changes in the tertiary structure of these mutant proteins. Activity measurements, using the spectrophotometric Kunitz assay, indicated that the Thr571Ala mutant was partially active as an endonuclease but the remaining alanine mutants were all completely inactive. All four mutant proteins, however, retained their ability to bind DNA in a gel shift assay, suggesting the mutations affect catalytic rather than substrate-binding residues. Searching the sequence databases for possible homology to other DNA-binding proteins revealed a significant match between residues 464 to 487 of the E9 DNase domain and helix IV of the POU domain of eukaryotic transcription factors.

• Barzilay G, Hickson ID
• Structure and function of apurinic/apyrimidinic endonucleases.
• Bioessays. 1995; 17: 713-9
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• The DNA of all species is constantly under threat from both endogenous and exogenous factors, which damage its chemical structure. Probably the most common lesion that arises in cellular DNA is the loss of a base to generate an abasic site, which is usually referred to as an apurinic or apyrimidinic (AP) site. Since these lesions are potentially both cytotoxic and mutagenic, cells of all organisms express dedicated repair enzymes, termed AP endonucleases, to counteract their damaging effects. Indeed, many organisms consider it necessary to express two or more of these lesion-specific endonucleases, underscoring the requirement that exists to remove AP sites for the maintenance of genome integrity and cell viability. Most AP endonucleases are very versatile enzymes, capable of performing numerous additional repair roles. In this article, we review the AP endonuclease class of repair enzymes, with emphasis on the evolutionary conservation of structural features, not only between prokaryotic and eukaryotic homologues, but also between these enzymes and the RNase H domain of one class of reverse transcriptase.

• Malone T, Blumenthal RM, Cheng X
• Structure-guided analysis reveals nine sequence motifs conserved among DNA amino-methyltransferases, and suggests a catalytic mechanism for these enzymes.
• J Mol Biol. 1995; 253: 618-32
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• Previous X-ray crystallographic studies have revealed that the catalytic domain of a DNA methyltransferase (Mtase) generating C5-methylcytosine bears a striking structural similarity to that of a Mtase generating N6-methyladenine. Guided by this common structure, we performed a multiple sequence alignment of 42 amino-Mtases (N6-adenine and N4-cytosine). This comparison revealed nine conserved motifs, corresponding to the motifs I to VIII and X previously defined in C5-cytosine Mtases. The amino and C5-cytosine Mtases thus appear to be more closely related than has been appreciated. The amino Mtases could be divided into three groups, based on the sequential order of motifs, and this variation in order may explain why only two motifs were previously recognized in the amino Mtases. The Mtases grouped in this way show several other group-specific properties, including differences in amino acid sequence, molecular mass and DNA sequence specificity. Surprisingly, the N4-cytosine and N6-adenine Mtases do not form separate groups. These results have implications for the catalytic mechanisms, evolution and diversification of this family of enzymes. Furthermore, a comparative analysis of the S-adenosyl-L-methionine and adenine/cytosine binding pockets suggests that, structurally and functionally, they are remarkably similar to one another.

• Knoop V, Brennicke A
• Evidence for a group II intron in Escherichia coli inserted into a highly conserved reading frame associated with mobile DNA sequences.
• Nucleic Acids Res. 1994; 22: 1167-71
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• The distribution of group II introns in the living world is an important aspect of the hypothesis which postulates their evolutionary relation to the nuclear spliceosome. As an alternative to the restricted experimental approaches towards their identification we devised a strategy to recognize group II introns in sequence data. By this approach we identified a locus on a plasmid in the bacterium Escherichia coli. Modelling of the derived RNA secondary structure reveals the presence of perfectly conserved domains V and VI as typical features of group II introns. An intron internal reading frame upstream of domain V is homologous to group II intron encoded maturases. A reading frame downstream of the predicted 3'-splice site is highly similar to a small polypeptide encoded in the central part of the Agrobacterium tumefaciens T-DNA. With the TBLASTN algorithm a set of plasmid-borne insertion sequences in Agrobacteria and Rhizobia and surprisingly also in a Yersinia pseudotuberculosis strain was identified which contain this highly conserved reading frame.

• Dalgaard JZ, Garrett RA, Belfort M
• A site-specific endonuclease encoded by a typical archaeal intron.
• Proc Natl Acad Sci U S A. 1993; 90: 5414-7
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• The protein encoded by the archaeal intron in the 23S rRNA gene of the hyperthermophile Desulfurococcus mobilis is a double-strand DNase that, like group I intron homing endonucleases, is capable of cleaving an intronless allele of the gene. This enzyme, I-Dmo I, is unusual among the intron endonucleases in that it is thermostable and is expressed only from linear and cyclized intron species and not from the precursor RNA. However, in analogy to its eukaryotic counterparts, but unlike the bacteriophage enzymes, I-Dmo I makes a staggered double-strand cut that generates 4-nt 3' extensions. Additionally, although the archaeal and group I introns have entirely different structural properties and splicing pathways, I-Dmo I shares sequence similarity, in the form of the LAGLI-DADG motif, with group I intron endonucleases of eukaryotes. These observations support the independent evolutionary origin of endonucleases and intron core elements and are consistent with the invasive potential of endonuclease genes.

• Mohr G, Perlman PS, Lambowitz AM
• Evolutionary relationships among group II intron-encoded proteins and identification of a conserved domain that may be related to maturase function.
• Nucleic Acids Res. 1993; 21: 4991-7
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• Many group II introns encode reverse transcriptase-like proteins that potentially function in intron mobility and RNA splicing. We compared 34 intron-encoded open reading frames and four related open reading frames that are not encoded in introns. Many of these open reading frames have a reverse transcriptase-like domain, followed by an additional conserved domain X, and a Zn(2+)-finger-like region. Some open reading frames have lost conserved sequence blocks or key amino acids characteristic of functional reverse transcriptases, and some lack the Zn(2+)-finger-like region. The open reading frames encoded by the chloroplast tRNA(Lys) genes and the related Epifagus virginiana matK open reading frame lack a Zn(2+)-finger-like region and have only remnants of a reverse transcriptase-like domain, but retain a readily identifiable domain X. Several findings lead us to speculate that domain X may function in binding of the intron RNA during reverse transcription and RNA splicing. Overall, our findings are consistent with the hypothesis that all of the known group II intron open reading frames evolved from an ancestral open reading frame, which contained reverse transcriptase, X, and Zn(2+)-finger-like domains, and that the reverse transcriptase and Zn(2+)-finger-like domains were lost in some cases. The retention of domain X in most proteins may reflect an essential function in RNA splicing, which is independent of the reverse transcriptase activity of these proteins.

• Bao Y, Herrin DL
• Nucleotide sequence and secondary structure of the chloroplast group I intron Cr.psbA-2: novel features of this self-splicing ribozyme.
• Nucleic Acids Res. 1993; 21: 1667-1667

• Freemont PS
• The RING finger. A novel protein sequence motif related to the zinc finger.
• Ann N Y Acad Sci. 1993; 684: 174-92

• Shub DA et al.
• Structural conservation among three homologous introns of bacteriophage T4 and the group I introns of eukaryotes.
• Proc Natl Acad Sci U S A. 1988; 85: 1151-5
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• Three group I introns of bacteriophage T4 have been compared with respect to their sequence and structural properties. The introns include the td intervening sequence, as well as the two newly described introns in the nrdB and sunY genes of T4. The T4 introns are very closely related, containing phylogenetically conserved sequence elements that allow them to be folded into a core structure that is characteristic of eukaryotic group IA introns. Similarities extend outward to the exon sequences surrounding the three introns. All three introns contain open reading frames (ORFs). Although the intron ORFs are not homologous and occur at different positions, all three ORFs are looped-out of the structure models, with only the 3' ends of each of the ORFs extending into the secondary structure. This arrangement invites interesting speculations on the regulation of splicing by translation. The high degree of similarity between the T4 introns and the eukaryotic group I introns must reflect a common ancestry, resulting either from vertical acquisition of a primordial RNA element or from horizontal transfer.