Sequence- and strand-specific cleavage in oligodeoxyribonucleotides and DNA containing 3'-thiothymidine.

Oligonucleotides containing a 3'-thiothymidine residue (T3's) at the cleavage site for the EcoRV restriction endonuclease (between the central T and A residues of the sequence GATATC) have been prepared on an automated DNA synthesizer using 5'-O-monomethoxytritylthymidine 3'-S-(2-cyanoethyl N,N-diisopropylphosphorothioamidite). The self-complementary sequence GACGAT3'sATCGTC was completely resistant to cleavage by EcoRV, while the heteroduplex composed of 5'-TCTGAT3'sATCCTC and 5'-GAGGATATCAGA (duplex 4) was cleaved only in the unmodified strand (5'-GAGGATATCAGA). In contrast, strands containing a 3'-S-phosphorothiolate linkage could be chemically cleaved specifically at this site with Ag+. A T3's residue has also been incorporated in the (-) strand of double-stranded closed circular (RF IV) M13mp18 DNA at the cleavage site of a unique EcoRV recognition sequence by using 5'-pCGAGCTCGAT3'sATCGTAAT as a primer for polymerization on the template (+) strand of M13mp18 DNA. On treatment of this substrate with EcoRV, only one strand was cleaved to produce the RF II or nicked DNA. Taken in conjunction with the cleavage studies on the oligonucleotides, this result demonstrates that the 3'-S-phosphorothiolate linkage is resistant to scission by EcoRV. Additionally, the phosphorothiolate-containing strand of the M13mp18 DNA could be cleaved specifically at the point of modification using iodine in aqueous pyridine. The combination of enzymatic and chemical techniques provides, for the first time, a demonstrated method for the sequence-specific cleavage of either the (+) or (-) strand.     

Investigation of the inhibitory role of phosphorothioate internucleotidic linkages on the catalytic activity of the restriction endonuclease EcoRV

The inhibitory effect of phosphorothioate residues, located within one strand of double-stranded DNA, on the hydrolytic activity of the restriction endonuclease EcoRV was investigated. Specific incorporation of a phosphorothioate group at the site of cleavage yielded the sequence 5'-GATsATC-3'. This modified sequence was cleaved at a relative rate of 0.1 compared to the unmodified substrate. Substrates 5'-GATsAsTC-3' and 5'-GsATsATC-3', both containing one additional phosphorothioate substitution, were linearized at a rate of 0.04 relative to unmodified DNA. However, under the same conditions, fully dAMPS-substituted DNA was found to be virtually resistant to the hydrolytic activity of EcoRV. Further experiments showed that double-stranded DNA fragments generated by PCR containing phosphorothioate groups within both strands are potent inhibitors of EcoRV catalysis. The inhibition was independent of whether the inhibitor fragment contained an EcoRV recognition site. We concluded that substitution of the phosphate group at the site of cleavage by a phosphorothioate residue decreases the rate of EcoRV-catalyzed hydrolysis most significantly. Substitution of other phosphate groups within the recognition sequence plays a limited role in enzyme inhibition. The presence of multiple dNMPS residues at regions of the DNA removed from the EcoRV recognition site may decrease the amount of enzyme available for catalysis by nonspecific binding to EcoRV.     

Study of the amino acid residue topography of the adenosine triphosphatase center of (Ca--Mg)-dependent sarcoplasmic reticulum adenosine triphosphatase by kinetic and spectral methods

The topography of HS- and NH2-groups and tryptophane residues in ATPase centre of (Ca--Mg)-ATPase on sarcoplasmic reticulum (SR) was investigated by kinetics, electron spectroscopy and spectrofluorimetry method. Both o-phthalaldehyde interacting with lysine or arginine residue or with end amino acid and fluorescein dimercuric acetate interaction with cysteine residue of HS-groups make (Ca--Mg)-ATPase both in SR and the pure enzyme completely inactive at molar ratio enzyme: inhibitor equal to 1 : 1. A 500 molar ATP surplus reduces drastically the enzyme inactivation rate by both inhibitors. The data supplied by the spectrofluorimetry and the induction-resonance theory were used to calculate the distances between nearest tryptophane residues and chromophore (o-FTC) generated by o-phthalaldehyde interaction with NH2-group the protein amino acid residue (17 A) and o-FTC and fluorescein dimercuric acetate (19 A) attached to enzyme HS-group. Because o-FTC is inside the protein pocket it is not accessible to J- ions up to 2.5 M KJ. However some tryptophane resudies and fluorescein dimercuric acetate attached to HS-group are near to the macromolecule surface. Lysine (or arginine residues) or end amino acid NH2-group and cysteine residues HS-group, and some tryptophane residues are at ATPase centre of (Ca--Mg)-ATPase from sarcoplasmic reticulum. Possible topography of the centre is discussed.     

Cleavage by restriction endonucleases MvaI and EcoRII of substrates modified in amino groups of heterocyclic bases

14-membered DNA-duplexes containing modified nucleoside residues, viz 4-N-methyldeoxycytidine (m4dC), 6-N-methyldeoxyadenosine (m6dA) or deoxyinosine (dI), in only one strand of the recognition site (CCA/TGG) of MvaI and EcoRII endonucleases were synthesized. It was shown that MvaI and EcoRII endonucleases interact with the exocyclic amino groups of the external dC residues and of the central dA residue of the recognition site exposed into the DNA major groove. These endonucleases which are isochizomers were found to possess different mechanisms of substrate cleavage. The ability of MvaI endonuclease to hydrolyze only unmodified strand of methylated duplexes allows one to make site-directed single-strand nicks in double-stranded DNA. Elimination of the 2-NH2-group located in the minor groove of DNA by substituting dI for dG had little, if any, effect on the hydrolytic activity of EcoRII and MvaI endonucleases.     

Role of thymidine residues in DNA recognition by the EcoRI and EcoRV restriction endonucleases.

We have synthesized a series of oligonucleotides containing the EcoRI (GAATTC) or EcoRV (GATATC) recognition site within which or adjacent to which thymidine was substituted by uridine or derivatives of uridine. The effects of these substitutions on the rate of the EcoRI and EcoRV catalyzed cleavage reaction were investigated. Our results show that most of the substitutions within the site are quite well tolerated by EcoRI, not, however, by EcoRV. We conclude that the thymin residues most likely are not directly involved in the recognition process of the EcoRI reaction. In contrast, they are major points of contact, between substrate and enzyme in the EcoRV reaction. The effects of substitutions in the position adjacent to the recognition site is also markedly different for EcoRI and EcoRV. Here, EcoRI seems to be considerably more selective than EcoRV.     

Linkage isomerization reaction of intrastrand cross-links in trans-diamminedichloroplatinum(II)-modified single-stranded oligonucleotides.

The stability of trans-(Pt(NH3)2[d(CGAG)-N7-G,N7-G]) adducts, resulting from cross-links between two guanine residues at d(CGAG) sites within single-stranded oligonucleotides by trans-diamminedichloro-platinum(II), has been studied under various conditions of temperature, salt and pH. The trans-(Pt(NH3)2[d(C GAG)-N7-G,N7-G]) cross-links rearrange into trans-(Pt(NH3)2[d(CGAG)-N3-C,N7-G]) cross-links. The rate of rearrangement is independent of pH, in the range 5-9, and of the nature and concentration of the salt (NaCl or NaCIO4) in the range 10-400 mM. The reaction rate depends upon temperature, the t1/2 values for the disappearance of the (G,G) intrastrand cross-link ranging from 120 h at 30 degrees C to 70 min at 80 degrees C. The linkage isomerization reaction occurs in oligonucleotides as short as the platinated tetramer d(CGAG). Replacement of the intervening residue A by T has no major effect on the reaction. The C residue adjacent to the adduct on the 5' side plays a key-role in the reaction; its replacement by a G, A or T residue prevents the reaction occuring. No rearrangement was observed with the C residue adjacent to the adduct on the 3' side. It is proposed that the linkage isomerization reaction results from a direct attack of the base residue on the platinum(II) square complex.     

On the possibilities and limitations of rational protein design to expand the specificity of restriction enzymes: a case study employing EcoRV as the target

The restriction endonuclease EcoRV has been characterized in structural and functional terms in great detail. Based on this detailed information we employed a structure-guided approach to engineer variants of EcoRV that should be able to discriminate between differently flanked EcoRV recognition sites. In crystal structures of EcoRV complexed with d(CGGGATATCCC)(2) and d(AAAGATATCTT)(2), Lys104 and Ala181 closely approach the two base pairs flanking the GATATC recognition site and thus were proposed to be a reasonable starting point for the rational extension of site specificity in EcoRV [Horton,N.C. and Perona,J.J. (1998) J. Biol. Chem., 273, 21721-21729]. To test this proposal, several single (K104R, A181E, A181K) and double mutants of EcoRV (K104R/A181E, K104R/A181K) were generated. A detailed characterization of all variants examined shows that only the substitution of Ala181 by Glu leads to a considerably altered selectivity with both oligodeoxynucleotide and macromolecular DNA substrates, but not the predicted one, as these variants prefer cleavage of a TA flanked site over all other sites, under all conditions tested. The substitution of Lys104 by Arg, in contrast, which appeared to be very promising on the basis of the crystallographic analysis, does not lead to variants which differ very much from the EcoRV wild-type enzyme with respect to the flanking sequence preferences. The K104R/A181E and K104R/A181K double mutants show nearly the same preferences as the A181E and A181K single mutants. We conclude that even for the very well characterized restriction enzyme EcoRV, properties that determine specificity and selectivity are difficult to model on the basis of the available structural information.     

Characterization of the hepatitis C virus-encoded serine proteinase: determination of proteinase-dependent polyprotein cleavage sites.

Processing of the hepatitis C virus (HCV) H strain polyprotein yields at least nine distinct cleavage products: NH2-C-E1-E2-NS2-NS3-NS4A-NS4B-NS5A-NS5B-CO OH. As described in this report, site-directed mutagenesis and transient expression analyses were used to study the role of a putative serine proteinase domain, located in the N-terminal one-third of the NS3 protein, in proteolytic processing of HCV polyproteins. All four cleavages which occur C terminal to the proteinase domain (3/4A, 4A/4B, 4B/5A, and 5A/5B) were abolished by substitution of alanine for either of two predicted residues (His-1083 and Ser-1165) in the proteinase catalytic triad. However, such substitutions have no observable effect on cleavages in the structural region or at the 2/3 site. Deletion analyses suggest that the structural and NS2 regions of the polyprotein are not required for the HCV NS3 proteinase activity. NS3 proteinase-dependent cleavage sites were localized by N-terminal sequence analysis of NS4A, NS4B, NS5A, and NS5B. Sequence comparison of the residues flanking these cleavage sites for all sequenced HCV strains reveals conserved residues which may play a role in determining HCV NS3 proteinase substrate specificity. These features include an acidic residue (Asp or Glu) at the P6 position, a Cys or Thr residue at the P1 position, and a Ser or Ala residue at the P1' position.     

Enzymatic synthesis of oligonucleotides of defined sequence. Addition of short blocks of nucleotide residues to oligonucleotide primers.

Polynucleotide phosphorylase from Escherichia coli can be used to catalyse the addition of short tracts of deoxyadenylate residues to the 3'-termini of deoxyribooligonucleotides of the type pdAn-dN (where dN = dC, dT or dG) using dADP as donor. Similarly, the enzyme can also be used to catalyse the addition of short tracts of adenylate residues to the 3'-termini of ribooligonucleotides of the type An-N (where N = C, U or G) using ADP as donor. In the ribooligonucleotide series, phosphorolytic cleavage of the primer oligonucleotides is significant and results in the concommitant production of oligoadenylates lacking the N residue. Oligomers of the same length, with and without the residue N, were readily separated by thermal elution from cellulose-pdT9 columns. This latter procedure therefore provides a simple method for purification of the oligoadenylates containing an internal base substitution and it also provides a convenient assay for oligonucleotide phosphorolysis.     

Quantitative amino acid makeup and the characteristics of the amino groups of ristomycin and the products of its partial acid hydrolysis]

The quantitative amino acid composition of ristomycin A, a glycopeptide antibiotic, peptides I-IV (from partial acid hydrolysis of the antibiotic) and their dinitrophenylic derivatives was determined. It was shown that both ristomycin and free peptides I-IV contained one residue of ristomycinic acid and one residue of actinoidinic acid, diamino-dicarbonic amino acids of the glycylphenolic type. Peptides I-IV had close molecular weights, i.e. 1100-1200 and differed from each other in the gradually increasing numbers of NH2- and COON- groups, from one in peptide I to four in peptide IV. The quantitative amino acid analysis of the dinitrophenylic derivatives of ristomycin and peptides I-IV showed that the free NH2-group in peptide I belonged to ristomycinic acid, the same as in the antibiotic, while in peptides III-IV at least one of the free NH2-groups belonged to ristomycinic acid and the other belonged to actinoidinic acid.     

Identification of methionine Nalpha-acetyltransferase from Saccharomyces cerevisiae

N alpha-Acetylation is the most frequently occurring chemical modification of the alpha-NH2 group of eukaryotic proteins and was believed until now to be catalyzed by a single N alpha-acetyltransferase. The transfer of an acetyl group from acetyl coenzyme A to the alpha-amino group of five NH2-terminal residues (serine, alanine, methionine, glycine, and threonine) in proteins accounts for approximately 95% of acetylated residues. We have found that a crude lysate from Saccharomyces cerevisiae mutant (aaa1) deficient in N alpha-acetyltransferase activity can effectively transfer an acetyl group to peptides containing NH2-terminal methionine but not to serine or alanine. This methionine N alpha-acetyltransferase has been extensively purified, and this purified enzyme can selectively transfer an acetyl group to various model peptides containing an NH2-terminal methionine residue and a penultimate aspartyl, asparaginyl, or glutamyl residue. Such specificity of N alpha-acetylation of methionine has been previously observed based on the analysis of eukaryotic protein sequences (Persson, B., Flinta, C., Heijne, G., and Jornvall, H. (1985) Eur. J. Biochem. 152, 523-527; Arfin, S.M., and Bradshaw, R. A. (1988) Biochemistry 27, 7979-7984). The indentification of this methionine N alpha-acetyltransferase provides an explanation as to why two distinct classes of N alpha-acetylated proteins exist in nature: (i) those whose initiator methionine is acetylated and (ii) those whose penultimate residue is acetylated after cleavage of the initiator methionine.     

Properties of 2'-fluorothymidine-containing oligonucleotides: interaction with restriction endonuclease EcoRV

2'-Fluorothymidine (Tf) was synthesized via an improved procedure with (diethylamino)sulfur trifluoride. The compatibility of the analogue with DNA synthesis via the phosphoramidite method was demonstrated after complete enzymatic digestion of the oligonucleotides d(Tf11T) and d(Tf3T), the sole products of which were 2'-fluorothymidine and thymidine in the expected ratio. The 2'-fluorothymidine was also incorporated into the EcoRV recognition sequence (underlined), within the complementary oligonucleotides d(CAAACCGATATCGTTGTG) and d(CACAACGATATCGGTTTG). Thermal melting characteristics of these duplexes showed a significant decrease in stability only when both of the thymidine residues in one of the strands were replaced. In contrast, when all of one strand of a duplex contained 2'-fluorothymidine, as in d(Tf11T).d(A12), a substantially higher Tm and cooperativity of melting was observed relative to the unmodified structure. EcoRV cleaved a duplex that contained a 2'-fluorothymidine at the scissile linkage in each strand at two-thirds of the rate obtained for the unmodified structure. A duplex containing two 2'-fluorothymidine residues in one strand and none in the other was cleaved at one-third of the rate in its unsubstituted strand, whereas the cleavage rate was reduced to 22% in its modified strand.     

The exocyclic amine at the RNase P cleavage site contributes to substrate binding and catalysis

Most tRNAs carry a G at their 5' termini, i.e. at position +1. This position corresponds to the position immediately downstream of the site of cleavage in tRNA precursors. Here we studied RNase P RNA-mediated cleavage of substrates carrying substitutions/modifications at position +1 in the absence of the RNase P protein, C5, to investigate the role of G at the RNase P cleavage site. We present data suggesting that the exocyclic amine (2NH2) of G+1 contributes to cleavage site recognition, ground state binding and catalysis by affecting the rate of cleavage. This is in contrast to O6, N7 and 2'OH that are suggested to affect ground state binding and rate of cleavage to significantly lesser extent. We also provide evidence that the effects caused by the absence of 2NH2 at position +1 influenced the charge distribution and conceivably Mg2+ binding at the RNase P cleavage site. These findings are consistent with models where the 2NH2 at the cleavage site (when present) interacts with RNase P RNA and/or influences the positioning of Mg2+ in the vicinity of the cleavage site. Moreover, our data suggest that the presence of the base at +1 is not essential for cleavage but its presence suppresses miscleavage and dramatically increases the rate of cleavage. Together our findings provide reasons why most tRNAs carry a guanosine at their 5' end.     

Post-translational addition of an arginine moiety to acidic NH2 termini of proteins is required for their recognition by ubiquitin-protein ligase

Recent studies have shown that selection of proteins for degradation by the ubiquitin system occurs most probably by binding to specific sites of the ubiquitin-protein ligase, E3. A free alpha-NH2 residue of the substrate is one important determinant recognized by the ligase. Selective binding sites have been described for basic and bulky-hydrophobic NH2 termini (Reiss, Y., Kaim, D., and Hershko, A. (1988) J. Biol. Chem. 263, 2693-2698) and for alanine, serine, and threonine at the NH2-terminal position (Gonda, D. K., Bachmair, A., Wünning, I., Tobias, J. W., Lane, W. S., and Varshavsky, A. (1989) J. Biol. Chem. 264, 16700-16712). Proteins with acidic NH2-terminal residues are degraded by the ubiquitin system only following conversion of the acidic residue to a basic residue by the addition of an arginine moiety (Ferber, S., and Ciechanover, A. (1987) Nature 326, 808-811). Although the enzymes involved in this post-translational modification have been characterized, the underlying mechanism has been obscure. By using a chemical cross-linking technique, we demonstrate that proteins with acidic NH2 termini do not bind to E3 without prior modification of this residue by the addition of arginine. In contrast, proteins with a basic NH2-terminal residue bind to the ligase without any modification. The recognition of acidic NH2-terminal substrates by E3 is dependent upon the addition of all the components of the modifying machinery, arginyl-tRNA-protein transferase, arginyl-tRNA synthetase, tRNA, and arginine. The ligase-bound modified proteins are converted to ubiquitin conjugates in a "pulse-chase" experiment, indicating that the binding is functional and that the enzyme-substrate complex is an obligatory intermediate in the conjugation process. Chemical modification of the carboxyl groups, which results in their neutralization, generates substrates that bind to E3 without modification. This finding suggests that the amino-terminal binding site of E3 is negatively charged, and only positively charged amino-terminal residues may bind to it. Negatively charged (acidic) NH2-terminal residues will bind only following neutralization or reversal of the charge.     

Site-directed mutagenesis studies with EcoRV restriction endonuclease to identify regions involved in recognition and catalysis.

Guided by the X-ray structure analysis of a crystalline EcoRV-d(GGGATATCCC) complex (Winkler, in preparation), we have begun to identify functionally important amino acid residues of EcoRV. We show here that Asn70, Asp74, Ser183, Asn185, Thr186, and Asn188 are most likely involved in the binding and/or cleavage of the DNA, because their conservative substitution leads to mutants of no or strongly reduced activity. In addition, C-terminal amino acid residues of EcoRV seem to be important for its activity, since their deletion inactivates the enzyme. Following the identification of three functionally important regions, we have inspected the sequences of other restriction and modification enzymes for homologous regions. It was found that two restriction enzymes that recognize similar sequences as EcoRV (DpnII and HincII), as well as two modification enzymes (M.DpnII and, in a less apparent form, M.EcoRV), have the sequence motif -SerGlyXXXAsnIleXSer- in common, which in EcoRV contains the essential Ser183 and Asn188 residues. Furthermore, the C-terminal region, shown to be essential for EcoRV, is highly homologous to a similar region in the restriction endonuclease SmaI. On the basis of these findings we propose that these restriction enzymes and to a certain extent also some of their corresponding modification enzymes interact with DNA in a similar manner.     

How are substrates recognized by the ubiquitin-mediated proteolytic system

A necessary step in the degradation of proteins by the ubiquitin system is recognition by the ubiquitin-protein ligases(s). Various structural features of the proteolytic substrate render it susceptible to conjugation with ubiquitin. The N-terminal residue plays a major role in this process, with distinct sites on the ligase(s) recognizing specific types of N-termini. Post-translational modification of some of these residues is required prior to their recognition. A free N terminus is not the only marker; proteins with either free or blocked N termini can be recognized via structural domains that are downstream and distinct from this residue.     

Substrate specificity of SpoIIGA, a signal-transducing aspartic protease in Bacilli

SpoIIGA is a novel type of membrane-associated aspartic protease that responds to a signal from the forespore by cleaving Pro-σ(E) in the mother cell during sporulation of Bacillus subtilis. Very little is known about how SpoIIGA recognizes Pro-σ(E). By co-expressing proteins in Escherichia coli, it was shown that charge reversal substitutions for acidic residues 24 and 25 of Pro-σ(E), and for basic residues 245 and 284 of SpoIIGA, impaired cleavage. These results are consistent with a model predicting possible electrostatic interactions between these residues; however, no charge reversal substitution for residue 245 or residue 284 of SpoIIGA restored cleavage of Pro-σ(E) with a charge reversal substitution for residue 24 or residue 25. Bacillus subtilis SpoIIGA cleaved Pro-σ(E) orthologs from Bacillus licheniformis and Bacillus halodurans, but not from Bacillus cereus. A triple substitution in the pro-sequence of B. cereus Pro-σ(E) allowed cleavage by B. subtilis SpoIIGA, indicating that residues distal from the cleavage site contribute to substrate specificity. Co-expression of SpoIIGA and Pro-σ(E) orthologs in different combinations suggested that B. licheniformis SpoIIGA has a relatively narrow substrate specificity as compared with B. subtilis SpoIIGA, whereas B. cereus SpoIIGA and B. halodurans SpoIIGA appear to have broader substrate specificity.     

Sequence-specific DNA cleavage by dipeptides disubstituted with chlorambucil and 2,6-dimethoxyhydroquinone-3-mercaptoacetic acid.

Two dipeptides, each containing a lysyl residue, were disubstituted with chlorambucil (CLB) and 2,6-dimethoxyhydroquinone-3-mercaptoacetic acid (DMQ-MA): DMQ-MA-Lys(CLB)-Gly-NH2 (DM-KCG) and DMQ-MA-beta-Ala-Lys(CLB)-NH2 (DM-BKC). These peptide-drug conjugates were designed to investigate sequence-specificity of DNA cleavage directed by the proximity effect of the DNA cleavage chromophore (DMQ-MA) situated close to the alkylating agent (CLB) inside a dipeptide moiety. Agarose electrophoresis studies showed that DM-KCG and DM-BKC possess significant DNA nicking activity toward supercoiled DNA whereas CLB and its dipeptide conjugate Boc-Lys(CLB)-Gly-NH2 display little DNA nicking activity. ESR studies of DMQ-MA and DM-KCG both showed five hyperfine signals centered at g = 2.0052 and are assigned to four radical forms at equilibrium, which may give rise to a semiquinone radical responsible for DNA cleavage. Thermal cleavage studies at 90 degrees C on a 265-mer test DNA fragment showed that besides alkylation and cleavage at G residues, reactions with DM-KCG and DM-BKC show a preference for A residues with the sequence pattern: 5'-G-(A)n-Pur-3' > 5'-Pyr-(A)n-Pyr-3' (where n = 2-4). By contrast, DNA alkylation and cleavage by CLB occurs at most G and A residues with less sequence selectivity than seen with DM-KCG and DM-BKC. Thermal cleavage studies using N7-deazaG and N7-deazaA-substituted DNA showed that strong alkylation and cleavage at A residues by DM-KCG and DM-BKC is usually flanked on the 3' side by a G residue whereas strong cleavage at G residues is flanked by at least one purine residue on either the 5' or 3' side. At 65 degrees C, it is notable that the preferred DNA cleavage by DM-KCG and DM-BKC at A residues is significantly more marked than for G residues in the 265-mer DNA; the strongest sites of A-specific reaction occur within the sequences 5'-Pyr-(A)n-Pyr-3'; 5'-Pur-(A)n-G-3' and 5'-Pyr-(A)n-G-3'. In pG4 DNA, cleavage by DM-KCG and DM-BKC is much greater than that by CLB at room temperature and at 65 degrees C. It was also observed that DM-KCG and DM-BKC cleaved at certain pyrimidine residues: C40, T66, C32, T34, and C36. These cleavages were also sequence selective since the susceptible pyrimidine residues were flanked by two purine residues on both the 5' and 3' sides or by a guanine residue on the 5' side. These findings strongly support the proposal that once the drug molecule is positioned so as to permit alkylation by the CLB moiety, the DMQ-MA moiety is held close to the alkylation site, resulting in markedly enhanced sequence-specific cleavage.     

Ubiquitination of p53 at multiple sites in the DNA-binding domain

The tumor suppressor p53 is negatively regulated by the ubiquitin ligase MDM2. The MDM2 recognition site is at the NH2-terminal region of p53, but the positions of the actual ubiquitination acceptor sites are less well defined. Lysine residues at the COOH-terminal region of p53 are implicated as sites for ubiquitination and other post-translational modifications. Unexpectedly, we found that substitution of the COOH-terminal lysine residues did not diminish MDM2-mediated ubiquitination. Ubiquitination was not abolished even after the entire COOH-terminal regulatory region was removed. Using a method involving in vitro proteolytic cleavage at specific sites after ubiquitination, we found that p53 was ubiquitinated at the NH2-terminal portion of the protein. The lysine residue within the transactivation domain is probably not essential for ubiquitination, as substitution with an arginine did not affect MDM2 binding or ubiquitination. In contrast, several conserved lysine residues in the DNA-binding domain are critical for p53 ubiquitination. Removal of the DNA-binding domain reduced ubiquitination and increased the stability of p53. These data provide evidence that in addition to the COOH-terminal residues, p53 may also be ubiquitinated at sites in the DNA-binding domain.     

A site-directed mutagenesis study to identify amino acid residues involved in the catalytic function of the restriction endonuclease EcoRV.

We have used site-directed mutagenesis of the EcoRV restriction endonuclease to change amino acid side chains that have been shown crystallographically to be in close proximity to the scissile phosphodiester bond of the DNA substrate. DNA cleavage assays of the resulting mutant proteins indicate that the largest effects on nucleolytic activity result from substitution of Asp74, Asp90, and Lys92. We suggest on the basis of structural information, mutagenesis data, and analogies with other nucleases that Asp74 and Asp90 might be involved in Mg2+ binding and/or catalysis and that Lys92 probably stabilizes the pentacovalent phosphorus in the transition state. These amino acids are part of a sequence motif, Pro-Asp...Asp/Glu-X-Lys, which is also present in EcoRI. In both enzymes, it is located in a structurally similar context near the scissile phosphodiester bond. A preliminary mutational analysis with EcoRI indicates that this sequence motif is of similar functional importance for EcoRI and EcoRV. On the basis of these results, a proposal is made for the mechanism of DNA cleavage by EcoRV and EcoRI.