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PubMed2001

Expression and processing of proteins encoded by the Saccharomyces retrotransposon Ty5.

Retroelements (retrotransposons and retroviruses) have two genes in common: gag, which specifies structural proteins that form a virus or virus-like particle, and pol, which specifies catalytic proteins required for replication. For many retroelements, gag and pol are present on separate reading frames. Their expression is highly regulated, and the ratio of Gag to Pol is critical for retroelement replication. The Saccharomyces retrotransposon Ty5 contains a single open reading frame, and we characterized Gag and Pol expression by generating transpositionally active Ty5 elements with epitope tags at the N terminus or C terminus or within the integrase coding region. Immunoblot analysis identified two Gag species (Gag-p27 and Gag-p37), reverse transcriptase (Pol-p59), and integrase (Pol-p80), all of which are largely insoluble in the absence of urea or ionic detergent. These proteins result from proteolytic processing of a polyprotein, because elements with mutations in the presumed active site of Ty5 protease express a single tagged protein (Gag-Pol-p182). Protease mutants are also transpositionally inactive. In a time course experiment, we monitored protein expression, proteolytic processing, and transposition of a Ty5 element with identical epitope tags at its N and C termini. Both transposition and the abundance of Gag-p27 increased over time. In contrast, the levels of Gag-p37 and reverse transcriptase peaked after approximately 14 h of induction and then gradually decreased. This may be due to differences in stability of Gag-p27 relative to Gag-p37 and reverse transcriptase. The ratio of Ty5 Gag to Pol averaged 5:1 throughout the time course experiment, suggesting that differential protein stability regulates the amounts of these proteins.

Fungal Proteins↗
PubMed2002

Transduction of interphase cells by avian sarcoma virus.

It has been generally believed that oncoretroviruses are dependent on mitosis for efficient nuclear entry of viral DNA. We previously identified a nuclear localization signal in the integrase protein of an oncoretrovirus, avian sarcoma virus (ASV), suggesting an active import mechanism for the integrase-DNA complex (G. Kukolj, R. A. Katz, and A. M. Skalka, Gene 223:157-163, 1998). Here, we have evaluated the requirement for mitosis in nuclear import and integration of ASV DNA. Using a modified ASV encoding a murine leukemia virus amphotropic env gene and a green fluorescent protein (GFP) reporter gene, DNA nuclear import was measured in cell cycle-arrested avian (DF-1) as well as human (HeLa) and mouse cells. The results showed efficient accumulation of nuclear forms of ASV DNA in gamma-irradiation-arrested cells. Efficient transduction of a GFP reporter gene was also observed after infection of cells that were arrested with gamma-irradiation, mitomycin C, nocodazole, or aphidicolin, confirming that nuclear import and integration of ASV DNA can occur in the absence of mitosis. By monitoring GFP expression in individual cells, we also obtained evidence for nuclear import of viral DNA during interphase in cycling cells. Lastly, we observed that ASV can transduce postmitotic mouse neurons. These results support an active nuclear import mechanism for the oncoretrovirus ASV and suggest that this mechanism can operate in both nondividing and dividing cells.

Active Transport, Cell Nucleus↗
PubMed2002

A sensitive, quantitative assay for human immunodeficiency virus type 1 integration.

Quantitative methods to measure human immunodeficiency virus type 1 (HIV-1) integration promise to be important tools in dissecting the mechanisms whereby latent reservoirs of provirus are established, most notably in the resting T cells of patients receiving antiretroviral therapy. Here we describe a fluorescence-monitored, nested PCR assay that is able to quantify the relatively rare integration events that occur within these cells. Following DNA extraction, a nonkinetic preamplification step is performed with primers that bind genomic Alu elements and HIV-1 gag sequences, under conditions where primers, deoxynucleoside triphosphates, and enzyme are not limiting. This is followed by a kinetic PCR that quantitates HIV-1 long terminal repeat sequences. A T-cell-based integration standard which reflects the randomness of HIV-1 integration is also described. The assay is 10 to 100 times more sensitive than previously reported quantitative Alu PCR-based integration assays. It is specific for integration events, since no proviruses are detected in cells infected either in the presence of an integrase inhibitor or with an integrase-deficient virus. This method promises to provide important new insights into the processes underlying the accumulation and persistence of latent HIV-1 reservoirs and may eventually be useful clinically in monitoring the eradication of latent virus by novel therapies.

CD4-Positive T-Lymphocytes↗
PubMed2004

Evidence that stable retroviral transduction and cell survival following DNA integration depend on components of the nonhomologous end joining repair pathway.

We have previously reported several lines of evidence that support a role for cellular DNA repair systems in completion of the retroviral DNA integration process. Failure to repair an intermediate in the process of integrating viral DNA into host DNA appears to trigger growth arrest or death of a large percentage of infected cells. Cellular proteins involved in the nonhomologous end joining (NHEJ) pathway (DNA-PK(CS)) and the damage-signaling kinases (ATM and ATR) have been implicated in this process. However, some studies have suggested that NHEJ proteins may not be required for the completion of lentiviral DNA integration. Here we provide additional evidence that NHEJ proteins are required for stable transduction by human immunodeficiency type 1 (HIV-1)-based vectors. Our analyses with two different reporters show that the number of stably transduced DNA-PK(CS)-deficient scid fibroblasts was reduced by 80 to 90% compared to the number of control cells. Furthermore, transduction efficiency can be restored to wild-type levels in scid cells that are complemented with a functional DNA-PK(CS) gene. The efficiency of stable transduction by an HIV-1-based vector is also reduced upon infection of Xrcc4 and ligase IV-deficient cells, implying a role for these components of the NHEJ repair pathway. Finally, we show that cells deficient in ligase IV are killed by infection with an integrase-competent but not an integrase-deficient HIV-1 vector. Results presented in this study lend further support to a general role for the NHEJ DNA repair pathway in completion of the retroviral DNA integration process.

Animals↗
PubMed2004

Effects of mutations in the G tract of the human immunodeficiency virus type 1 polypurine tract on virus replication and RNase H cleavage.

The RNase H cleavages that generate and remove the polypurine tract (PPT) primer during retroviral reverse transcription must be specific in order to create a linear viral DNA that is suitable for integration. Lentiviruses contain a highly conserved sequence consisting of six guanine residues at the 3' end of the PPT (hereafter referred to as the G tract). We introduced mutations into the G tract of a human immunodeficiency virus type 1-based vector and determined the effects on the virus titer and RNase H cleavage specificity. Most mutations in the G tract had little or no effect on the virus titer. Mutations at the second and fifth positions of the G tract increased the proportion of two-long-terminal-repeat (2-LTR) circle junctions with one or two nucleotide insertions. The second and fifth positions of the G tract make specific contacts with amino acids in the RNase H domain that are important for RNase H cleavage specificity. These complementary data define protein-nucleic acid interactions that help control the specificity of RNase H cleavage. When the G-tract mutants were analyzed in a viral background that was deficient in integrase, in most cases the proportion of consensus 2-LTR circle junctions increased. However, in the case of a mutant with Ts at the second and fifth positions of the G tract, the proportion of 2-LTR circle junctions containing the one-nucleotide insertion increased, suggesting that linear viral DNAs containing an extra base are substrates for integration. This result is consistent with the idea that the 3' end-processing reactions of retroviral integrases may help to generate defined ends from a heterogenous population of linear viral DNAs.

Base Sequence↗
PubMed2005

Specific recognition and cleavage of the plus-strand primer by reverse transcriptase.

Reverse transcriptases (RTs) of retroviruses and long terminal repeat (LTR)-retrotransposons possess DNA polymerase and RNase H activities. During reverse transcription these activities are necessary for the programmed sequence of events that include template switching and primer processing. Integrase then inserts the completed cDNA into the genome of the host cell. The RT of the LTR-retrotransposon Tf1 was subjected to random mutagenesis, and the resulting transposons were screened with genetic assays to test which mutations reduced reverse transcription and which inhibited integration. We identified a cluster of mutations in the RNase H domain of RT that were surprising because they blocked integration without reducing cDNA levels. The results of immunoblots demonstrated that these mutations did not reduce levels of RT or integrase. DNA blots showed that the mutations did not lower the amounts of full-length cDNA. The sequences of the 3' ends of the cDNA revealed that mutations within the cluster in RNase H specifically reduced the removal of the polypurine tract (PPT) primer from the ends of the cDNA. These results indicate that primer removal is not a necessary component of reverse transcription. The residues mutated in Tf1 RNase H are conserved in human immunodeficiency virus type 1 and make direct contact with DNA opposite the PPT. Thus, our results identify a conserved element in RT that contacts the PPT and is specifically required for PPT removal.

DNA, Viral↗
PubMed2005

Weak palindromic consensus sequences are a common feature found at the integration target sites of many retroviruses.

Integration into the host genome is one of the hallmarks of the retroviral life cycle and is catalyzed by virus-encoded integrases. While integrase has strict sequence requirements for the viral DNA ends, target site sequences have been shown to be very diverse. We carefully examined a large number of integration target site sequences from several retroviruses, including human immunodeficiency virus type 1, simian immunodeficiency virus, murine leukemia virus, and avian sarcoma-leukosis virus, and found that a statistical palindromic consensus, centered on the virus-specific duplicated target site sequence, was a common feature at integration target sites for these retroviruses.

Base Sequence↗
PubMed2006

Mutations in the U5 sequences adjacent to the primer binding site do not affect tRNA cleavage by rous sarcoma virus RNase H but do cause aberrant integrations in vivo.

In most retroviruses, the first nucleotide added to the tRNA primer becomes the right end of the U5 region in the right long terminal repeat (LTR); the removal of this tRNA primer by RNase H defines the right end of the linear double-stranded DNA. Most retroviruses have two nucleotides between the 5' end of the primer binding site (PBS) and the CA dinucleotide that will become the end of the integrated provirus. However, human immunodeficiency virus type 1 (HIV-1) has only one nucleotide at this position, and HIV-2 has three nucleotides. We changed the two nucleotides (TT) between the PBS and the CA dinucleotide of the Rous sarcoma virus (RSV)-derived vector RSVP(A)Z to match the HIV-1 sequence (G) and the HIV-2 sequence (GGT), and we changed the CA dinucleotide to TC. In all three mutants, RNase H removes the entire tRNA primer. Sequence analysis of RSVP(HIV2) proviruses suggests that RSV integrase can remove three nucleotides from the U5 LTR terminus of the linear viral DNA during integration, although this mutation significantly reduced virus titer, suggesting that removing three nucleotides is inefficient. However, the results obtained with RSVP(HIV1) and RSVP(CATC) show that RSV integrase can process and integrate the normal U3 LTR terminus of a linear DNA independently of an aberrant U5 LTR terminus. The aberrant end can then be joined to the host DNA by unusual processes that do not involve the conserved CA dinucleotide. These unusual events generate either large duplications or, less frequently, deletions in the host genomic DNA instead of the normal 5- to 6-base duplications.

Avian Sarcoma Viruses↗
PubMed1992

Posttranslational control of Ty1 retrotransposition occurs at the level of protein processing.

High-level expression of a transpositionally competent Ty1 element fused to the inducible GAL1 promoter on a 2 microns plasmid (pGTy1) overcomes transpositional dormancy in Saccharomyces cerevisiae. To investigate the mechanisms controlling the rate of Ty1 retrotransposition, we quantitated transposition and Ty1 gene products in cells induced and uninduced for expression of pGTy1. The increase in Ty1 transposition was 45- to 125-fold greater than the increase in Ty1 RNA effected by pGTy1 induction. Translational efficiency of Ty1 RNA was not altered in transposition-induced cells, since p190TYA1-TYB1 protein synthesis increased in proportion to steady-state Ty1 RNA levels. Therefore, expression of a pGTy1 element increases the efficiency of Ty1 transposition at a posttranslational level. Galactose induction of pGTy1 enhanced TYA1 protein processing and allowed detection of processed TYB1 proteins, which are normally present at very low levels in uninduced cells. When the ability of genomic Ty1 elements to complement defined mutations in HIS3-marked pGTy1 elements was examined, mutations in the protease domain or certain mutations in the integrase domain failed to be complemented, but mutations in the reverse transcriptase domain were partially complemented by genomic Ty1 elements. Therefore, the activity of Ty1 elements in yeast cells may be limited by the availability of Ty1 protease and possibly integrase. These results suggest that Ty1 transposition is regulated at the level of protein processing and that this regulation is overcome by expression of a pGTy1 element.

Base Sequence↗
PubMed1992

Functional analysis of box I mutations in yeast site-specific recombinases Flp and R: pairwise complementation with recombinase variants lacking the active-site tyrosine.

The site-specific recombinases Flp and R from Saccharomyces cerevisiae and Zygosaccharomyces rouxii, respectively, are related proteins that belong to the yeast family of site-specific recombinases. They share approximately 30% amino acid matches and exhibit a common reaction mechanism that appears to be conserved within the larger integrase family of site-specific recombinases. Two regions of the proteins, designated box I and box II, also harbor a significantly high degree of homology at the nucleotide sequence level. We have analyzed the properties of Flp and R variants carrying point mutations within the box I segment in substrate-binding, DNA cleavage, and full-site and half-site strand transfer reactions. All mutations abolish or seriously diminish recombinase function either at the substrate-binding step or at the catalytic steps of strand cleavage or strand transfer. Of particular interest are mutations of Arg-191 of Flp and R, residues which correspond to one of the two invariant arginine residues of the integrase family. These variant proteins bind substrate with affinities comparable to those of the corresponding wild-type recombinases. Among the binding-competent variants, only Flp(R191K) is capable of efficient substrate cleavage in a full recombination target. However, this protein does not cleave a half recombination site and fails to complete strand exchange in a full site. Strikingly, the Arg-191 mutants of Flp and R can be rescued in half-site strand transfer reactions by a second point mutant of the corresponding recombinase that lacks its active-site tyrosine (Tyr-343). Similarly, Flp and R variants of Cys-189 and Flp variants at Asp-194 and Asp-199 can also be complemented by the corresponding Tyr-343-to-phenylalanine recombinase mutant.

Binding Sites↗
PubMed1993

Mechanism of cleavage and ligation by FLP recombinase: classification of mutations in FLP protein by in vitro complementation analysis.

The FLP recombinase of the 2 microns plasmid of Saccharomyces cerevisiae is a member of the integrase family of site-specific recombinases. Recombination catalyzed by members of this family proceeds via the ordered cleavage and religation of four strands of DNA. Although the amino acid sequences of integrase family members are quite different, each recombinase maintains an absolutely conserved tetrad of amino acids (R-191, H-305, R-308, Y-343; numbers are those of the FLP protein). This tetrad is presumed to reflect a common chemical mechanism for cleavage and ligation that has evolved among all family members. The tyrosine is the nucleophile that causes phosphodiester bond cleavage and covalently attaches to the 3'-PO4 terminus, whereas the other three residues have been implicated in ligation of strands. It has recently been shown that cleavage by FLP takes place in trans; that is, a FLP molecule binds adjacent to the site of cleavage but receives the nucleophilic tyrosine from a molecule of FLP that is bound to another FLP-binding element (J.-W. Chen, J. Lee, and M. Jayaram, Cell 69:647-658, 1992). These studies led us to examine whether the ligation step of the FLP reaction is performed by the FLP molecule bound adjacent to the cleavage site (ligation in cis). We have found that FLP promotes ligation in cis. Furthermore, using in vitro complementation analysis, we have classified several mutant FLP proteins into one of two groups: those proteins that are cleavage competent but ligation deficient (group I) and those that are ligation competent but cleavage defective (group II). This observation suggests that the active site of FLP is composed of several amino acid residues from each of two FLP molecules.

Amino Acid Sequence↗
PubMed1994

Active-site assembly and mode of DNA cleavage by Flp recombinase during full-site recombination.

A combination of half-site substrates and step arrest mutants of Flp, a site-specific recombinase of the integrase family, had earlier revealed the following features of the half-site recombination reaction. (i) The Flp active site is assembled by sharing of catalytic residues from at least two monomers of the protein. (ii) A Flp monomer does not cleave the half site to which it is bound (DNA cleavage in cis); rather, it cleaves a half site bound by a second Flp monomer (DNA cleavage in trans). For the lambda integrase (Int protein), the prototype member of the Int family, catalytic complementation between two active-site mutants has been observed in reactions with a suicide attL substrate. By analogy with Flp, this observation is strongly suggestive of a shared active site and of trans DNA cleavage. However, reactions with linear suicide attB substrates and synthetic Holliday junctions are more compatible with cis than with trans DNA cleavage. These Int results either argue against a common mode of active-site assembly within the Int family or challenge the validity of Flp half sites as mimics of the normal full-site substrates. We devised a strategy to assay catalytic complementation between Flp monomers in full sites. We found that the full-site reaction follows the shared active-site paradigm and the trans mode of DNA cleavage. These results suggest that within the Int family, a unitary chemical mechanism of recombination is achieved by more than one mode of physical interaction among the recombinase monomers.

Base Sequence↗
PubMed1998

Schizosaccharomyces pombe retrotransposon Tf2 mobilizes primarily through homologous cDNA recombination.

The Tf2 retrotransposon, found in the fission yeast Schizosaccharomyces pombe, is nearly identical to its sister element, Tf1, in its reverse transcriptase-RNase H and integrase domains but is very divergent in the gag domain, the protease, the 5' untranslated region, and the U3 domain of the long terminal repeats. It has now been demonstrated that a neo-marked copy of Tf2 overexpressed from a heterologous promoter can mobilize into the S. pombe genome and produce true transposition events. However, the Tf2-neo mobilization frequency is 10- to 20-fold lower than that of Tf1-neo, and 70% of the Tf2-neo events are homologous recombination events generated independently of a functional Tf2 integrase. Thus, the Tf2 element is primarily dependent on homologous recombination with preexisting copies of Tf2 for its propagation. Finally, production of Tf2-neo proteins and cDNA was also analyzed; surprisingly, Tf2 was found to produce its reverse transcriptase as a single species in which it is fused to protease, unlike all other retroviruses and retrotransposons.

Cloning, Molecular↗
PubMed2001

Asymmetric processing of human immunodeficiency virus type 1 cDNA in vivo: implications for functional end coupling during the chemical steps of DNA transposition.

Retroviral integration, like all forms of DNA transposition, proceeds through a series of DNA cutting and joining reactions. During transposition, the 3' ends of linear transposon or donor DNA are joined to the 5' phosphates of a double-stranded cut in target DNA. Single-end transposition must be avoided in vivo because such aberrant DNA products would be unstable and the transposon would therefore risk being lost from the cell. To avoid suicidal single-end integration, transposons link the activity of their transposase protein to the combined functionalities of both donor DNA ends. Although previous work suggested that this critical coupling between transposase activity and DNA ends occurred before the initial hydrolysis step of retroviral integration, work in the related Tn10 and V(D)J recombination systems had shown that end coupling regulated transposase activity after the initial hydrolysis step of DNA transposition. Here, we show that integrase efficiently hydrolyzed just the wild-type end of two different single-end mutants of human immunodeficiency virus type 1 in vivo, which, in contrast to previous results, proves that two functional DNA ends are not required to activate integrase's initial hydrolysis activity. Furthermore, despite containing bound protein at their processed DNA ends, these mutant viruses did not efficiently integrate their singly cleaved wild-type end into target DNA in vitro. By comparing our results to those of related DNA recombination systems, we propose the universal model that end coupling regulates transposase activity after the first chemical step of DNA transposition.

Base Sequence↗
PubMed2004

A direct repeat sequence associated with the centromeric retrotransposons in wheat.

A high-density BAC filter of Triticum monococcum was screened for the presence of a centromeric retrotransposon using the integrase region as a probe. Southern hybridization to the BAC digests using total genomic DNA probes of Triticum monococcum, Triticum aestivum, and Hordeum vulgare detected differentially hybridizing restriction fragments between wheat and barley. The fragments that hybridized to genomic DNA of wheat but not to that of barley were subcloned. Fluorescence in situ hybridization (FISH) analysis indicated that the clone pHind258 hybridized strongly to centromeric regions in wheat and rye and weakly to those in barley. The sequence of pHind258 was homologous to integrase and long terminal repeats of centromeric Ty3-gypsy retrotransposons of cereal species. Additionally, pHind258 has a pair of 192-bp direct repeats. FISH analysis indicated that the 192-bp repeat probe hybridized to centromeres of wheat and rye but not to those of barley. We found differential FISH signal intensities among wheat chromosomes using the 192-bp probe. In general, the A-genome chromosomes possess strong FISH signals, the B-genome chromosomes possess moderate signals, and the D-genome chromosomes possess weak signals. This was consistent with the estimated copy numbers of the 192-bp repeats in the ancestral species of hexaploid wheat.

Base Sequence↗
PubMed2026

Systematic profiling of nudivirus-like genes reveals conserved and differentiated roles in a domesticated endogenous virus.

Cotesia vestalis bracovirus (CvBV) is a type of domesticated endogenous virus (DEV) derived from ancestral nudiviruses that is integrated into the genome of the parasitoid wasp Cotesia vestalis. The CvBV proviral genome is composed of two distinct components: one encoding genes associated with virion morphogenesis and assembly, and the other harboring virulence genes that are excised, circularized, and packaged into virions. CvBV replication and particle assembly occur exclusively in the ovaries of female wasps. While prior studies have largely focused on the function of virulence genes during parasitization, the molecular mechanisms underlying CvBV replication and assembly remain poorly understood. Here, we identified 71 nudivirus-like genes in the C. vestalis genome through integrated transcriptomic and proteomic analyses. Using gene silencing and microscopy-based imaging approaches, we functionally characterized 24 key genes involved in DNA replication (helicase, integrase-1, and integrase-2), transcriptional regulation (p47, lef-5, and lef-9), capsid formation (vp39, PmV, HzNVorf9-1, HzNVorf9-2, HzNVorf106, 38k, 27b, and K425_459), envelope formation (11k, 17a-1, 35a-1, 35a-2, and K425_461), virion assembly (vlf-1, HzNVorf140-1, and HzNVorf140-2), and viral infectivity (pif-0 and vp91). Although the functions of most nudivirus-like genes are generally conserved among baculoviruses, nudiviruses, and bracoviruses, lef-5, K425_459, 11k, and vp91 appear to have undergone functional divergence relative to their homologs in baculoviruses, nudiviruses, and Microplitis demolitor bracovirus, highlighting lineage-specific adaptations in CvBV. Collectively, our work provides a molecular framework for understanding CvBV assembly and serves as a valuable resource for investigating bracovirus evolution.

Animals↗
PubMed2004

Integron analysis and genetic mapping of antimicrobial resistance genes in Salmonella enterica serotype Typhimurium.

Antimicrobial resistance determinants may be transferred among bacteria via mobile genetic elements including plasmids, transposons, and the more recently explored integrons. Integrons are naturally occurring genetic elements found as part of the Tn21 transposon family or located on various broad host-range plasmids. The fundamental integron structure consists of a 5'-conserved segment (5'-CS) of 1.4-kbp and a 2-kbp 3'-CS. Between these conserved regions are DNA sequences of variable length and molecular complexity. These intervening sequences are known as gene cassettes, and several have now been characterized. Acquisition and dissemination of these genes located within the integron structure, results in an increase in antimicrobial resistance. Three classes of integron structure have been described. Class 1 integrons are of principal importance in clinical isolates. The 5'-CS of class 1 integrons includes an intI 1 gene of 1358 bp, which encodes a specific recombinase, a member of the DNA integrase family. This gene contains the att1 recombination site, required for specifically integrating gene cassettes. Classes 2 and 3 also contain integrase genes (intI 2 and intI 3), with the former showing 40% sequence identity to those of class 1, and the latter showing 61%. All three classes of integrons contain similar gene cassettes from the same families, which suggests the existence of a common pool of gene cassette with cross-specificity between the classes. When the 3'-CS region is examined in detail, it contains several open reading frames (ORFs). These include qacEDelta1, which confers resistance to quaternary ammonium compounds, often associated with antiseptics, along with a sul1 gene expressing resistance to sulphonamide antimicrobial agents. The sul1 gene encodes the enzyme hydopteroate synthase. Transcription of the sul1 gene begins at a promoter located in the 5'-CS. The latter is also responsible for the transcription of the inserted gene cassette(s). Two additional ORFs, ORF-5 and -6, are located toward the distal end of the 3'-CS. The gene product of ORF 5 appears to share some sequence similarity with puromycin acetyltransferase, and this feature suggests a possible role in antimicrobial resistance. A biological function has yet to be ascribed to ORF-6.

Base Sequence↗
PubMedDate not supplied

Sequence comparison of the EcoHK31I and EaeI restriction-modification systems suggests an intergenic transfer of genetic material.

The genes coding for the EcoHK31I and EaeI restriction-modification (R-M) systems from Escherichia coli strain HK31 and Enterobacter aerogenes, respectively, have been cloned and sequenced. Both ENases recognize and cleave Y/GGCCR leaving 4 nucleotide 5'-protruding ends, while the MTases modify the internal cytosine. The systems were isolated on a 2.3kb AseI fragment for EcoHK31I, and a 4.6 kb HindIII fragment for EaeI. The R and M genes of both systems converge and overlap by 14 nucleotides. Previously, we found that M.EcoHK31I consisted of two subunits, (alpha and beta), with the beta subunit being translated from an alternative open reading frame within the gene encoding the alpha subunit. Sequence comparison between the EcoHK31I and EaeI systems reveals striking similarity. The eaeIM gene also encodes alpha and beta polypeptides of 309 and 176 amino acids which share 96% and 97% identity, respectively, with those of ecoHK31IM. ecoHK31IR and eaeIR encode proteins of 318 and 315 aa, respectively, which share 92% identity but are otherwise unique in the GenBank database. The EaeI and the EcoHK31I R-M systems were found to be flanked by genes coding for integrases. It is possible that these integrases have facilitated the transfer of this system among different bacterial species.

Amino Acid Sequence↗