Biomedical subjects

U H Mortensen

Publications and source records attributed to U H Mortensen.

15 recordsLinked to original sources

PubMed2001

Rad52 forms DNA repair and recombination centers during S phase.

Maintenance of genomic integrity and stable transmission of genetic information depend on a number of DNA repair processes. Failure to faithfully perform these processes can result in genetic alterations and subsequent development of cancer and other genetic diseases. In the eukaryote Saccharomyces cerevisiae, homologous recombination is the major pathway for repairing DNA double-strand breaks. The key role played by Rad52 in this pathway has been attributed to its ability to seek out and mediate annealing of homologous DNA strands. In this study, we find that S. cerevisiae Rad52 fused to green fluorescent protein (GFP) is fully functional in DNA repair and recombination. After induction of DNA double-strand breaks by gamma-irradiation, meiosis, or the HO endonuclease, Rad52-GFP relocalizes from a diffuse nuclear distribution to distinct foci. Interestingly, Rad52 foci are formed almost exclusively during the S phase of mitotic cells, consistent with coordination between recombinational repair and DNA replication. This notion is further strengthened by the dramatic increase in the frequency of Rad52 focus formation observed in a pol12-100 replication mutant and a mec1 DNA damage checkpoint mutant. Furthermore, our data indicate that each Rad52 focus represents a center of recombinational repair capable of processing multiple DNA lesions.

Cell Nucleus↗

PubMed1998

Reversed-flow affinity elution applied to the purification of carboxypeptidase Y.

In the present study we describe a novel method for obtaining highly pure carboxypeptidase Y, or derivatives thereof, in a single-step purification procedure. The method is based on affinity chromatography and the results demonstrate that an efficient method is obtained only when the affinity gel is fully saturated with enzyme. Thus, pilot experiments are required to determine the binding capacity of the resin with respect to a given enzyme. To avoid this additional experimental effort, we have developed a method utilizing reversed-flow affinity elution. The method has been successfully employed to purify hundreds of carboxypeptidase Y mutant enzymes.

Carboxypeptidases↗

PubMed1997

Cloning-free PCR-based allele replacement methods.

Efficient homologous recombination permits the directed introduction of specific mutations into the yeast genome. Here we describe a cloning-free, PCR-based allele replacement method that simplifies allele transfer between yeast strains. The desired allele from one strain is amplified by PCR, along with a selectable/counterselectable marker. After transformation, the resident allele in the target strain is replaced by creating a duplication of the new allele. Selection for direct repeat recombinants results in a single copy of the new allele in the target strain. Specifically, the desired allele is amplified by PCR with a pair of adaptamers, which are chimeric oligonucleotides that are used to amplify the allele and differentially tag its 5' and 3' ends. These tags allow the directed fusion to two different, but overlapping, regions of an appropriately tagged selectable/counterselectable marker after a second round of PCR amplification. Following cotransformation of the two fusion fragments into yeast, homologous recombination efficiently generates a duplication of the amplified allele flanking the intact selectable marker in the genome. After counterselection, only the desired allele is retained as a result of direct repeat recombination. A simple modification of this method allows the creation of de novo mutations in the genome.

Alleles↗

PubMed1996

Direct association between the yeast Rad51 and Rad54 recombination proteins.

The RAD54 and RAD51 genes are involved in genetic recombination and double-strand break repair in the yeast Saccharomyces cerevisiae. The Rad51 protein is thought to be a yeast analogue of the Eschericia coli recA gene product and catalyzes strand exchange between homologous single- and double-stranded DNAs in vitro. RAD54 exhibits homologies to several known ATPases and is a member of the SWI2/MOT1 family. We show here that the Rad54 protein interacts with the Rad51 protein in vivo and in vitro and that the NH2-terminal 115 residues of the Rad54 protein are necessary for this interaction. Combined with previously reported results, these data imply that the Rad54 protein is part of a multiprotein yeast recombination complex.

Binding Sites↗

PubMed1996

DNA strand annealing is promoted by the yeast Rad52 protein.

The Saccharomyces cerevisiae RAD52 gene plays a pivotal role in genetic recombination. Here we demonstrate that yeast Rad52 is a DNA binding protein. To show that the interaction between Rad52 and DNA is direct and not mediated by other yeast proteins and to facilitate protein purification, a recombinant expression system was developed. The recombinant protein can bind both single- and double-stranded DNA and the addition of either Mg2+ or ATP does not enhance the binding of single-stranded DNA. Furthermore, a DNA binding domain was found in the evolutionary conserved N terminus of the protein. More importantly, we show that the protein stimulates DNA annealing even in the presence of a large excess of nonhomologous DNA. Rad52-promoted annealing follows second-order kinetics and the rate is 3500-fold faster than that of the spontaneous reaction. How this annealing activity relates to the genetic phenotype associated with rad52 mutant cells is discussed.

DNA Repair↗

PubMed1996

Studies on the hydrolytic properties of (serine) carboxypeptidase Y.

The activity of serine carboxypeptidases is dependent on a catalytic triad, an oxyanion hole, and a binding site equivalent to those found in the serine endopeptidases. The action of carboxypeptidase Y on substrates containing amino acids, alcohols, and amines as leaving groups is described. It is demonstrated that the features common to serine endopeptidases and carboxypeptidases are sufficient for hydrolysis of ester bonds. However, rapid hydrolysis of amide bonds is dependent on interactions between the C-terminal carboxylate group of the substrate and the C-terminal recognition site of the enzyme. Furthermore, on the basis of the pH dependencies of wild-type and mutant enzyme, combined with the ability of the enzyme to utilize binding energy to promote catalysis, alternative models for the high activity of carboxypeptidase Y at low pH are discussed. They describe how the catalytically essential histidine is maintained in its active deprotonated state through perturbation of its pKa value in the enzyme-substrate complex.

Amino Acid Sequence↗

PubMed1994

The activity of carboxypeptidase Y toward substrates with basic P1 amino acid residues is drastically increased by mutational replacement of leucine 178.

A random mutagenesis study on carboxypeptidase Y has previously suggested that Leu178 is situated in the S1 binding pocket, and this has later been confirmed by the three-dimensional structure. We here report the mutational replacement of Leu178 with Trp, Phe, Ala, Ser, Cys, Asn, Asp, or Lys and the kinetic characterization of each mutant, using substrates systematically varied at the P1 position. The general effect of these substitutions is a reduced kcat/Km for substrates with uncharged amino acid residues in the P1 position, little effect on those with acidic residues, and an increased kcat/Km for those with basic amino acid residues. There is a clear correlation between the reduction in kcat/Km for substrates with uncharged P1 side chains and the nature of the residue at position 178. A small reduction is observed when Leu178 is replaced by another hydrophobic amino acid residue, a larger reduction when it is replaced by a polar residue, and a very large reduction when it is replaced by a charged residue. When Leu178 is replaced by Asp, kcat/Km is reduced by a factor of 2200 for a substrate with Val in the P1 position. The kcat/Km values for the hydrolysis of substrates with charged P1 side chains are increased when Leu178 is replaced by an amino acid residue with the opposite charge, and they are decreased when it is replaced by a residue with the same charge. Surprisingly, all mutants (except L178K) exhibit increased activity with substrates with basic P1 side chains.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Recognition of C-terminal amide groups by (serine) carboxypeptidase Y investigated by site-directed mutagenesis.

Serine carboxypeptidases have the ability to hydrolyze peptides as well as peptide amides. Previously, it has been demonstrated that Asn51 and Glu145 (in the protonated form) each donate a hydrogen bond to the alpha-carboxylate of peptide substrate. It is here demonstrated by characterization of carboxypeptidase Y derivatives, mutationally altered at positions 51 and 145, that the same groups are involved in the interaction with the C-terminal carboxyamide group of peptide amides. Asn51 donates a hydrogen bond to the C = O group of the substrate, and Glu145 (in the charged form) accepts one from the NH2 group of the substrate. Thus, the ionic state of Glu145 is different when peptides are hydrolyzed as compared with when peptide amides are hydrolyzed. This explains why Km for the hydrolysis of peptides increases with pH, whereas it remains constant for peptide amides. As a consequence, kcat/Km for the hydrolysis of peptide amides is higher than for the hydrolysis of peptides at pH > 8. At physiological pH, peptides and peptide amides are hydrolyzed with rates of the same order of magnitude; this is in accordance with reports describing that serine carboxypeptidases are involved in the degradation of biologically active peptide amides.

Amidohydrolases↗

PubMed1994

Site-directed mutagenesis on (serine) carboxypeptidase Y. A hydrogen bond network stabilizes the transition state by interaction with the C-terminal carboxylate group of the substrate.

The three-dimensional structure of (serine) carboxypeptidase Y suggests that the side chains of Trp49, Asn51, Glu65, and Glu145 could be involved in the recognition of the C-terminal carboxylate group of peptide substrates. The mutations Trp49-->Phe; Asn51-->Ala, Asp, Glu, Gln, Ser, or Thr; Glu65-->Ala; and Glu145-->Ala, Asp, Asn, Gln, or Ser have been performed. Enzymes with Ala at these positions were also produced as double and triple mutations. These mutations have only little effect on the esterase activity of the enzyme, consistent with the absence of a hydrogen bond acceptor in the P1' position of such substrates. On the other hand, removal of the hydrogen-bonding capacity by incorporation of Ala at any of these four positions results in reduced peptidase activity, in particular when Asn51 and Glu145 are replaced. The results are consistent with Trp49 and Glu65 orienting Asn51 and Glu145 by hydrogen bonds, such that these can function as hydrogen bond donors (Glu145 only in its protonated carboxylic acid form) with the C-terminal alpha-carboxylate group of the peptide substrate as acceptor. However, it appears that strong interactions are formed only in the transition state since the combined removal of Asn51 and Glu145 reduces kcat about 100-fold and leaves KM practically unchanged. The results obtained with enzymes in which Asn51 or Glu145 has been replaced with other residues possessing the capacity to donate a hydrogen bond demonstrate that there is no flexibility with respect to the nature of the hydrogen bond donor at position 145, whereas enzymes with Gln, Ser, or Thr at position 51 exhibit much higher activity than N51A, although none of them reaches the wild-type level. With carboxypeptidase Y as well as other serine carboxypeptidases the binding of peptide substrates in the ground state (KM) is adversely affected by an increase in pH. It is shown that deprotonation of a single ionizable group with a pKa of 4.3 on the enzyme is responsible for this pH effect. The results show that the group involved is either Glu65 or Glu145, the latter being the more probable. The effect of this ionization on KM is explained by charge repulsion between the carboxylate group of the substrate and that of Glu145, hence preventing substrate from binding.

Base Sequence↗

PubMed1994

A conserved glutamic acid bridge in serine carboxypeptidases, belonging to the alpha/beta hydrolase fold, acts as a pH-dependent protein-stabilizing element.

Serine endopeptidases of the chymotrypsin family contain a salt bridge situated centrally within the active site, the acidic component of the salt bridge being adjacent to the catalytically essential serine. Serine carboxypeptidases also contain an acidic residue in this position but it interacts through a short hydrogen bond, probably of low-barrier type, with another acidic residue, hence forming a "glutamic acid bridge." In this study, the residues constituting this structural element in carboxypeptidase Y have been replaced by site-specific mutagenesis. It is demonstrated that the glutamic acid bridge contributes significantly to the stability of the enzyme below pH 6.5 and has an adverse effect at pH 9.5. Carboxypeptidase WII from wheat contains 2 such bridges, and it is more stable than carboxypeptidase Y at acidic pH.

Amino Acid Sequence↗

PubMed1994

Effects of introduced aspartic and glutamic acid residues on the P'1 substrate specificity, pH dependence and stability of carboxypeptidase Y.

Carboxypeptidase Y is a serine carboxypeptidase isolated from Saccharomyces cerevisiae with a preference for C-terminal hydrophobic amino acid residues. In order to alter the inherent substrate specificity of CPD-Y into one for basic amino acid residues in P'1, we have introduced Asp and/or Glu residues at a number of selected positions within the S'1 binding site. The effects of these substitutions on the substrate specificity, pH dependence and protein stability have been evaluated. The results presented here demonstrate that it is possible to obtain significant changes in the substrate preference by introducing charged amino acids into the framework provided by an enzyme with a quite different specificity. The introduced acidic amino acid residues provide a marked pH dependence of the (kcat/Km)FA-A-R-OH/(kcat/Km)FA-A-L-OH ratio. The change in stability upon introduction of Asp/Glu residues can be correlated to the difference in the mean buried surface area between the substituted and the substituting amino acid. Thus, the effects of acidic amino acid residues on the protein stability depend upon whether the introduced amino acid protrudes from the solvent accessible surface as defined by the surrounding residues in the wild type enzyme or is submerged below.

Amino Acid Sequence↗

PubMed1991

Eukaryotic topoisomerase I-DNA interaction is stabilized by helix curvature.

The influence of DNA structure on topoisomerase I-DNA interaction has been investigated using a high affinity binding site and mutant derivatives thereof. Parallel determinations of complex formation and helix structure in the absence of superhelical stress suggest that the interaction is intensified by stable helix curvature. Previous work showed that a topoisomerase I binding site consists of two functionally distinct subdomains. A region located 5' to the topoisomerase I cleavage site is essential for binding. The region 3' to the cleavage site is covered by the enzyme, but not essential. We report here that the helix conformation of the latter region is an important modulator of complex formation. Thus, complex formation is markedly stimulated, when an intrinsically bent DNA segment is installed in this region. A unique pattern of phosphate ethylation interferences in the 3'-part of the binding site indicates that sensing of curvature involves backbone contacts. Since dynamic curvature in supercoiled DNA may substitute for stable curvature, our findings suggest that topoisomerase I is able to probe DNA topology by assessment of writhe, rather than twist.

Base Sequence↗

PubMed1990

Distamycin inhibition of topoisomerase I-DNA interaction: a mechanistic analysis.

Inhibition of eukaryotic DNA topoisomerase I by the minor groove binding ligand, distamycin A, was investigated. Low concentrations of the ligand selectively prevented catalytic action at a high affinity topoisomerase I binding sequence. A restriction enzyme protection assay indicated that the catalytic cycle was blocked at the binding step. Distamycin binding sites on DNA were localized by hydroxyl radical footprinting. A strongly preferred site mapped to a homopolymeric (dA).(dT)-tract partially included in the essential topoisomerase I binding region. Mutational elimination of the stable helix curvature associated with this ligand binding site demonstrated that (i) the intrinsic bend was unessential for efficient binding of topoisomerase I, and (ii) distamycin inhibition did not occur by deformation of a stable band. Alternative modes of inhibition are discussed.

Animals↗

PubMed1989

Interactions between eukaryotic DNA topoisomerase I and a specific binding sequence.

The interaction between eukaryotic DNA topoisomerase I and a high affinity binding sequence was investigated. Quantitative footprint analysis demonstrated that the substrate preference results from strong specific binding of topoisomerase I to the sequence. The specificity was conferred by a tight noncovalent association between the enzyme and its target DNA, whereas the transient formation of a covalently bound enzyme.nicked DNA intermediate contributed insignificantly to the overall affinity. Topoisomerase I protected both strands over a 20-base pair region in which the cleavage site was centrally located. DNA modification interference analysis revealed a 16-base pair interference region on the scissile strand. Essential bases were confined to the 5' side of the cleavage site. The 6-base pair interference region observed on the complementary strand did not contain essential bases.

Animals↗

PubMedDate not supplied

Improvement of the applicability of carboxypeptidase Y in peptide synthesis by protein engineering.

Asn51 and Glu145 of (serine) carboxypeptidase Y function as binding sites for the C-terminal carboxylate group of peptide substrates, and Glu65 is involved in orienting these two amino acid residues. A series of mutants of carboxypeptidase Y where these three amino acid residues have been replaced were investigated for their applicability in transacylation reactions with amino acid esters as acceptors. With H-Val-OMethyl as the nucleophile, the fraction of aminolysis is significantly higher than with the corresponding amino acid, suggesting a beneficial effect of blocking the alpha-carboxylate group. Increasing the size of the alcohol moiety, i.e., -OEthyl, -OPropyl or OButyl, has an adverse effect on the binding of the nucleophile and on the maximum yield of aminolysis. Replacement of Asn51 and Glu145 with Ala or Gly has a pronounced beneficial effect both on binding and the maximum fraction of aminolysis. However, the results do not establish a specific type of interaction between the enzyme and these valine esters. It is probable that the rotational freedom around the ester bond allows multiple binding modes, depending on both the leaving group and type of structural change within the binding site. From a synthetic point of view, some of the mutant enzymes are much better than the wildtype enzyme when amino acid esters are used as nucleophiles.

Acylation↗