[go: up one dir, main page]

WO1997015657A1 - Reparation de l'adn - Google Patents

Reparation de l'adn Download PDF

Info

Publication number
WO1997015657A1
WO1997015657A1 PCT/GB1996/002595 GB9602595W WO9715657A1 WO 1997015657 A1 WO1997015657 A1 WO 1997015657A1 GB 9602595 W GB9602595 W GB 9602595W WO 9715657 A1 WO9715657 A1 WO 9715657A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide according
hmsh
polypeptide
binding
fragment
Prior art date
Application number
PCT/GB1996/002595
Other languages
English (en)
Inventor
Alexander Fred Markham
Adrian Whitehouse
Original Assignee
The University Of Leeds
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The University Of Leeds filed Critical The University Of Leeds
Priority to AU73165/96A priority Critical patent/AU7316596A/en
Publication of WO1997015657A1 publication Critical patent/WO1997015657A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • the invention relates to products and methods for use in identifying mismatched oligonucleotides in at least a fragment of genetic material for use, particularly but not exclusively, in the diagnosis of genetic disease.
  • HNPCC hereditary non-polyposis colorectal cancer
  • mismatch repair pathway is the methyl-directed long patch repair pathway in E. coli (1), which is involved in increasing the fidelity of replication by specific repair of DNA polymerase incorporation errors. Initiation of heteroduplex repair is dependent on the product of the MutS gene, which binds to base mispairs and loops of up to four unpaired nucleotides. After this, the MutL gene product binds to the DNA-MutS complex, initiating excision of a tract of single-stranded DNA that contains the mismatched residue(s). The repair process is completed by resynthesis of the excised DNA strand and ligation of the remaining nick.
  • the human proteins hMSH-2 and the recently discovered G T binding protein (GTBP), which are believed to form a heterodimer (hMutS ⁇ ) are homologs of MutS (4, 5), whereas the human proteins hMLH-1, hPMS-1 and hPMS-2 are analogous to MutL. Mutations in any of these genes are believed to inactivate mismatch repair in man and yield a mutator phenotype which can destroy the normal functioning of critical genes and lead to tumour formation (6).
  • MutS has been used in methods involving proof-reading or repair enzymes. Binding of this enzyme to mismatches has been shown to be sensitive and specific (7). However, MutS which has been expressed in bacterial systems and purified is unstable.
  • hMSH-2 The cDNA of hMSH-2 is known (12) furthermore hMSH-2 has been shown to bind to DNA containing nucleotide mismatches in vitro (8,9).
  • hMSH-2 it is difficult to achieve acceptable levels of expression of hMSH-2 in expression systems.
  • Our own work to express hMSH-2 in baculovirus expression systems has not been successful. This is probably a consequence of the overexpression of full length hMSH-2 being deleterious to the growth of the insect virus in cell culture.
  • fragments of hMSH-2 can be successfully expressed in bacteria and we have identified a domain of the hMSH-2 enzyme which when expressed displays mismatch binding activity in vitro.
  • hMS-2 and its homologues are very highly conserved over their carboxy terminal domains.
  • an isolated polypeptide showing mismatch nucleotide binding activity in vitro said polypeptide comprising at least a part of the C terminal domain of a nucleotide binding protein, or a type A nucleotide binding motif, which domain, or motif, further exhibits ATPase activity.
  • a type A nucleotide binding motif includes reference to a motif that has been identified following structural studies and shown to comprise a type A sequence including a flexible loop bounded by a ⁇ sheet with an ⁇ helix on either side (22-26).
  • polypeptide is a part of an enzyme whose functions in vitro is the recognition of mismatches such as a proof-reading enzyme or repair enzyme and ideally a C-terminal domain of said enzyme.
  • said polypeptide comprises approximately 300, and more preferably 297, amino acids and ideally the last 270 amino acids of said enzyme.
  • polypeptide comprises amino acids 637 to 877 of the protein hMSH-2. Even more preferably still said polypeptide comprises amino acids 664 to 877. More preferably further still said polypeptide comprises amino acids 664 to 805 of hMSH-2.
  • polypeptide comprises the amino acids shown in the alignment sequence of Figure 1, or at least a substantial part thereof, which part whose runction in vitro is the recognition of mismatch binding as herein broadly described.
  • polypeptide comprises the nucleotide binding domain of hMSH-2, or a homologue or analogue thereof, comprising lysine at nucleotide position 675.
  • an expression system for the manufacture of a protein fragment in accordance with the invention which system comprises a host cell comprising a fragment of DNA encoding the protein fragment of the invention which DNA is functionally coupled to the replication system of the host cell whereby the protein fragment of the invention can be made.
  • a vector for transforming a host cell whereby the protein fragment of the invention can be made is provided.
  • a method for obtaining the protein fragment of the invention comprising:
  • the host cell is ideally a bacterial host cell.
  • said protein fragment is provided with a tag for example a C-terminal Flag peptide such as (Asp Tyr Lys Asp Asp Asp Asp Lys).
  • a tag for example a C-terminal Flag peptide such as (Asp Tyr Lys Asp Asp Asp Asp Lys).
  • any other tag such as a fluorescent marker or radioactive marker may be used.
  • an antibody ideally monoclonal, which specifically binds to the Flag can be used to identify the fragment.
  • oligonucleotides for amplifying said DNA.
  • a method for identifying mismatched oligonucleotides comprising exposing strands of oligonucleotides to the protein fragment of the invention under conditions which promote binding; and determining the amount of binding taking place.
  • the said oligonucleotides can be tagged using, for example, a radio label.
  • kit for determining mismatch binding comprising at least the protein fragment of the invention.
  • said kit comprises a control comprising at least one mismatched binding pair of oligonucleotides and ideally at least one matched complementary binding pair of oligonucleotides.
  • a fragment of a nucleotide binding protein for detection of mismatched complementary oligonucleotide pairs or of mismatches in double-stranded nucleic acid fragments or in double-stranded PCR products.
  • nucleotide binding protein or a fragment thereof, comprising the substitution, or deletion, of a critical codon, or amino acid, in the nucleotide binding domain thereof.
  • said substitution or deletion comprises a manipulation of: the codon encoding the amino acid lysine at codon 675 of hMSH-2, or its equivalent in a homologous or analogous protein; or the corresponding lysine amino acid, so that lysine is either substituted or deleted in the relevant protein, or a fragment thereof.
  • Figure 1 Shows alignment of amino acid sequences of the conserved COOH terminal region of hMSH-2, and MSH-2 and MutS.
  • Figure 2 Shows how a DNA fragment containing the carboxy terminal domain of hMSH-2 was generated using PCR. This fragment contained amino acids 611 to 852 of the published sequence (ii). The domain was ligated to pFlag.CTC to derive phMSH2.Flag.
  • Figure 3 Shows analysis of hMSH-2 flag fusion protein.
  • Figs. 5&5A Show functional analysis of the bacterial fusion protein.
  • Oligonucleotides containing either a perfect match a selected single mismatch were radiolabelled using primer extension.
  • One pmole of labelled DNA was incubated for 1 hour with: Figure 5 E. coli MutS (Lane 1), or protein extracts of Flag (Lane 2), or hMSH-2.Flag (Lane 3-4); or Figure 5A protein extracts of hMSH-2.Flag or Flag. After the incubation period the mixtures were slowly filtered over preset nitrocellulose, washed and bound DNA was detected using autoradiography.
  • Figs 6&6A Shows quantification of the binding assay.
  • Figures 6 and 6A correspond to Figures 5 and 5A, respectively.
  • Radioactive spots on the nitrocellulose filter were excised and quantified using a scintillation counter. The results are shown as counts per minute and variations between three replicated assays are indicated.
  • Figure 8 Analysis of expression of mutant fusion proteins. Coomassie blue stained SDS PAGE gel showing protein extracts analysed 4 hours post induction with ImM IPTG. Lane 1-2, hMSH-2 domain - uninduced and induced; Lane 3-4, ⁇ 1 - uninduced and induced; Lane 5-6, ⁇ 2 - uninduced and induced; Lane 7-8, ⁇ 3 - uninduced and induced; Lane 9-10, ⁇ 4 - uninduced and induced; Lane 11-12, ⁇ 5 - uninduced and induced, respectively.
  • Figure 10 Functional analysis of the mutant bacterial fusion proteins. Oligonucleotides containing either a perfect match or a range of single mismatches were radiolabelled using polynucleotide kinase. One pmole of labelled DNA was incubated for 1 hour with protein extracts of hMSH-2, pET and ⁇ l-5. After the incubation period the mixtures were slowly filtered over prewet nitrocellulose. Washed and bound DNA was detected using autoradiography.
  • hMSH-2 C-terminal domain expression vector A DNA fragment containing the cDNA fragment required from hMSH-2 was generated by polymerase chain reaction (PCR) using lOng of plasmid pBShMSH2 DNA, and 250ng of the oligonucleotides dCCG AAG CTT AGG CAT GCT TGT GTT GAA GTT CAA GAT and dGCG GGA TCC TCT TTC CAG ATA GCA CTT CTT TGC TGC. These oligonucleotides inco ⁇ orated BamHI and Hindlll restriction sites respectively, for convenient cloning of the PCR product.
  • the reaction was performed with 4 units of Taq polymerase (Promega) in the buffer recommended by the supplier. After 30 cycles (1 minute, 92° C, 1 minute 60° C, 1 minute 72° C), the DNA produced was phenol/chloroform extracted, ethanol precipitated, digested with BamHI and Hindlll and cloned into the corresponding sites of pFlag.CTC (IBI) to derive phMSH2.Flag. The integrity of the insert was checked by DNA sequencing (data not shown).
  • a fresh overnight culture of transformed E. coli was diluted 1 in 20 with LB medium containing ampicillin (100 ⁇ g/ml). After growth at 37° C for 2 hours, the culture was induced with IPTG (1 mM) and grown at 37° C for a further 5 hours.
  • the cells were harvested by centrifugation at 3200g for 10 minutes and resuspended in 0.1 volume lysis buffer (lOOmM Tris-HCl, pH 8.0, ImM EDTA) and incubated on ice with 3 mg/ml of lysozyme for 30 minutes.
  • the cells were then sonicated and lysed by the addition of Tween 20 lysis buffer (100 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.3 mg/ml phenylmethylsulphonyl fluoride, 0.8 ⁇ g/ml pepstatin, 1 mM DTT, 1 % Tween 20).
  • Tween 20 lysis buffer 100 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.3 mg/ml phenylmethylsulphonyl fluoride, 0.8 ⁇ g/ml pepstatin, 1 mM DTT, 1 % Tween 20.
  • Protein extracts were mixed with 2x reducing sample buffer (50mM Tris-HCl, pH 6.8, 4% sodium dodecyl sulphate (SDS), 5 mM EDTA, 10% ⁇ - mercapthoethanol, 1 mM DTT and 0.01% bromophenol blue). After boiling for 3 minutes, samples were fractionated on a 12% SDS polyacrylamide gel. After electrophoresis the gel was soaked for 10 minutes in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol (v:v), and 0.1 % SDS), and the proteins were transferred to nitrocellulose membranes by electroblotting for 3 hours at 250 mA.
  • the membranes were soaked in PBS and incubated for 2 hours in blocking buffer (PBS contauiing 5% nonfat dry milk).
  • PBS contauiing 5% nonfat dry milk After transfer, the membranes were soaked in PBS and incubated for 2 hours in blocking buffer (PBS contauiing 5% nonfat dry milk).
  • Membranes were incubated with a 1/100 dilution of the M2 monoclonal antibody (IgG,, IBI), washed with PBS and incubated for 1 hour at 37° C with a 1/1000 dilution of rabbit anti-mouse immunoglobulin conjugated with horseradish peroxidase in blocking buffer.
  • the nitrocellulose membranes were developed in PBS containing 0.02% l-chloro-4-naphthol and 0.006% hydrogen peroxide.
  • the assay was performed at 37° C in 20 mM Tris-HCl pH 7.6, 0.5 mM CaCl 2 , 5mM MgCl 2 , 1 mM DTT, 100 ⁇ g ml BSA, 0.1 mM EDTA and 150 ng of hMSH-2 domain. Assays were performed using 2, 2.5, 3.3, 5 and 10 ⁇ M ATP. Hydrolysis of [ ⁇ - 32 P]ATP by the carboxy terminal domain of hMSH-2 was assayed by thin layer chromatography. The radioactive counts for ATP and its hydrolysis products were quantified using a scintillation counter (Packard).
  • Oligonucleotides (dCGG ATC CGG AXG TCA TGG AAT TCC and dGGA ATT CCA TGA CXT CCG GAT CCG) were synthesised and annealed to produce either a perfect matched double-stranded molecule or a single G:T mismatch (position shown in bold type).
  • Oligonucleotides were mixed to a final concentration of 100 pmole/ ⁇ l each in 100 ⁇ l STM (100 mM NaCl, 10 mM Tris-HCl, pH 7.0, 10 mM MgCI 2 , 5 mM DTT) heated to 95° C and cooled to 25° C over 2 hours. The annealed products were then stored in 50% glycerol at -20° C until required. End-labelling of double-stranded DNA (100 pmole) in STM buffer was by polynucleotide kinase. After incubating at 20° C for 10 minutes the unincorporated label was removed using a Sephadex NAP 5 column.
  • STM 100 mM NaCl, 10 mM Tris-HCl, pH 7.0, 10 mM MgCI 2 , 5 mM DTT
  • the labelled DNA was diluted to 0.2 pmole/ ⁇ l.
  • the binding assay used 1 pmole of DNA with 150 ng hMSH-2 domain in a total volume of 10 ⁇ l. After 1 hour on ice the mixture was slowly filtered over pure prewetted nitrocellulose (Millipore, 0.45 ⁇ m) and washed in STM buffer. The filter was then allowed to air dry and bound DNA was detected by autoradiography. Bound material was quantified using a scintillation counter (Packard).
  • DNA fragments expressing the C-terminal DNA binding domain sufficient to bind specific mismatched oligonucleotides and mutants 1-5 shown in Figure 7 were generated by polymerase chain reaction (PCR) using lOng of plasmid pBShMSH-2 DNA, and 250ng of the following forward primers dCCG GGA TCC TTC CAC ATC ATT ACT GGC CCC AAT ATG GGA GGT AAA TCA; dCCG GGA TCC TTC CAC ATC GGT ACT GGC CCC AAT ATG GGA GGT AAA TCA; dCCG GGA TCCTTC CAC ATC ATT ACT GCC CCC AAT ATG GGA GGT AAA TCA; dCCG GGA TCC TTC CAC ATC ATT ACT GGC CCC AAT ATG GGA GCT AAA TCA; dCCG GGA TCC TTC CAC ATC ATT ACT GGC CCC AAT ATG GGA GCT AAA TCA; dCCG GGA TCC
  • oligonucletoides incorporated BamHI restriction sites for convenient cloning of the PCR products.
  • the reaction was performed with 4 units of Pfu DNA Polymerase (Stratagene) in the buffer recommended by the supplier. After 30 cycles (1 min, 92° C, 1 min 60° C, 1 min 72° C), the DNA produced was phenol/chloroform extracted, ethanol precipitated, digested with BamHI and cloned into the corresponding site of pET21a (Novagen) to derive pEThMSH-2 and PET ⁇ 1-5 respectively. The integrity of each insert was confirmed by DNA sequencing (data not shown).
  • the wild type and mutant proteins encoding the amino acid 663-877 hMSH-2 domain in the pET bacterial expression vector were used to transform E. coli strain BL21(DE3).
  • a fresh overnight culture of transformed E. coli was diluted 1 in 20 with LB medium containing ampicillin (100 ⁇ g/ml). After growth at 37° C for 2 hours, the culture was induced with IPTG (1 mM) and grown at 37° C for a further 5 hours.
  • the cells were harvested by centrifugation at 3200g for 10 minutes and resuspended in 0.1 volume lysis buffer (lOOmM Tris-HCl, pH 8.0, ImM EDTA) and incubated on ice with 3 mg/ml of lysozyme for 30 minutes.
  • the cells were than sonicated and lysed by the addition of Tween 20 lysis buffer (100 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.3 mg/ml phenylmethylsulphonyl fluoride, 0.8 ⁇ .0, 200 mM NaCl, 1 mM EDTA, 0.3 mg/ml phenylmethylsulphonyl fluoride, 0.8 ⁇ g/ml pepstatin, 1 mM DTT, 1 % Tween 20).
  • Tween 20 lysis buffer 100 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.3 mg/ml phenylmethylsulphonyl fluoride, 0.8 ⁇ g/ml pepstatin, 1 mM DTT, 1 % Tween 20.
  • Protein extracts were mixed with 2x reducing sample buffer (50mM Tris-HCl, pH 6.8, 4% sodium dodecyl sulphate (SDS), 5mM EDTA, 10% ⁇ - mercapthoethanol, 1 mM DTT and 0.01 % bromophenol blue). After boiling for 3 minutes, samples were fractionated on a 12% SDS polyacrylamide gel. Following electrophoresis the gel was stained with Coomassie blue solution (25% v/v isopropyl alcohol, 10% v/v acetic acid and 0.25% w/v Coomassie blue).
  • 2x reducing sample buffer 50mM Tris-HCl, pH 6.8, 4% sodium dodecyl sulphate (SDS), 5mM EDTA, 10% ⁇ - mercapthoethanol, 1 mM DTT and 0.01 % bromophenol blue.
  • the assay was performed at 37° C in 20mM Tris-HCl, pH 7.6, 0.5 mM CaCl 2 , 5mM MgCl 2 , 1 mM DTT, 100 ⁇ g/ml BSA, 0.1 mM EDTA with 150 ng of wild type or mutant hMSH-2 domains. Assays were performed using 2, 2.5, 3.3, 5 and 10 ⁇ M ATP. Hydrolysis of [ ⁇ - 32 P]ATP by the wild type and each mutant carboxy terminal domain was assayed by thin layer chromatography. The radioactive counts for ATP and its hydrolysis products were quantified using a scintillation counter (Packard). Functional binding assay
  • oligonucleotides (dCGG ATC CGG AXG TCA TGG AAT TCC and dGGA ATT CCA TXA CAT CCG GAT CCG) were annealed to produce either a perfect matched double-stranded molecule or a single mismatch (position shown in bold type).
  • Oligonucleotides were mixed to a final concentration of 100 pmole/ ⁇ l each in 100 ⁇ l STM (100 mM NaCl, 10 mM Tris-HCl, pH 7.0, 10 mM MgCl 2 , 5 mM DTT (heated to 95° C and cooled to 25° C over 2 hours. End-labelling of double-stranded DNA (100 pmole) in STM buffer was performed with polynucleotide kinase. After incubation at 20° C for 10 minutes the unincorporated label was removed using a Sephadex NAP 5 column. The labelled DNA was diluted to 0.2 pmole/ ⁇ l.
  • the binding assay used 1 pmole of DNA with 150 ng of wild type or each mutant hMSH-2 domain in a total volume of 10 ⁇ l. After 1 hour on ice the mixture was slowly filtered over pure prewetted nitrocellulose (Millipore, 0.45 ⁇ m) and washed in STM buffer. The filter was then allowed to air dry and bound DNA was detected by autoradiography.
  • the PCR product was ligated to pFlag.CTC, in phase with respect to the ATG translational start codon immediately upstream of the multiple cloning site (MCS) and also in frame with the C-terminal coding sequence immediately downstream of the MCS to ensure proper fusion to the C-terminal Flag peptide (Asp Tyr Lys Asp Asp Asp Asp Lys).
  • the hMSH-2 domain was thus cloned into the bacterial expression vector Flag (IBI). Expression of the hMSH-2 Flag fusion protein resulted in a 30 kDa species detected by Western blot analysis on SDS-PAGE ( Figure 3).
  • the anti-Flag M2 monoclonal (IgGI) mouse antibody (IBI) was used to specifically bind to the eight amino acid Flag peptide, which identified the 249 amino acid recombinant protein comprising the hMSH-2 domain (containing a type A nucleotide binding site consensus sequence) coupled to the Flag peptide at its carboxy terminus.
  • the Walkers A-type nucleotide binding motif conserved in MutS proteins has been shown to have ATPase activity (21).
  • [ ⁇ 32p]ATP was incubated with the fusion protein and separated using TLC.
  • K m and fc cat values of the hMSH-2 domain ATPase activity was measured in the presence of various concentrations of ATP (Fig. 4). At 37° C the K m and k cat were calculated to be 6.6 ⁇ M and 0.5 s "1 , respectively.
  • nonenzymatic hydrolysis of ATP in the absence of the expressed domain was less than 5%.
  • a mismatch binding assay was developed to measure the hMSH-2 C-terminal domain's activity. Mismatch binding was detected by nitrocellulose binding of labelled oligonucleotides containing a mismatch at position 11 within the context of a double-stranded 24mer oligonucleotide pair. The binding of the hMSH-2 domain to an G-T mismatch containing oligonucleotides is shown in Figure 5. The binding of the hMSH-2 domain to a range of mismatch containing oligonucleotides is shown in Figures 5 A.
  • Radiolabelled oligonucleotides containing a perfect match or a single mismatch were incubated with purified MutS or protein extracts containing hMSH-2.Flag or Flag alone.
  • the binding of proteins to mismatched or matched oligonucleotides was quantified using a scintillation counter ( Figures 6 and 6A).
  • Figure 6 it can be seen that the MutS and hMSH-2 domain selectively bound to the oligonucleotides containing the mismatch, but not the perfectly matched oligonucleotides.
  • hMSH-2 domain selectively bound to the oligonucleotides containing all possible mismatches, apart from C/C and A/A mismatches.
  • the flag control bound to no oligonucleotide, showing the hMSH-2 domain alone is sufficient to bind oligonucleotides containing a G/T mismatch.
  • a fragment of the hMSH-2 cDNA sequence which encodes amino acid residues 637 to 877 has been shown to bind oligonucleotides containing mismatches (27).
  • mutant proteins have been produced which alter specific residues within the putative nucleotide binding region (Fig. 7).
  • DNA fragments which encode the nucleotide binding domain of hMSH-2 were amplified using PCR, inco ⁇ orating BamHI restriction sites for convenient cloning.
  • Each product was ligated to the expression vector pET21a, in phase with respect to the ATG translational start codon immediately upstream of the multiple cloning site (MCS) and also in frame with the C-terminal coding sequence immediately downstream of the MCS to ensure proper fusion to the C-terminal HisTag.
  • MCS multiple cloning site
  • each mutant hMSH-2 nucleotide binding domain was cloned into the bacterial expression vector pET21a.
  • Expression of the mutant hMSH-2 fusion proteins resulted in 30 kDa species detected using SDS-PAGE comprising the hMSH-2 domain (containing a type A nucleotide binding site consensus sequence) coupled to the HisTag peptide at its carboxy terminus. (Fig. 8).
  • Fig. 8 We designate these mutant fusion proteins ⁇ l to ⁇ 5. All mutant proteins were expressed at comparable levels to the wild type fusion protein.
  • ⁇ 5 had a reduced activity with K m and k ⁇ , values of 3.6 ⁇ M and 0.65 S '1 , and ⁇ 4 which alters the codon 675 from a lysine to an alanine has a marked effect upon ATP hydrolysis, effectively reducing it to zero.
  • nonenzymatic hydrolysis of ATP in the absence of the wild type expressed domain was less than 5%.
  • a mismatch binding assay was developed to measure the hMSH-2 C-terminal domains activity (27). Mismatch recognition was detected by nitrocellulose binding of labelled oligonucleotides containing a mismatch at position 11 within the context of a double-stranded 24-mer oligonucleotide pair. We found that the wild type C-terminal domain of hMSH-2 selectively bound all specific mismatches apart from A/A and C/C, in agreement with results described previously (27). The pET control did not bind to any labelled oligonucleotide pair.
  • hMSH-2 and its homologues around the domain of the type A nucleotide binding motif bind mismatched oligonucleotides.
  • this domain we have expressed this domain as a bacterial fusion protein and shown that mismatch-containing oligonucleotides are selectively bound.
  • a 'type A' sequence is a flexible loop bounded by a ⁇ sheet with an ⁇ helix on either side (22-26). This flexible loop allows the protein to undergo conformational change, thus controlling the accessibility of substrate binding or binding site affinities (27). Further studies have shown that an analogous lysine plays an important role in ATP-dependent function of these proteins (27-28).
  • Lys 675 The key role of Lys 675 is also emphasised by the fact that mutations at residues 666, 668 and indeed 674 (the residue next to the critical lys residue) have minimal effects on mismatch recognition and ATPase activity. Furthermore, mutation of the conserved Ser 676, the residue immediately C- terminal of Lys 675 still provides a protein with retains 40% of its normal activity.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne l'identification d'un polypeptide présentant une activité de fixation avec mésappariement in vitro et s'utilisant dans des dosages de fixation avec mésappariement, en vue de détecter des mutations et/ou des polymorphismes dans du matériel génétique.
PCT/GB1996/002595 1995-10-24 1996-10-23 Reparation de l'adn WO1997015657A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU73165/96A AU7316596A (en) 1995-10-24 1996-10-23 Dna repair

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB9521788.1A GB9521788D0 (en) 1995-10-24 1995-10-24 DNA repair
GB9521788.1 1995-10-24

Publications (1)

Publication Number Publication Date
WO1997015657A1 true WO1997015657A1 (fr) 1997-05-01

Family

ID=10782840

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1996/002595 WO1997015657A1 (fr) 1995-10-24 1996-10-23 Reparation de l'adn

Country Status (3)

Country Link
AU (1) AU7316596A (fr)
GB (1) GB9521788D0 (fr)
WO (1) WO1997015657A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6248541B1 (en) 2000-04-21 2001-06-19 Genencor International, Inc. Screening under nutrient limited conditions
WO2001073079A3 (fr) * 2000-03-28 2002-05-16 Univ California Proteines chimeres permettant de detecter et de quantifier des mutations d'adn, des variations de sequences d'adn, des dommages a l'adn et des incompatibilites d'adn
US6534292B1 (en) 2000-05-08 2003-03-18 Genencor International, Inc. Methods for forming recombined nucleic acids
US6582914B1 (en) 2000-10-26 2003-06-24 Genencor International, Inc. Method for generating a library of oligonucleotides comprising a controlled distribution of mutations

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
A. WHITEHOUSE ET AL.: "A carboxy terminal domain of the hMSH2 gene product is sufficient for binding specific mismatched oligonucleotides", BIOCHEMICAL BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 225, 1996, pages 289 - 295, XP002027431 *
J. H. BAYLE ET AL.: "The carboxy-terminal domain of the p53 protein regulates sequence-speific DNA binding through its nonspecific nucleic acid-binding activity", PROCEEDINGS OF NATIONAL ACADEMY OF SCIENCES USA, vol. 92, 1995, pages 5729 - 5733, XP002027433 *
L.T. HABER ET AL.: "Altering the conserved nucleotide binding motif in the S.typhimurium MutS mismatch repair protein affects both its ATPase and mismatch binding activities", EMBO JOURNAL, vol. 10, no. 9, 1991, pages 2707 - 2715, XP002027435 *
M.J. HUGHES ET AL.: "The purification of a human mismatch-binding protein and identification of its associated ATPase and helicase activities", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 267, no. 33, 1992, pages 23876 - 23882, XP000615520 *
NAI-WEN CHI ET AL.: "The effect of DNA mismatches on the ATPase activity of MSH1, a protein in yeast mitochondria that recognizes DNA mismatches", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 269, no. 47, 1994, pages 29993 - 29997, XP002027434 *
R. FISHEL ET AL.: "Binding of mismatched microasatellite DNA sequences by the human MSH2 protein", SCIENCE, vol. 266, 1994, pages 1403 - 1405, XP002027436 *
S. LEE ET AL.: "p53 and its 14kDa C-terminal domain recognizes primary DNA damage in the form of insertion/deletion mismatches", CELL, vol. 81, 1995, pages 1013 - 1020, XP002027432 *
S. SHARMA ET AL.: "Cloning, overexpression, purification and characterization of the carboxy-terminal nucleotide binding domain of the P-glycoprotein", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 270, no. 23, 1995, pages 14085 - 14093, XP000651087 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001073079A3 (fr) * 2000-03-28 2002-05-16 Univ California Proteines chimeres permettant de detecter et de quantifier des mutations d'adn, des variations de sequences d'adn, des dommages a l'adn et des incompatibilites d'adn
US6248541B1 (en) 2000-04-21 2001-06-19 Genencor International, Inc. Screening under nutrient limited conditions
US6534292B1 (en) 2000-05-08 2003-03-18 Genencor International, Inc. Methods for forming recombined nucleic acids
US7037726B2 (en) 2000-05-08 2006-05-02 Genencor International, Inc. Methods for forming recombined nucleic acids
US6582914B1 (en) 2000-10-26 2003-06-24 Genencor International, Inc. Method for generating a library of oligonucleotides comprising a controlled distribution of mutations

Also Published As

Publication number Publication date
GB9521788D0 (en) 1996-01-03
AU7316596A (en) 1997-05-15

Similar Documents

Publication Publication Date Title
Ogawa et al. RecA-like recombination proteins in eukaryotes: functions and structures of RAD51 genes
US5474896A (en) Nucleotide sequence encoding the enzyme I-SceI and the uses thereof
US5877280A (en) Thermostable muts proteins
US6365355B1 (en) Chimeric proteins for detection and quantitation of DNA mutations, DNA sequence variations, DNA damage and DNA mismatches
Nicoletti et al. Molecular characterization of the human gene encoding an abundant 61 kDa protein specific to the retinal pigment epithelium
AU716362B2 (en) Compositions and methods to promote homologous recombination in eukaryotic cells and organisms
Plass et al. Identification of Grf1 on mouse chromosome 9 as an imprinted gene by RLGS–M
Moncalian et al. The rad50 signature motif: essential to ATP binding and biological function
Macina et al. Genomic organization of the human PAX3 gene: DNA sequence analysis of the region disrupted in alveolar rhabdomyosarcoma
Buckholz et al. The allantoinase (DAL1) gene of Saccharomyces cerevisiae
Sander et al. Cloning and characterization of Rrp1, the gene encoding Drosophila strand transferase: carboxy-terminal homology to DNA repair endo/exonucleases
Wilson STUDIES OF MAMMALIAN DNA POLYMERASE B
Saxe et al. SAS1 and SAS2, GTP-binding protein genes in Dictyostelium discoideum with sequence similarities to essential genes in Saccharomyces cerevisiae
US6492168B1 (en) Methyltransferase gene and enzyme
WO1997015657A1 (fr) Reparation de l'adn
Scott et al. Structure and expression of the Drosophila melanogaster gene encoding 6-phosphogluconate dehydrogenase
Yang et al. Molecular cloning and functional expression of cDNA encoding a mammalian inorganic pyrophosphatase.
JPH04504208A (ja) 組換えトリコサンチンおよびコード配列
WO1996040882A1 (fr) Endonuclease artificielle de restriction
Whitehouse et al. A carboxy terminal domain of the hMSH-2 gene product is sufficient for binding specific mismatched oligonucleotides
JPH07505057A (ja) プロテインキナーゼ
Whitehouse et al. Mutational analysis of the nucleotide binding domain of the mismatch repair enzyme hMSH-2
Whitehouse et al. Mapping the minimal domain of hMSH-2 sufficient for binding mismatched oligonucleotides
JP2000050882A (ja) 核移行活性を有するペプチド
JP3498808B2 (ja) Ｄｎａポリメラーゼ遺伝子

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE HU IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TR TT UA UG US UZ VN AM AZ BY KG KZ MD RU TJ TM

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE LS MW SD SZ UG AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

NENP Non-entry into the national phase

Ref country code: JP

Ref document number: 97516395

Format of ref document f/p: F

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA