Invariant Tyr Research Articles

Diverse roles of the metal binding domains and transport mechanism of copper transporting P-type ATPases

Copper transporting P-type (P1B-1-) ATPases are essential for cellular homeostasis. Nonetheless, the E1-E1P-E2P-E2 states mechanism of P1B-1-ATPases remains poorly understood. In particular, the role of the intrinsic metal binding domains (MBDs) is enigmatic. Here, four cryo-EM structures and molecular dynamics simulations of a P1B-1-ATPase are combined to reveal that in many eukaryotes the MBD immediately prior to the ATPase core, MBD−1, serves a structural role, remodeling the ion-uptake region. In contrast, the MBD prior to MBD−1, MBD−2, likely assists in copper delivery to the ATPase core. Invariant Tyr, Asn and Ser residues in the transmembrane domain assist in positioning sulfur-providing copper-binding amino acids, allowing for copper uptake, binding and release. As such, our findings unify previously conflicting data on the transport and regulation of P1B-1-ATPases. The results are critical for a fundamental understanding of cellular copper homeostasis and for comprehension of the molecular bases of P1B-1-disorders and ongoing clinical trials.

Tenuous Transcriptional Threshold of Human Sex Determination. I. SRY and Swyer Syndrome at the Edge of Ambiguity.

Male sex determination in mammals is initiated by SRY, a Y-encoded transcription factor. The protein contains a high-mobility-group (HMG) box mediating sequence-specific DNA bending. Mutations causing XY gonadal dysgenesis (Swyer syndrome) cluster in the box and ordinarily arise de novo. Rare inherited variants lead to male development in one genetic background (the father) but not another (his sterile XY daughter). De novo and inherited mutations occur at an invariant Tyr adjoining the motif’s basic tail (box position 72; Y127 in SRY). In SRY-responsive cell lines CH34 and LNCaP, de novo mutations Y127H and Y127C reduced SRY activity (as assessed by transcriptional activation of principal target gene Sox9) by 5- and 8-fold, respectively. Whereas Y127H impaired testis-specific enhancer assembly, Y127C caused accelerated proteasomal proteolysis; activity was in part rescued by proteasome inhibition. Inherited variant Y127F was better tolerated: its expression was unperturbed, and activity was reduced by only twofold, a threshold similar to other inherited variants. Biochemical studies of wild-type (WT) and variant HMG boxes demonstrated similar specific DNA affinities (within a twofold range), with only subtle differences in sharp DNA bending as probed by permutation gel electrophoresis and fluorescence resonance-energy transfer (FRET); thermodynamic stabilities of the free boxes were essentially identical. Such modest perturbations are within the range of species variation. Whereas our cell-based findings rationalize the de novo genotype-phenotype relationships, a molecular understanding of inherited mutation Y127F remains elusive. Our companion study uncovers cryptic biophysical perturbations suggesting that the para-OH group of Y127 anchors a novel water-mediated DNA clamp.

An Overview of the Proofreading Functions in Bacteria and in Severe Acute Respiratory Syndrome-Coronaviruses

Aim: To understand the structure-function relationship of the proofreading (PR) functions in eubacteria and viruses with special reference to Severe Acute Respiratory Syndrome-Coronaviruses (SARS-CoVs) and propose a plausible mechanism of action for PR exonucleases of SARS-CoVs.
 Study Design: Bioinformatics, biochemical, site-directed mutagenesis (SDM), X-ray crystallographic data were used to study the structure-function relationships of the PR exonucleases from bacteria and CoVs.
 Methodology: The protein sequences of the PR exonucleases of various DNA polymerases, and RNA polymerases of SARS, SARS-related and human CoVs (HCoVs) were obtained from PUBMED and SWISS-PROT databases. The advanced version of Clustal Omega was used for protein sequence analysis. Along with the conserved motifs identified by the bioinformatics analysis, the data already available by biochemical, SDM experiments and X-ray crystallographic analysis on these enzymes were used to arrive at the possible active amino acids in the PR exonucleases of these crucial enzymes.
 Results: A complete analysis of the active sites of the PR exonucleases from various bacteria and CoVs were done. The multiple sequence alignment (MSA) analysis showed many conserved amino acids, small and large peptide regions among them. Based on the conserved motifs, the PR exonucleases are found to fit broadly into two superfamilies, viz. DEDD and polymerase-histidinol phosphatase (PHP) superfamilies. The bacterial DNA polymerases I and II, RNase D, RNase T and ε-subunit of DNA polymerases III belong to the DEDD superfamily. The PR enzymes from SARS, SARS-related CoVs and other HCoVs also essentially belong to the DEDD superfamily. The DEDD superfamily either uses an invariant Tyr or a His as proton acceptor during catalysis. Depending on the proton acceptor, they are further classified into DEDHD and DEDYD subfamilies. RNase T, ε-subunit of DNA polymerases III and the SARS, SARS-related CoVs and other HCoVs belong to DEDHD subfamily. However, the SARS, SARS-related CoVs and other HCoVs showed additional zinc finger motifs (ZFMs) in their active sites. DNA polymerases I, II and RNase D belong to DEDYD subfamily. The bacterial DNA polymerases X, YcdX phosphoesterases and the co-editing exonuclease of DNA polymerases III belong to the PHP superfamily. Based on the MSA, X-ray crystallographic analyses and SDM experiments, the proposed active-site proton acceptor is Tyr/His in DEDDY/H subfamilies and His in PHP superfamily of PR exonucleases. 
 Conclusions: Based on the similarities of active site amino acids/motifs, it may be concluded that the DEDD and PHP superfamilies of PR exonucleases should have evolved from a common ancestor but diverged very long ago. The biochemical properties of these enzymes, including the four conserved acidic amino acid residues in the catalytic core, suggest that the CoVs might have acquired the exonuclease function, possibly from a prokaryote. However, the presence of two zinc fingers in the PR active site of the SARS, SARS-related CoVs and other HCoVs sets their PR exonucleases apart from other homologues.

Structural Basis for Pore Blockade of the Human Cardiac Sodium Channel Nav1.5 by the Antiarrhythmic Drug Quinidine**

AbstractNav1.5, the primary voltage‐gated Na+ (Nav) channel in heart, is a major target for class I antiarrhythmic agents. Here we present the cryo‐EM structure of full‐length human Nav1.5 bound to quinidine, a class Ia antiarrhythmic drug, at 3.3 Å resolution. Quinidine is positioned right beneath the selectivity filter in the pore domain and coordinated by residues from repeats I, III, and IV. Pore blockade by quinidine is achieved through both direct obstruction of the ion permeation path and induced rotation of an invariant Tyr residue that tightens the intracellular gate. Structural comparison with a truncated rat Nav1.5 in the presence of flecainide, a class Ic agent, reveals distinct binding poses for the two antiarrhythmics within the pore domain. Our work reported here, along with previous studies, reveals the molecular basis for the mechanism of action of class I antiarrhythmic drugs.

Structural Basis for Pore Blockade of the Human Cardiac Sodium Channel Nav 1.5 by the Antiarrhythmic Drug Quinidine*.

Nav 1.5, the primary voltage-gated Na+ (Nav ) channel in heart, is a major target for class I antiarrhythmic agents. Here we present the cryo-EM structure of full-length human Nav 1.5 bound to quinidine, a class Ia antiarrhythmic drug, at 3.3 Å resolution. Quinidine is positioned right beneath the selectivity filter in the pore domain and coordinated by residues from repeats I, III, and IV. Pore blockade by quinidine is achieved through both direct obstruction of the ion permeation path and induced rotation of an invariant Tyr residue that tightens the intracellular gate. Structural comparison with a truncated rat Nav 1.5 in the presence of flecainide, a class Ic agent, reveals distinct binding poses for the two antiarrhythmics within the pore domain. Our work reported here, along with previous studies, reveals the molecular basis for the mechanism of action of class I antiarrhythmic drugs.

An Invariant Arginine in Common with MHC Class II Allows Extension at the C-Terminal End of Peptides Bound to Chicken MHC Class I

MHC molecules are found in all jawed vertebrates and are known to present peptides to T lymphocytes. In mammals, peptides can hang out either end of the peptide-binding groove of classical class II molecules, whereas the N and C termini of peptides are typically tightly bound to specific pockets in classical class I molecules. The chicken MHC, like many nonmammalian vertebrates, has a single dominantly expressed classical class I molecule encoded by the BF2 locus. We determined the structures of BF2*1201 bound to two peptides and found that the C terminus of one peptide hangs outside of the groove with a conformation much like the peptides bound to class II molecules. We found that BF2*1201 binds many peptides that hang out of the groove at the C terminus, and the sequences and structures of this MHC class I allele were determined to investigate the basis for this phenomenon. The classical class I molecules of mammals have a nearly invariant Tyr (Tyr84 in humans) that coordinates the peptide C terminus, but all classical class I molecules outside of mammals have an Arg in that position in common with mammalian class II molecules. We find that this invariant Arg residue switches conformation to allow peptides to hang out of the groove of BF2*1201, suggesting that this phenomenon is common in chickens and other nonmammalian vertebrates, perhaps allowing the single dominantly expressed class I molecule to bind a larger repertoire of peptides.

Substitution of Tyrosine 146 in the dI Component of Proton-translocating Transhydrogenase Leads to Reversible Dissociation of the Active Dimer into Inactive Monomers

Transhydrogenase couples the redox reaction between NADH and NADP+ to proton translocation across a membrane. The protein has three components: dI binds NADH, dIII binds NADP+, and dII spans the membrane. Transhydrogenase is a "dimer" of two dI-dII-dIII "monomers"; x-ray structures suggested that the two catalytic sites alternate during turnover. Invariant Tyr146 in recombinant dI of Rhodospirillum rubrum transhydrogenase was substituted with Phe and Ala (proteins designated dI.Y146F and dI.Y146A, respectively). Analytical ultracentrifuge experiments and differential scanning calorimetry show that dI.Y146A more readily dissociates into monomers than wild-type dI. Analytical ultracentrifuge and Trp fluorescence experiments indicate that the dI.Y146A monomers bind NADH much more weakly than dimers. Wild-type dI and dI.Y146F reconstituted activity to dI-depleted membranes with similar characteristics. However, dI.Y146A reconstituted activity in its dimeric form but not in its monomeric form, this despite monomers retaining their native fold and binding to the dI-depleted membranes. It is suggested that transhydrogenase reconstructed with monomers of dI.Y146A is catalytically compromised, at least partly as a consequence of the lowered affinity for NADH, and this results from lost interactions between the nucleotide binding site and the protein beta-hairpin upon dissociation of the dI dimer. The importance of these interactions and their coupling to dI domain rotation in the mechanism of action of transhydrogenase is emphasized. Two peaks in the 1H NMR spectrum of wild-type dI are broadened in dI.Y146A and are tentatively assigned to S-methyl groups of Met resonances in the beta-hairpin, consistent with the segmental mobility of this feature in the structure.

Structure-Function Analysis of the Human Sialyltransferase ST3Gal I: ROLE OF N-GLYCOSYLATION AND A NOVEL CONSERVED SIALYLMOTIF

All eukaryotic sialyltransferases have in common the presence in their catalytic domain of several conserved peptide regions (sialylmotifs L, S, and VS). Functional analysis of sialylmotifs L and S previously demonstrated their involvement in the binding of donor and acceptor substrates. The region comprised between the sialylmotifs S and VS contains a stretch of four highly conserved residues, with the following consensus sequence (H/y)Y(Y/F/W/h)(E/D/q/g). (Capital letters and lowercase letters indicate a strong or low occurrence of the amino acid, respectively.) The functional importance of these residues and of the conserved residues of motif VS (HX(4)E) was assessed using as a template the human ST3Gal I. Mutational analysis showed that residues His(299) and Tyr(300) of the new motif, and His(316) of the VS motif, are essential for activity since their substitution by alanine yielded inactive enzymes. Our results suggest that the invariant Tyr residue (Tyr(300)) plays an important conformational role mainly attributable to the aromatic ring. In contrast, the mutants W301F, E302Q, and E321Q retained significant enzyme activity (25-80% of the wild type). Kinetic analyses and CDP binding assays showed that none of the mutants tested had any significant effect in nucleotide donor binding. Instead the mutant proteins were affected in their binding to the acceptor and/or demonstrated lower catalytic efficiency. Although the human ST3Gal I has four N-glycan attachment sites in its catalytic domain that are potentially glycosylated, none of them was shown to be necessary for enzyme activity. However, N-glycosylation appears to contribute to the proper folding and trafficking of the enzyme.

Mutational and Kinetic Evaluation of Conserved His-123 in Dual Specificity Protein-tyrosine Phosphatase Vaccinia H1-related Phosphatase: PARTICIPATION OF TYR-78 AND THR-73 RESIDUES IN TUNING THE ORIENTATION OF HIS-123

Active-site cysteine strategically positioned in the P-loop of protein-tyrosine phosphatases has been suggested to be further stabilized by hydrogen bonding arrays radiating out from the P-loop to neighboring residues. In this work, we investigated the structural role of histidine array in HC(X)(5)RS motif of the vaccinia H1-related protein phosphatase (VHR), using site-directed mutagenesis in conjunction with an extensive kinetic analysis. Conserved His-123 was mutated along with neighboring residues Tyr-78 and Thr-73. The increased pK(a) values of active-site Cys-124 found in Y78F and T73A mutants (6.51 and 6.75, respectively) were comparable to those of H123A and H123F mutants. Kinetic evaluation of Y78F and T73A mutants further implicates that the mutations perturb the relative position of Cys-124 within the P-loop. These results imply that Tyr-78 and Thr-73 make up an essential part of the His-123 array and structurally tune the Cys-124 position. Tyr-78 of VHR turns out to be the invariant Tyr reported in several protein-tyrosine phosphatases by a structure-based sequence alignment. Therefore, orientation of the imidazole ring of His-123 by the invariant Tyr-78 is crucial for maintaining the proper position of Cys-124 in the P-loop.

The Crystal Structure of Nitrophorin 2: A TRIFUNCTIONAL ANTIHEMOSTATIC PROTEIN FROM THE SALIVA OF RHODNIUS PROLIXUS

Nitrophorin 2 (NP2) (also known as prolixin-S) is a salivary protein that transports nitric oxide, binds histamine, and acts as an anticoagulant during blood feeding by the insect Rhodnius prolixus. The 2.0-A crystal structure of NP2 reveals an eight-stranded antiparallel beta-barrel containing a ferric heme coordinated through His(57), similar to the structures of NP1 and NP4. All four Rhodnius nitrophorins transport NO and sequester histamine through heme binding, but only NP2 acts as an anticoagulant. Here, we demonstrate that recombinant NP2, but not recombinant NP1 or NP4, is a potent anticoagulant; recombinant NP3 also displays minor activity. Comparison of the nitrophorin structures suggests that a surface region near the C terminus and the loops between beta strands B-C and E-F is responsible for the anticoagulant activity. NP2 also displays larger NO association rates and smaller dissociation rates than NP1 and NP4, which may result from a more open and more hydrophobic distal pocket, allowing more rapid solvent reorganization on ligand binding. The NP2 protein core differs from NP1 and NP4 in that buried Glu(53), which allows for larger NO release rates when deprotonated, hydrogen bonds to invariant Tyr(81). Surprisingly, this tyrosine lies on the protein surface in NP1 and NP4.

Replacement of the invariant tyrosine in the CAGY region of the human chorionic gonadotropin β subunit

The mammalian glycoprotein hormone β subunits contain a highly conserved amino acid sequence, Cys-Ala-Gly-Tyr-Cys (residues 34–38 of human chorionic gonadotropin β), that is denoted as the ‘CAGY region’. Using site-directed mutagenesis we have replaced Tyr-37 in hCGβ, i.e., the invariant Tyr in all known mammalian CG, LH, FSH, and TSH β subunits, with two hydrophobic amino acid residues, Phe and Leu. The resultant mutant forms were characterized for α subunit binding and the resulting heterodimers were analyzed for biological activity using two in vitro assays with transformed murine Leydig cells (MA-10). Chinese hamster ovary cells containing a stably integrated gene for bovine α were transiently transfected with a eukaryotic expression vector containing a Rous sarcoma viral promoter and the wild-type and mutant cDNAs. The hCGβ(Phe-37) mutant bound to α essentially to the same extent as hCGβ wild-type, while the hCGβ(Leu-37) mutant formed somewhat less heterodimer. The heterologous heterodimeric mutant and wild-type gonadotropins were equipotent in a competitive binding assay with [ 125I]hCG. In a steroidogenic assay, the mutant hormones were active, but they appeared slightly less potent than the wild-type form. Thus, this invariant Tyr can be replaced with another aromatic amino acid residue or with a hydrophobic, but not aromatic amino acid residue in hCGβ without any dramatic effect on function. These results indicate that Tyr-37 in hCGβ, while not obligatory, may participate, either directly or indirectly, in subunit assembly and that the hydroxyl group may function in a modulatory role in signaling.

Hydrolysis of Micellar Diheptanoylphosphatidylcholine Catalyzed by Bovine Pancreatic Phospholipase A2: Kinetic Characterization of Group I and II Enzymes1

Initial velocity data for the hydrolysis of micellar 1,2-diheptanoyl-sn-glycero-3-phosphorylcholine (diC7PC) catalyzed by bovine pancreatic PLA2 (Group I) were analyzed using the Michaelis-Menten equation. The Km value for the micellar substrate was found to be independent of Ca2+ concentration, as was the Km value for the monodispersed substrate. The pH dependence curve of Km in the presence of saturating amounts of Ca2+ showed two transitions reflecting large pK shifts of two ionizable groups from 5.0 to 5.45 and from 9.5 to 10.25, whereas the Km value for the monodispersed substrate was independent of pH [Fujii et al. (1991) J. Biochem. 110, 1008-1015]. The pH dependence curve of kcat showed three transitions, indicating the participation of three ionizable groups with pK values of 5.45, 8.4, and 10.25. Deprotonation of the first group and protonation of the third group were found to be essential for catalysis. The respective groups were assigned as the catalytic group His 48, the N-terminal alpha-amino group, and invariant Tyr 52. The present results as well as those for another Group I PLA2 (Naja naja atra) are very different from those for Group II PLA2s (Agkistrodon halys blomhoffii and Trimeresurus flavoviridis), which showed Ca(2+)-dependent substrate binding and no participation of the alpha-amino group in catalysis [Teshima et al. (1989) J. Biochem. 106, 518-527; Nishimura et al. (1992) J. Biochem. 111, 210-218].(ABSTRACT TRUNCATED AT 250 WORDS)

Computer-assisted dissection of rolling circle DNA replication

A comparative analysis of the proteins involved in initiation and termination of rolling circle replication (RCR) was performed using computer-assisted methods of data based screening, motif search and multiple amino acid sequence alignment. Two vast classes of such proteins were delineated, one of these being associated with RCR proper, and the other with mobilization (conjugal transfer) of plasmid DNA. The common denominator of the two classes was found to be a conserved amino acid motif that consists of the sequence HisUHisUUU (U — bulky hydrophobic residue; hereafter HUH motif). Based on analogies with metalloenzymes, it is hypothesized that the two conserved His residues this motif may be involved in metal ion coordination required for the activity of the RCR and mobilization proteins. The proteins of the replication (Rep) class contained two additional conserved motifs, with the motif around the Tyr residue(s) forming the covalent link with nicked DNA being located C-proximally of the HUH motif. This class further split into two large superfamilies and several smaller families, with the proteins belonging to a single but not to different (super)families demonstrating statistically significant similarity to each other. Superfamily I, prototyped by the gene A proteins of small isometric single-stranded (ss) DNA bacteriophages, included also Rep proteins of P2-related double-stranded (ds) DNA bacteriophages, the small phage-plasmid hybrid phasyl, and several cyanobacterial and archaebacterial plasmids. These proteins contained two invariant Tyr residues separated by three partially conserved amino acids, suggesting that they all may share the cleavage-ligation mechanism proposed for φX174 A protein and involving alternate covalent binding of both tyrosines to DNA (Van Mansfeld, A.D., Van Teeffelen, H.A., Baas, P.D., Jansz, H.S., 1986. Nucl. Acids Res. 14, 4229–4238). Superfamily II included Rep proteins of a number of ssDNA plasmids replicating mainly in gram-positive bacteria that unexpectedly were shown to be related to the Rep proteins of plant geminiviruses. Conservation of the “HUH” motif and a motif around the putative DNA-linking Tyr residue was observed also in the Rep proteins of animal parvoviruses containing linear ssDNA with a terminal hairpin and replicating via the rolling hairpin mechanism. The class of plasmid mobilization (Mob) proteins was characterized by the opposite orientation of the conserved motifs, with the (putative) DNA-linking Tyr being located N-proximally of the “HUH” motif. This class also separated into several distinct families, the largest of which was comprised by the Mob (Tral) proteins of promiscuous IncP and IncI plasmids, VirD2 endonucleases of Agrobacterium TI plasmids, and Mob proteins of a group of gram-positive bacterial ssDNA plasmids. The majority of ssDNA plasmid Mob proteins constituted another family, whereas the Mob domains of TraI proteins of F factor and related plasmids formed a separate group that was only distantly related to the former two families. Additionally, a family of plasmid Rep proteins was analyzed that are unrelated to the above two classes and do not contain the HUH motif but possess instead several distinct conserved motifs. A protein encoded by an archaebacterial virus gene was shown to be distantly related to this family, with significant sequence conservation observed around the putative DNA-linked tyrosine residue. This analysis allowed the prediction of the amino acid residues involved in DNA nicking, which is required for the initiation of RCR or conjugational transfer of ssDNA, in the Rep and Mob proteins encoded by a number of replicons of highly diverse size, structure and origin. It is conjectured that recombination has played a major part in the dissemination of genes encoding related Rep or Mob proteins among these replicons. It is speculated that the eukaryotic replicons encoding proteins with the conserved RCR motifs and replicating via RCR-related mechanisms, such as geminiviruses and parvoviruses, may have evolved from eubacterial ssDNA replicons. Analysis of the nucleotide sequences of the replication and transfer origins ( ori) of various replicons allowed the tentative identification of several previously uncharacterized ori sites but showed that only partial correlation exists between the sequence conservation in the replication (transfer) initiation proteins, and in ori sites.

Kinetics of the Hydrolysis of Monodispersed Dihexanoylphosphatidylcholine Catalyzed by Bovine Pancreatic Phospholipase A2: Roles of Ca2+ Binding and Ionizations of Amino Acid Residues in the Active Site1

Phospholipases A2 (PLA2s) are classified into two groups, I and II, according to differences in the polypeptide-chain length and intramolecular disulfide bondings. The hydrolysis of monodispersed 1,2-dihexanoyl-sn-glycero-3-phosphorylcholine (diC6PC), catalyzed by bovine pancreas PLA2 (Group I) was studied at 25 degrees C and ionic strength 0.2, and the initial velocity data were analyzed by means of the Michaelis-Menten equation. The Michaelis constant, Km, was found to be practically independent of Ca2+ concentration and also of pH value. The latter result indicates no participation of the ionizable groups in the active site in the substrate binding. The pH-dependence curve of the logarithm of the catalytic center activity, kcat, obtained in the presence of a practically saturating amount of Ca2+, showed three transitions ascribable to the participation of three ionizable groups with pK values of 5.00, 8.40, and 9.50. The respective groups were tentatively assigned to the catalytic group His 48, the N-terminal alpha-amino group, and invariant Tyr 52, which is located in close proximity to the imidazole ring of His 48. Deprotonation of His 48 and protonation of Tyr 52 were shown to be essential to the catalysis. The importance of the ionization state of the alpha-amino group was also indicated.(ABSTRACT TRUNCATED AT 250 WORDS)

Author index for volume 72, number 1

A comparative analysis of the proteins involved in initiation and termination of rolling circle replication (RCR) was performed using computer-assisted methods of data based screening, motif search and multiple amino acid sequence alignment. Two vast classes of such proteins were delineated, one of these being associated with RCR proper, and the other with mobilization (conjugal transfer) of plasmid DNA. The common denominator of the two classes was found to be a conserved amino acid motif that consists of the sequence HisUHisUUU (U — bulky hydrophobic residue; hereafter HUH motif). Based on analogies with metalloenzymes, it is hypothesized that the two conserved His residues this motif may be involved in metal ion coordination required for the activity of the RCR and mobilization proteins. The proteins of the replication (Rep) class contained two additional conserved motifs, with the motif around the Tyr residue(s) forming the covalent link with nicked DNA being located C-proximally of the HUH motif. This class further split into two large superfamilies and several smaller families, with the proteins belonging to a single but not to different (super)families demonstrating statistically significant similarity to each other. Superfamily I, prototyped by the gene A proteins of small isometric single-stranded (ss) DNA bacteriophages, included also Rep proteins of P2-related double-stranded (ds) DNA bacteriophages, the small phage-plasmid hybrid phasyl, and several cyanobacterial and archaebacterial plasmids. These proteins contained two invariant Tyr residues separated by three partially conserved amino acids, suggesting that they all may share the cleavage-ligation mechanism proposed for φX174 A protein and involving alternate covalent binding of both tyrosines to DNA (Van Mansfeld, A.D., Van Teeffelen, H.A., Baas, P.D., Jansz, H.S., 1986. Nucl. Acids Res. 14, 4229–4238). Superfamily II included Rep proteins of a number of ssDNA plasmids replicating mainly in gram-positive bacteria that unexpectedly were shown to be related to the Rep proteins of plant geminiviruses. Conservation of the “HUH” motif and a motif around the putative DNA-linking Tyr residue was observed also in the Rep proteins of animal parvoviruses containing linear ssDNA with a terminal hairpin and replicating via the rolling hairpin mechanism.The class of plasmid mobilization (Mob) proteins was characterized by the opposite orientation of the conserved motifs, with the (putative) DNA-linking Tyr being located N-proximally of the “HUH” motif. This class also separated into several distinct families, the largest of which was comprised by the Mob (Tral) proteins of promiscuous IncP and IncI plasmids, VirD2 endonucleases of Agrobacterium TI plasmids, and Mob proteins of a group of gram-positive bacterial ssDNA plasmids. The majority of ssDNA plasmid Mob proteins constituted another family, whereas the Mob domains of TraI proteins of F factor and related plasmids formed a separate group that was only distantly related to the former two families.Additionally, a family of plasmid Rep proteins was analyzed that are unrelated to the above two classes and do not contain the HUH motif but possess instead several distinct conserved motifs. A protein encoded by an archaebacterial virus gene was shown to be distantly related to this family, with significant sequence conservation observed around the putative DNA-linked tyrosine residue. This analysis allowed the prediction of the amino acid residues involved in DNA nicking, which is required for the initiation of RCR or conjugational transfer of ssDNA, in the Rep and Mob proteins encoded by a number of replicons of highly diverse size, structure and origin.It is conjectured that recombination has played a major part in the dissemination of genes encoding related Rep or Mob proteins among these replicons. It is speculated that the eukaryotic replicons encoding proteins with the conserved RCR motifs and replicating via RCR-related mechanisms, such as geminiviruses and parvoviruses, may have evolved from eubacterial ssDNA replicons.Analysis of the nucleotide sequences of the replication and transfer origins (ori) of various replicons allowed the tentative identification of several previously uncharacterized ori sites but showed that only partial correlation exists between the sequence conservation in the replication (transfer) initiation proteins, and in ori sites.