Effects on NaeI-DNA recognition of the leucine to lysine substitution that transforms restriction endonuclease NaeI to a topoisomerase: a model for restriction endonuclease evolution.

Substituting lysine for leucine at position 43 (L43K) transforms NaeI from restriction endonuclease to topoisomerase and makes NaeI hypersensitive to intercalative anticancer drugs. Here we investigated DNA recognition by Nael-L43K. Using DNA competition and gel retardation assays, NaeI-L43K showed reduced affinity for DNA substrate and the ability to bind both single- and double-stranded DNA with a definite preference for the former. Sedimentation studies showed that under native conditions NaeI-L43K, like NaeI, is a dimer. Introduction of mismatched bases into double-stranded DNA significantly increased that DNA's ability to inhibit NaeI-L43K. Wild-type NaeI showed no detectable binding of either single-stranded DNA or mismatched DNA over the concentration range studied. These results demonstrate that the L43K substitution caused a significant change in recognition specificity by NaeI and imply that NaeI-L43K's topoisomerase activity is related to its ability to bind single-stranded and distorted regions in DNA. A mechanism is proposed for the evolution of the NaeI restriction-modification system from a topoisomerase/ligase by a mutation that abolished religation activity and provided a needed change in DNA recognition.     

Alkylation of cysteine 41, but not cysteine 200, decreases the ADP-ribosyltransferase activity of the S1 subunit of pertussis toxin

Sulfhydryl-alkylating reagents are known to inactivate the NAD glycohydrolase and ADP-ribosyltransferase activities of the S1 subunit of pertussis toxin, a protein which contains two cysteines at positions 41 and 200. It has been proposed that NAD can retard alkylation of one of the two cysteines of this protein (Kaslow, H.R., and Lesikar, D.D. (1987) Biochemistry 26, 4397-4402). We now report that NAD retards the ability of these alkylating reagents to inactivate the S1 subunit. In order to determine which cysteine is protected by NAD, we used site-directed mutagenesis to construct analogs of the toxin with serines at positions 41 and/or 200. Sulfhydryl-alkylating reagents reduced the ADP-ribosyltransferase activity of the analog with a single cysteine at position 41; NAD retarded this inactivation. In contrast, sulfhydryl-alkylating reagents did not inactivate analogs with serine at position 41. An analog with alanine at position 41 possessed substantial ADP-ribosyltransferase activity. We conclude that alkylation of cysteine 41, and not cysteine 200, inactivates the S1 subunit of pertussis toxin, but that the sulfhydryl group of cysteine 41 is not essential for the ADP-ribosyltransferase activity of the toxin. These results suggest that the region near cysteine 41 contributes to features of the S1 subunit important for ADP-ribosyltransferase activity. Using site-directed mutagenesis, we found that changing aspartate 34 to asparagine, arginine 39 to lysine, and glutamine 42 to glutamate had little effect on ADP-ribosyltransferase activity. However, substituting an asparagine for the histidine at position 35 markedly decreased, but did not eliminate, ADP-ribosyltransferase activity. Chou-Fasman analysis predicted no significant modifications in secondary structure of the S1 peptide with the change of histidine 35 to asparagine. Thus, histidine 35 may interact with a substrate of the S1 subunit without being essential for catalysis.     

Structure of NaeI-DNA complex reveals dual-mode DNA recognition and complete dimer rearrangement

NaeI, a novel DNA endonuclease, shows topoisomerase and recombinase activities when a Lys residue is substituted for Leu 43. The NaeI-DNA structure demonstrates that each of the two domains of NaeI recognizes one molecule of DNA duplex. DNA recognition induces dramatic rearrangements: narrowing the binding site of the Topo domain 16 A to grip DNA, widening that of the Endo domain 8 A to encircle and bend DNA 45 degrees for cleavage, and completely rebuilding the homodimer interface. The NaeI-DNA structure presents the first example of novel recognition of two copies of one DNA sequence by two different amino acid sequences and two different structural motifs in one polypeptide.     

Evidence for mutations that break communication between the Endo and Topo domains in NaeI endonuclease/topoisomerase

NaeI is a type IIe endonuclease that interacts with two DNA recognition sequences to cleave DNA. One DNA sequence serves as a substrate and the other serves to activate cleavage. NaeI is divided into two domains whose structures parallel the two functionalities recognized in NaeI, endonuclease and topoisomerase. In this study, we report evidence for mutations that break interdomain functional communication in a NaeI-DNA complex. Deletion of the initial 124 amino acids of the N-terminal domain of NaeI converted NaeI to a monomer, consistent with self-association being mediated by the Endo domain. Deletions within a small region of the C-terminal DNA binding domain of NaeI (amino acids 182-192) altered the recognition by NaeI of sequences flanking the NaeI recognition sequence. Substituting Ala for Arg182 within this region had no apparent effect on DNA binding but greatly reduced the extent of DNA cleavage even though it is not part of the catalytic Endo domain. Substituting Ala for Ile185 reduced the extent of DNA binding about 1000-fold. Substituting Ala for Lys189 altered flanking sequence recognition. Residues 182-192 are away from the Endo domain responsible for cleavage and also face away from the modeled DNA binding faces of the apoprotein crystal structure. We propose that residues 182-192 are part of a web that mediates the flow of information between the NaeI Endo and Topo domains.     

NaeI endonuclease binding to pBR322 DNA induces looping.

Previous work has demonstrated the existence of both resistant and cleavable NaeI sites. Cleavable sites introduced on exogenous DNA can act in trans to increase the catalysis of NaeI endonuclease cleavage at resistant sites without affecting the apparent binding affinity of the enzyme for the resistant site [Conrad, M., & Topal, M. D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9707-9711]. This activation suggests allosteric regulation of NaeI cleavage by distant cis- and trans-acting sites in DNAs containing both resistant and cleavable sites. Plasmid pBR322 contains four NaeI sites, at least one of which is resistant to cleavage. Electron microscopy is used here to demonstrate that NaeI endonuclease simultaneously binds to multiple recognition sites in pBR322 DNA to form loops with NaeI protein bound at the loop's base. The maximum number of loops formed with a common base suggests four binding sites per enzyme molecule. Looping was inhibited by addition of enzyme-saturating amounts of double-stranded oligonucleotide containing an NaeI site, whereas another double-strand oligonucleotide without the NaeI site had no effect. The number of loops seen was not above background when double-stranded M13 DNA, which contains only a single NaeI recognition site, was used as substrate.     

Step-wise DNA relaxation and decatenation by NaeI-43K.

Nae I protein was originally isolated for its restriction endonuclease properties. Nae I was later discovered to either relax or cleave supercoiled DNA, depending upon whether Nae I position 43 contains a lysine (43K) or leucine (43L) respectively. Nae I-43K DNA relaxation activity appears to be the product of coupling separate endonuclease and ligase domains within the same polypeptide. Whereas Nae I relaxes supercoiled DNA like a topoisomerase, even forming a transient covalent intermediate with the substrate DNA, Nae I shows no obvious sequence similarity to the topoisomerases. To further characterize the topoisomerase activity of Nae I, we report here that Nae I-43K changes the linking number of a single negatively supercoiled topoisomer of pBR322 by units of one and therefore is a type I topoisomerase. Positively supercoiled pBR322 was resistant to Nae I-43K. At low salt concentration Nae I-43K was processive; non-saturating amounts of enzyme relaxed a fraction of the DNA. At high salt concentration the same non-saturating amounts of Nae I-43K partially relaxed all the DNA in a step-wise fashion to give a Gaussian distribution of topoisomers, demonstrating a switch from a processive to a distributive mode of action. Nae I-43K decatenated kinetoplast DNA containing nicked circles, implying that Nae I-43K can cleave opposite a nick. The products of the reaction are decatenated nicked circles under both processive and distributive conditions. The behavior of Nae I-43K is consistent with that of a prokaryotic type I topoisomerase.     

Biochemical characterization of an invariant histidine involved in Escherichia coli DNA topoisomerase I catalysis.

An invariant histidine residue, His-365 in Escherichia coli DNA topoisomerase I, is located at the active site of type IA DNA topoisomerases and near the active site tyrosine. Its ability to participate in the multistep catalytic process of DNA relaxation was investigated. His-365 was mutated to alanine, arginine, asparagine, aspartate, glutamate, and glutamine to study its ability to participate in general acid/base catalysis and bind DNA. The mutants were examined for pH-dependent DNA relaxation and cleavage, salt-dependent DNA relaxation, and salt-dependent DNA binding affinity. The mutants relax DNA in a pH-dependent manner and at low salt concentrations. The pH dependence of all mutants is different from the wild type, suggesting that His-365 is responsible for the pH dependence of the enzyme. Additionally, whereas the wild type enzyme shows pH-dependent oligonucleotide cleavage, cleavage by both H365Q and H365A is pH-independent. H365Q cleaves DNA with rates similar to the wild type enzyme, whereas H365A has a slower rate of DNA cleavage than the wild type but can cleave more substrate overall. H365A also has a lower DNA binding affinity than the wild type enzyme. The binding affinity was determined at different salt concentrations, showing that the alanine mutant displaces half a charge less upon binding DNA than an inactive form of topoisomerase I. These observations indicate that His-365 participates in DNA binding and is responsible for optimal catalysis at physiological pH.     

Amino Acid transport and metabolism in relation to the nitrogen economy of a legume leaf

Net balances of amino acids were constructed for stages of development of a leaf of white lupin (Lupinus albus L.) using data on the N economy of the leaf, its exchanges of amino acids through xylem and phloem, and net changes in its soluble and protein-bound amino acids. Asparagine, aspartate, and γ-aminobutyrate were delivered to the leaf in excess of amounts consumed in growth and/or phloem export. Glutamine was supplied in excess until full leaf expansion (20 days) but was later synthesized in large amounts in association with mobilization of N from the leaf. Net requirements for glutamate, threonine, serine, proline, glycine, alanine, valine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, and arginine were met mainly or entirely by synthesis within the leaf. Amides furnished the bulk of the N for amino acid synthesis, asparagine providing from 24 to 68%. In vitro activity of asparaginase (EC 3.5.1.1) exceeded that of asparagine:pyruvate aminotransferase (EC 2.6.1.14) during early leaf expansion, when in vivo estimates of asparagine metabolism were highest. Thereafter, aminotransferase activity greatly exceeded that of asparaginase. Rates of activity of one or both asparagine-utilizing enzymes exceeded estimated rates of asparagine catabolism throughout leaf development. In vitro activities of glutamine synthetase (EC 6.3.1.2) and glutamate synthase (EC 1.4.7.1) were consistently much higher than that of glutamate dehydrogenase (EC 1.4.1.3), and activities of the former two enzymes more than accounted for estimated rates of ammonia release in photorespiration and deamidation of asparagine.     

Chemotaxis toward amino acids in Escherichia coli

Escherichia coli cells are shown to be attracted to the l-amino acids alanine, asparagine, aspartate, cysteine, glutamate, glycine, methionine, serine, and threonine, but not to arginine, cystine, glutamine, histidine, isoleucine, leucine, lysine, phenylalanine, tryptophan, tyrosine, or valine. Bacteria grown in a proline-containing medium were, in addition, attracted to proline. Chemotaxis toward amino acids is shown to be mediated by at least two detection systems, the aspartate and serine chemoreceptors. The aspartate chemoreceptor was nonfunctional in the aspartate taxis mutant, which showed virtually no chemotaxis toward aspartate, glutamate, or methionine, and reduced taxis toward alanine, asparagine, cysteine, glycine, and serine. The serine chemoreceptor was nonfunctional in the serine taxis mutant, which was defective in taxis toward alanine, asparagine, cysteine, glycine, and serine, and which showed no chemotaxis toward threonine. Additional data concerning the specificities of the amino acid chemoreceptors with regard to amino acid analogues are also presented. Finally, two essentially nonoxidizable amino acid analogues, alpha-aminoisobutyrate and alpha-methylaspartate, are shown to be attractants for E. coli, demonstrating that extensive metabolism of attractants is not required for amino acid taxis.     

Oligonucleotide site directed mutagenesis of all histidine residues within the T4 endonuclease V gene: role in enzyme-nontarget DNA binding

In order to evaluate the contributions that histidine residues might play both in the catalytic activities of endonuclease V and in binding to nontarget DNA, the technique of oligonucleotide site directed mutagenesis was used to create mutations at each of the four histidine residues in the endonuclease V gene. Although none of the histidines were shown to be absolutely required for the pyrimidine dimer specific DNA glycosylase activity or the apurinic lyase activity, conservative amino acid changes at His16 produced enzymes with little or no catalytic activity. In addition, the evaluation of conservative and radical amino acid substitutions at positions 34, 56, and 107 is consistent with the interpretation that each of these histidines may be involved in nontarget DNA binding. The data supporting this conclusion are that histidine changes to lysine at positions 34 and 107 enhance the nontarget DNA binding activity of the mutant enzymes while neutralization of charge at His56 reduces nontarget DNA binding.     

Characterization of human UDP-glucose dehydrogenase reveals critical catalytic roles for lysine 220 and aspartate 280

Human UDP-glucose dehydrogenase (UGDH) is a homohexameric enzyme that catalyzes two successive oxidations of UDP-glucose to yield UDP-glucuronic acid, an essential precursor for matrix polysaccharide and proteoglycan synthesis. We previously used crystal coordinates for Streptococcus pyogenes UGDH to generate a model of the human enzyme active site. In the studies reported here, we have used this model to identify three putative active site residues: lysine 220, aspartate 280, and lysine 339. Each residue was site-specifically mutagenized to evaluate its importance for catalytic activity and maintenance of hexameric quaternary structure. Alteration of lysine 220 to alanine, histidine, or arginine significantly impaired enzyme function. Assaying activity over longer time courses revealed a plateau after reduction of a single equivalent of NAD+ in the alanine and histidine mutants, whereas turnover continued in the arginine mutant. Thus, one role of this lysine may be to stabilize anionic transition states during substrate conversion. Mutation of aspartate 280 to asparagine was also severely detrimental to catalysis. The relative position of this residue within the active site and dependence of function on acidic character point toward a critical role for aspartate 280 in activation of the substrate and the catalytic cysteine. Finally, changing lysine 339 to alanine yielded the wild-type Vmax, but a 165-fold decrease in affinity for UDP-glucose. Interestingly, gel filtration of this substrate-binding mutant also determined it was a dimer, indicating that hexameric quaternary structure is not critical for catalysis. Collectively, this analysis has provided novel insights into the complex catalytic mechanism of UGDH.     

Cloning the FnuDI, NaeI, NcoI and XbaI restriction-modification systems

Methyltransferase genes from the FnuDI, NaeI, NcoI, and XbaI restriction-modification systems have been isolated in Escherichia coli by 'shot-gun' cloning bacterial DNA fragments into plasmid vectors and selecting for protectively modified molecules that resist digestion by the corresponding restriction endonuclease.     

Enhancement of the catalytic properties of human carbonic anhydrase III by site-directed mutagenesis

Among the seven known isozymes of carbonic anhydrase in higher vertebrates, isozyme III is the least efficient in catalytic hydration of CO2 and the least susceptible to inhibition by sulfonamides. We have investigated the role of two basic residues near the active site of human carbonic anhydrase III (HCA III), lysine 64 and arginine 67, to determine whether they can account for some of the unique properties of this isozyme. Site-directed mutagenesis was used to replace these residues with histidine 64 and asparagine 67, the amino acids present at the corresponding positions of HCA II, the most efficient of the carbonic anhydrase isozymes. Catalysis by wild-type HCA III and mutants was determined from the initial velocity of hydration of CO2 at steady state by stopped-flow spectrophotometry and from the exchange of 18O between CO2 and water at chemical equilibrium by mass spectrometry. We have shown that histidine 64 functions as a proton shuttle in carbonic anhydrase by substituting histidine for lysine 64 in HCA III. The enhanced CO2 hydration activity and pH profile of the resulting mutant support this role for histidine 64 in the catalytic mechanism and suggest an approach that may be useful in investigating the mechanistic roles of active-site residues in other isozyme groups. Replacing arginine 67 in HCA III by asparagine enhanced catalysis of CO2 hydration 3-fold compared with that of wild-type HCA III, and the pH profile of the resulting mutant was consistent with a proton transfer role for lysine 64. Neither replacement enhanced the weak inhibition of HCA III by acetazolamide or the catalytic hydrolysis of 4-nitrophenyl acetate.     

On the role of conserved histidine 106 in 10-formyltetrahydrofolate dehydrogenase catalysis: connection between hydrolase and dehydrogenase mechanisms

The enzyme, 10-formyltetrahydrofolate dehydrogenase (FDH), converts 10-formyltetrahydrofolate (10-formyl-THF) to tetrahydrofolate in an NADP(+)-dependent dehydrogenase reaction or an NADP(+)-independent hydrolase reaction. The hydrolase reaction occurs in a 310-amino acid long amino-terminal domain of FDH (N(t)-FDH), whereas the dehydrogenase reaction requires the full-length enzyme. The amino-terminal domain of FDH shares some sequence identity with several other enzymes utilizing 10-formyl-THF as a substrate. These enzymes have two strictly conserved residues, aspartate and histidine, in the putative catalytic center. We have shown recently that the conserved aspartate is involved in FDH catalysis. In the present work we studied the role of the conserved histidine, His(106), in FDH function. Site-directed mutagenesis experiments showed that replacement of the histidine with alanine, asparagine, aspartate, glutamate, glutamine, or arginine in N(t)-FDH resulted in expression of insoluble proteins. Replacement of the histidine with another positively charged residue, lysine, produced a soluble mutant with no hydrolase activity. The insoluble mutants refolded from inclusion bodies adopted a conformation inherent to the wild-type N(t)-FDH, but they did not exhibit any hydrolase activity. Substitution of alanine for three non-conserved histidines located close to the conserved one did not reveal any significant changes in the hydrolase activity of N(t)-FDH. Expressed full-length FDH with the substitution of lysine for the His(106) completely lost both the hydrolase and dehydrogenase activities. Thus, our study showed that His(106), besides being an important structural residue, is also directly involved in both the hydrolase and dehydrogenase mechanisms of FDH. Modeling of the putative hydrolase catalytic center/folate-binding site suggested that the catalytic residues, aspartate and histidine, are unlikely to be adjacent to the catalytic cysteine in the aldehyde dehydrogenase catalytic center. We hypothesize that 10-formyl-THF dehydrogenase reaction is not an independent reaction but is a combination of hydrolase and aldehyde dehydrogenase reactions.     

Real-time observation of DNA looping dynamics of Type IIE restriction enzymes NaeI and NarI

Many restriction enzymes require binding of two copies of a recognition sequence for DNA cleavage, thereby introducing a loop in the DNA. We investigated looping dynamics of Type IIE restriction enzymes NaeI and NarI by tracking the Brownian motion of single tethered DNA molecules. DNA containing two endonuclease recognition sites spaced a few 100 bp apart connect small polystyrene beads to a glass surface. The position of a bead is tracked through video microscopy. Protein-mediated looping and unlooping is then observed as a sudden specific change in Brownian motion of the bead. With this method we are able to directly follow DNA looping kinetics of single protein–DNA complexes to obtain loop stability and loop formation times. We show that, in the absence of divalent cations, NaeI induces DNA loops of specific size. In contrast, under these conditions NarI mainly creates non-specific loops, resulting in effective DNA compaction for higher enzyme concentrations. Addition of Ca²⁺ increases the NaeI-DNA loop lifetime by two orders of magnitude and stimulates specific binding by NarI. Finally, for both enzymes we observe exponentially distributed loop formation times, indicating that looping is dominated by (re)binding the second recognition site.     

Modified DNA fragments activate NaeI cleavage of refractory DNA sites.

Endonuclease NaeI cleaves DNA using a two-site mechanism. The DNA-binding sites are nonidentical: they recognize different families of flanking sequences. A unique NaeI site that is resistant to cleavage resides in M13 double-stranded DNA. NaeI can be activated to cleave this site by small DNA fragments containing one or more NaeI sites. These activators are not practical for genetic engineering because unphosphorylated activators that are consumed during the cleavage of substrate give ends that may interfere with subsequent ligations. We show that a DNA fragment containing phosphorothioate linkages at the NaeI scissile bonds (S-activator) is not cleaved by NaeI, even though this S-activator binds to the substrate site. The S-activator activates NaeI to cleave M13 DNA under conditions that completely exhaust unsubstituted activator. These results demonstrate that activation is not coupled to cleavage of activator, that NaeI reverts to its inactive state soon after dissociation of the EA complex, and that S-activator makes for a nondepletable activator during prolonged incubations.     

Changes in plasma amino acid concentrations with increasing age in patients with propionic acidemia

The objective of the study is to analyze plasma amino acid concentrations in propionic acidemia (PA) for the purpose of elucidating possible correlations between propionyl-CoA carboxylase deficiency and distinct amino acid behavior. Plasma concentrations of 19 amino acids were measured in 240 random samples from 11 patients (6 families) with enzymatically and/or genetically proven propionic acidemia (sampling period, January 2001–December 2007). They were compared with reference values from the literature and correlated with age using the Pearson correlation coefficient test. Decreased plasma concentrations were observed for glutamine, histidine, threonine, valine, isoleucine, leucine, phenylalanine and arginine. Levels of glycine, alanine and aspartate were elevated, while values of serine, asparagine, ornithine and glutamate were normal. For lysine, proline and methionine a clear association was not possible. Significant correlations with age were observed for 13 amino acids (positive correlation: asparagine, glutamine, proline, alanine, histidine, threonine, methionine, arginine; negative correlation: leucine, phenylalanine, ornithine, glutamate and aspartate). This study gives new insight over long-term changes in plasma amino acid concentrations and may provide options for future therapies (e.g., substitution of anaplerotic substances) in PA patients.     

Yeast methionine aminopeptidase I. Alteration of substrate specificity by site-directed mutagenesis

In eukaryotes, two isozymes (I and II) of methionine aminopeptidase (MetAP) catalyze the removal of the initiator methionine if the penultimate residue has a small radius of gyration (glycine, alanine, serine, threonine, proline, valine, and cysteine). Using site-directed mutagenesis, recombinant yeast MetAP I derivatives that are able to cleave N-terminal methionine from substrates that have larger penultimate residues have been expressed. A Met to Ala change at 329 (Met206 in Escherichia coli enzyme) produces an average catalytic efficiency 1.5-fold higher than the native enzyme on normal substrates and cleaves substrates containing penultimate asparagine, glutamine, isoleucine, leucine, methionine, and phenylalanine. Interestingly, the native enzyme also has significant activity with the asparagine peptide not previously identified as a substrate. Mutation of Gln356 (Gln233 in E. coli MetAP) to alanine results in a catalytic efficiency about one-third that of native with normal substrates but which can cleave methionine from substrates with penultimate histidine, asparagine, glutamine, leucine, methionine, phenylalanine, and tryptophan. Mutation of Ser195 to alanine had no effect on substrate specificity. None of the altered enzymes produced cleaved substrates with a fully charged residue (lysine, arginine, aspartic acid, or glutamic acid) or tyrosine in the penultimate position.     

Critical functional requirement for the guanidinium group of the arginine 41 side chain of human epidermal growth factor as revealed by mutagenic inactivation and chemical reactivation.

In a preliminary study we demonstrated that the formation of the epidermal growth factor (EGF) receptor-ligand complex requires the participation of the highly conserved arginine 41 side chain of the growth factor peptide (Engler, D.A., Montelione, G.T., and Niyogi, S.K. (1990) FEBS Lett. 271, 47-50). In an attempt to gain further insight into the nature of this interaction(s), we used both site-directed mutagenesis and chemical modification reagents to produce human EGF (hEGF) analogues with altered chemical properties of the residue 41 side chain. Eight mutant analogues of hEGF were generated, substituting arginine 41 with lysine, glutamine, isoleucine, tyrosine, glycine, alanine, aspartate, or glutamate. Although each of the mutant analogues was able to displace wild-type hEGF fully in receptor competition binding assays, affinity of the receptor for the mutants was substantially reduced, varying from 0.4 to less than 0.01% of that observed for wild-type growth factor. At sufficiently high concentrations these mutants were able to stimulate DNA synthesis in mouse keratinocytes. Substitution of lysine for arginine 41 reduced the receptor affinity 250-fold from that observed for wild type, despite retention of the positive electrostatic charge. The lysine substitution leaves a reactive amine at position 41 and made it possible, using amine-specific chemical modification reagents, to produce selected arginine homologues that were tested for their effects on receptor binding, receptor tyrosine kinase activation, and stimulation of DNA synthesis in mouse keratinocytes. The reaction of lysine 41 with methyl acetimidate resulted in a lysineacetamidine product which only partially restored activity of the lysine hEGF mutant. However, reaction with O-methylisourea resulted in generation of an arginine 41 homologue (homoarginine) which restored full activity. The results indicate that the chemical properties inherent in the guanidinium group of the arginine 41 side chain of hEGF are responsible for optimal receptor-ligand association.     

Effect of amino acid substitutions in the rad50 ATP binding domain on DNA double strand break repair in yeast

The Saccharomyces cerevisiae Rad50-Mre11-Xrs2 complex plays a central role in the cellular response to DNA double strand breaks. Rad50 has a globular ATPase head domain with a long coiled-coil tail. DNA binding by Rad50 is ATP-dependent and the Rad50-Mre11-Xrs2 complex possesses DNA unwinding and endonuclease activities that are regulated by ATP. Here we have examined the role of the Rad50 Walker type A ATP binding motif in DNA double strand break repair by a combination of genetic and biochemical approaches. Replacement of the conserved lysine residue within the Walker A motif with alanine, glutamate, or arginine results in the same DNA damage sensitivity and homologous recombination defect as the rad50 deletion mutation. The Walker A mutations also cause a deficiency in non-homologous end-joining. As expected, complexes containing the rad50 Walker A mutant proteins are defective in ATPase, ATP-dependent DNA unwinding, and ATP-stimulated endonuclease activities. Although the DNA end-bridging activity of the Rad50-Mre11-Xrs2 complex is ATP-independent, the end-bridging activity of complexes containing the rad50 Walker A mutant proteins is salt-sensitive. These results provide a molecular explanation for the observed in vivo defects of the rad50 Walker mutant strains and reveal a novel ATP-independent function for Rad50 in DNA end-bridging.