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WO1997015657A1 - Dna repair - Google Patents

Dna repair Download PDF

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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
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Prior art keywords
polypeptide according
hmsh
polypeptide
binding
fragment
Prior art date
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PCT/GB1996/002595
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French (fr)
Inventor
Alexander Fred Markham
Adrian Whitehouse
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The University Of Leeds
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Priority to AU73165/96A priority Critical patent/AU7316596A/en
Publication of WO1997015657A1 publication Critical patent/WO1997015657A1/en

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    • 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.

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Abstract

The invention relates to the identification of a polypeptide which shows mismatch binding activity in vitro for use in mismatch binding assays with a view to detecting mutations and/or polymorphisms in genetic material.

Description

DNA Repair
Field of Invention
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.
The diagnosis of genetic disease using DNA markers linked to disease genes or, increasingly, the direct analysis of the relevant gene for mutations of the DNA sequence is of enormous clinical importance. If it were possible to simplify the detection of DNA sequence mutations a common approach could be adopted for the diagnosis of, or prediction of, predisposition to a wide range of inherited and acquired conditions. The most sensitive method currently available for the detection of mutations is probably that of Chemical Cleavage of Mutations (CCM). This method can theoretically detect all mismatches and small insertions and deletions and provide an approximate location of the mismatch. However, this approach is cumbersome and difficult to use in practice; still requires confirmation by DNA sequencing; and to batch large sample numbers this method would become very time- consuming. New methods are now being investigated based on the use of proof-reading enzymes or repair enzymes where function in vitro is the recognition of mismatches.
Just one example of a disease characterised by a high incidence of mutation is hereditary non-polyposis colorectal cancer (HNPCC). This is one of the most common human autosomal dominant diseases. Estimations are that up to one in two hundred of the population may be heterozygotes for mutant alleles and be predisposed to develop this form of cancer (13). HNPCC kindreds have a high incidence of mutation in tumour micro-satellite sequences when these are compared with the normal repeats occurring in non- malignant tissues from the same individuals (14). Also, cell lines derived from HNPCC tumours are genetically unstable (15). These observations suggest that a possible causative factor for HNPCC is a failure in DNA repair. Indeed, it has now been shown that cancer predisposition in these patients is attributable to defects in any one of four human genes involved in mismatch repair, hMSH-2, hMLH-1, hPMS-1 or hPMS-2 (12, 16-20).
The best studied 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.
Mutations of MutS and MutL, or their homologues in yeast, lead to increases in the rate of spontaneous mutation resulting in a mutator phenotype (2, 3).
The human proteins hMSH-2 and the recently discovered G T binding protein (GTBP), which are believed to form a heterodimer (hMutSα) 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.
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). However, 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. However, we have shown that 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. We have examined the domain and we have found that it demonstrates homology to a type A consensus sequence found in many proteins that bind and hydrolyse nucleotides (10). It has been shown that MutS displays a weak ATPase activity in the presence and absence of DNA and genetic alteration of this ATP binding site results in a protein which is defective in mismatch repair. It is therefore notable that we demonstrate herein that the aforementioned domain of hMSH-2 also exhibits ATPase activity.
It is therefore an object of the invention to provide a protein fragment that shows mismatch binding activity in vitro.
According to a first aspect of the invention there is provided 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.
Reference herein to 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 β sheet with an α helix on either side (22-26).
Ideally the 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.
More preferably still said polypeptide comprises approximately 300, and more preferably 297, amino acids and ideally the last 270 amino acids of said enzyme.
More preferably further still said 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.
More preferably yet still said 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.
More preferably still said polypeptide comprises the nucleotide binding domain of hMSH-2, or a homologue or analogue thereof, comprising lysine at nucleotide position 675.
According to a second aspect of the invention there is provided 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.
According to a third aspect of the invention there is provided a vector for transforming a host cell whereby the protein fragment of the invention can be made.
According to a fourth aspect of the invention there is provided a method for obtaining the protein fragment of the invention comprising:
a) inserting a fragment of DNA encoding the protein fragment of the invention and any necessary transcriptional/translational control elements into a suitable host cell expression system;
b) providing conditions which favour transcription and translation of said DNA in said host cells; c) harvesting said host cells;
d) lysing said host cells;
e) collecting the lysate; purifying the fragment.
In each of the above expression systems and methods the host cell is ideally a bacterial host cell.
Although obtaining the protein fragment of the invention has been described with reference to a biological expression system the said fragment may also be synthetically produced.
In yet a further preferred aspect of the invention 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). However, any other tag such as a fluorescent marker or radioactive marker may be used.
In the instance where said Flag is used an antibody, ideally monoclonal, which specifically binds to the Flag can be used to identify the fragment.
According to a further aspect of the invention there is provided DNA sequence encoding the protein fragment of the invention.
According to a further aspect of the invention there is provided oligonucleotides for amplifying said DNA.
According to a yet further aspect of the invention there is provided 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.
If preferred, the said oligonucleotides can be tagged using, for example, a radio label.
According to a yet further aspect of the invention there is provided a kit for determining mismatch binding comprising at least the protein fragment of the invention.
Ideally 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.
According to a yet further aspect of the invention there is provided the use of 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.
According to a yet further aspect of the invention there is provided means for regulating the activity of a 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.
Preferably, 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.
An embodiment of the invention will now be described by way of example only with reference to the following figures, materials and methods wherein:
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.
Transformed E. coli were grown at 37° C for 2 hours, the cultures were grown for a further 5 hours (Lane 1), or induced with IPTG (1 mM) and grown for 0 hours (Lane 2), 2 hours
(Lane 3) and 5 hours (Lane 4). Extracts were resolved by SDS PAGE, transferred to nitrocellulose, and incubated with M2 monoclonal antibody (IgG,) to the flag epitope and immune complexes were detected by using rabbit anti mouse immunoglobulin conjugated with horseradish peroxidase. The nitrocellulose membranes were developed in PBS containing 0.02% l-chloro-4-naphtol and 0.006% hydrogen peroxide. Figure 4 Shows ATPase analysis of the bacterial fusion. Hydrolysis of various substrate concentrations of [α-32P]ATP by the carboxy terminal domain of hMSH-2 was assayed by thin layer chromatography, and quantified using a scintillation counter.
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 7 Homology between hMSH-2 DNA binding domain and the
'Type A' consensus sequence. Bold type indicates conserved residues between both proteins. The mutants produced are indicated showing the alteration of each conserved residue.
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, Δ 1 - uninduced and induced; Lane 5-6, Δ2 - uninduced and induced; Lane 7-8, Δ3 - uninduced and induced; Lane 9-10, Δ4 - uninduced and induced; Lane 11-12, Δ5 - uninduced and induced, respectively.
Figure 9 ATPase analysis of the mutant bacterial fusion proteins. Hydrolysis of various substrate concentrations of [α-32=P]ATP by the carboxy terminal domain of hMSH-2, pET and Δ l-5 were assayed by thin layer chromatography, and quantified using a scintillation counter.
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 Δ l-5. After the incubation period the mixtures were slowly filtered over prewet nitrocellulose. Washed and bound DNA was detected using autoradiography.
Materials and Methods
Construction of a 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 incoφorated BamHI and Hindlll restriction sites respectively, for convenient cloning of the PCR product. They generate a PCR fragment which encodes amino acid 637 to amino acid 877 in the published sequence (11). 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).
Expression of hMSH-2 C-terminal domain as a bacterial fusion protein
The PCR product encoding the 637 to 877 amino acid hMSH-2 domain in the Flag bacterial expression vector (IBI), was used to transform E. coli strain DH5α . A fresh overnight culture of transformed E. coli was diluted 1 in 20 with LB medium containing ampicillin (100 μg/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 μg/ml pepstatin, 1 mM DTT, 1 % Tween 20). Cellular debris was pelleted by centrifugation at 4,000g.
Detection of fusion protein by Western blot analysis
Protein extracts were mixed with 2x reducing sample buffer (50mM Tris-HCl, pH 6.8, 4% sodium dodecyl sulphate (SDS), 5 mM EDTA, 10% β- 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. 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. After five washes with PBS the nitrocellulose membranes were developed in PBS containing 0.02% l-chloro-4-naphthol and 0.006% hydrogen peroxide.
ATPase Assay
The assay was performed at 37° C in 20 mM Tris-HCl pH 7.6, 0.5 mM CaCl2, 5mM MgCl2, 1 mM DTT, 100 μg 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 μM ATP. Hydrolysis of [α-32P]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).
Functional binding assay
Mismatch binding was detected by a nitrocellulose binding assay of labelled oligonucleotides followed by autoradiography. 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/ μl each in 100 μl STM (100 mM NaCl, 10 mM Tris-HCl, pH 7.0, 10 mM MgCI2, 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. The labelled DNA was diluted to 0.2 pmole/ μl. The binding assay used 1 pmole of DNA with 150 ng hMSH-2 domain in a total volume of 10 μl. After 1 hour on ice the mixture was slowly filtered over pure prewetted nitrocellulose (Millipore, 0.45 μm) 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).
Construction of mutant hMSH-2 nucleotide binding domain expression vectors
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 GGT GCA TCA; dCCG GGA TCC TTC CAC ATC ATT ACT GGC CCC AAT ATG GGA GGT AAA GCA and the reverse primer dGCG GGA TCC TCT TTC CAG ATA GCA CTT CTT TGC TGC (changes shown in bold type). These 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 PETΔ 1-5 respectively. The integrity of each insert was confirmed by DNA sequencing (data not shown).
Production ofhMSH-2 nucleotide binding domain mutants as bacterial fusion proteins
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 μg/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 μ.0, 200 mM NaCl, 1 mM EDTA, 0.3 mg/ml phenylmethylsulphonyl fluoride, 0.8 μg/ml pepstatin, 1 mM DTT, 1 % Tween 20). Cellular debris was pelleted by centrifugation at 4,000g.
Detection of fusion protein by SDS-PAGE
Protein extracts were mixed with 2x reducing sample buffer (50mM Tris-HCl, pH 6.8, 4% sodium dodecyl sulphate (SDS), 5mM EDTA, 10% β- 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).
ATPase assay
The assay was performed at 37° C in 20mM Tris-HCl, pH 7.6, 0.5 mM CaCl2, 5mM MgCl2, 1 mM DTT, 100 μg/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 μM ATP. Hydrolysis of [α-32P]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
Mismatch recognition was detected by a nitrocellulose binding assay of labelled oligonucleotides followed by autoradiography as described previously (1). Briefly, 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/ μl each in 100 μl STM (100 mM NaCl, 10 mM Tris-HCl, pH 7.0, 10 mM MgCl2, 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/μl. 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 μl. After 1 hour on ice the mixture was slowly filtered over pure prewetted nitrocellulose (Millipore, 0.45 μm) and washed in STM buffer. The filter was then allowed to air dry and bound DNA was detected by autoradiography.
Results
PCR amplification and Cloning
Protein sequence alignments of hMSH-2 and its homologues, MutS, MSH-2 and GTBP revealed a highly conserved region at the COOH terminus (Figure 1). This region contains a type A nucleotide binding site consensus sequence. A 720 bp fragment was amplified using PCR, incoφorating BamHI and Hindlll restriction sites for convenient cloning. This fragment of the hMSH-2 cDNA sequence encodes amino acid residues 637 to 877. 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). [Figure 2]
Expression of the hMSH-2 Q-terminal domain
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.
ATPase Analysis of Bacterial Fusion Protein
The Walkers A-type nucleotide binding motif conserved in MutS proteins has been shown to have ATPase activity (21). In order to determine whether the carboxy terminal domain of hMSH-2 hydrolyses ATP to ADP and Pi, [α32p]ATP was incubated with the fusion protein and separated using TLC. To determine Km and fccat values of the hMSH-2 domain, ATPase activity was measured in the presence of various concentrations of ATP (Fig. 4). At 37° C the Km and kcat were calculated to be 6.6 μM and 0.5 s"1, respectively. In a control experiment, nonenzymatic hydrolysis of ATP in the absence of the expressed domain was less than 5%.
Functional analysis of the bacterial fusion protein
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). In 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. In Figure 6A it can be seen that 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.
PCR amplification and cloning
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). In order to determine which specific residues are important in this domain, 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, incoφorating 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.
Expression of the hMSH-2 nucleotide binding domain mutants
To confirm their integrity, 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). We designate these mutant fusion proteins Δ l to Δ5. All mutant proteins were expressed at comparable levels to the wild type fusion protein.
ATPase analysis of mutant fusion proteins
It has been shown that the carboxy terminal domain of hMSH02 contains ATPase activity (27). In order to determine whether these mutants hydrolyse ATP to ADP and Pi, [α32P]ATP was incubated with each mutant fusion protein and separated using TLC. To determine Km and k^, values for the mutants, ATPase activity was measured in the presence of various concentrations of ATP (Fig. 9). The results show that Δ l, Δ2, Δ3 have limited effects on the ATPase activity of the domain. Wild type Km and k,.., values were 8.33 μM and 0.55 S"1, respectively, compared to Δ2 values of 5.88 μM and 0.633 S"1, respectively. However, Δ5 had a reduced activity with Km and k^, values of 3.6 μM and 0.65 S'1, and Δ4 which alters the codon 675 from a lysine to an alanine has a marked effect upon ATP hydrolysis, effectively reducing it to zero. In a control experiment, nonenzymatic hydrolysis of ATP in the absence of the wild type expressed domain was less than 5%.
Functional analysis of the mutant fusion proteins
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. Applying this assay to the mutant proteins, we found that Δ l, Δ2 and Δ 3 bound the same specific mismatches as the wild type domain albeit to a somewhat lesser extent. This may be due to the amino acid substitutions reducing recognition of the mismatches or a reduced affinity of these proteins once bound to a mismatch resulting in separation from the mismatch in the washing procedures of the assay. Δ5 which alters Ser 676 to an Ala has further reduced affinity for these mismatches and Δ4 was found to have no selective binding to any of the specific mismatches (Fig. 10).
Discussion
hMSH-2 and its homologues around the domain of the type A nucleotide binding motif, bind mismatched oligonucleotides. Herein we have expressed this domain as a bacterial fusion protein and shown that mismatch-containing oligonucleotides are selectively bound.
The inability of Δ4, which alters the codon 675 from a lysine to an alanine, to identify DNA containing mismatches suggests that this Lys 675 residue is important for the binding function. It is unlikely that the mutation alters the structure of this domain significantly so as to reduce stability in E. coli as the expression level of this mutant is comparable to that of wild type hMSH-2 nucleotide binding domain. Thus the deficiency is not due to a gross structural instability. At present the role of Lys 675 within the nucleotide binding site is hMSH-2 is not known. However, similar motifs in other proteins have been analysed. Structural studies have shown that a 'type A' sequence is a flexible loop bounded by a β sheet with an α 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).
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. These observations suggest that structural factors alone may not fully explain the importance of Lys 675 and perhaps this basic cationic residue is involved more directly, for example in recognising the phosphate backbone at the mismatch point in mispaired DNA.
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Claims

1. 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.
2. A polypeptide according to claim 1 wherein said enzyme is hMSH-2 or a homologue or analogue thereof.
3. A polypeptide according to claims 1 or 2 wherein said fragment comprises no more than the last 300 amino acids of said enzyme.
4. A polypeptide according to claim 3 wherein said fragment comprises no more than the last 270 amino acids of said enzyme.
5. A polypeptide according to any preceding claim which comprises amino acids 637-877 of the protein hMSH-2.
6. A polypeptide according to claim 2 wherein said fragment comprises amino acids 644-877 of the protein hMSH-2.
7. A polypeptide according to claim 2 wherein said fragment comprises amino acids 664-805 of the protein hMSH-2.
8. A polypeptide according to any preceding claim wherein said polypeptide is provided with a tag so as to enable the binding of same to be detected.
9. A polypeptide according to claim 8 wherein said tag is a C-terminal flag peptide.
10. A polypeptide according to claim 9 wherein said tag further includes an antibody adapted to bind to said C-terminal flag peptide.
11. An expression system for the manufacture of a polypeptide according to any of the proceeding claims which system comprises a host cell including a fragment of DNA encoding the polypeptide according to any of the preceding claims which DNA is functionally coupled to the replication system of the host cell whereby said polypeptide can be made.
12. A vector for transforming a host cell whereby the polypeptide according to any one of claims 1-10 of the invention can be made.
13. A method for obtaining a polypeptide according to claims 1 to 10 comprising;
a) inserting a fragment of DNA encoding the said polypeptide and any necessary transcriptional/translational control elements into a suitable host cell expression system;
b) providing conditions which favour transcription and translation of said DNA in said host cell;
c) harvesting said host cells;
d) lysing said host cells; e) collecting the lysates; and
f) purifying the polypeptide fragment.
14. A DNA sequence encoding the polypeptide according to any one of claims 1-10.
15. Oligonucleotides for amplifying the DNA encoding the polypeptide according to anyone of claims 1-10.
16. A method for identifying mismatched oligonucleotides comprising exposing strands of oligonucleotides to the polypeptide of any one of claims 1-10 under conditions which promote binding and determining the amount of binding taking place.
17. A kit for determining mismatch binding comprising at least a polypeptide according to any one of claims 1-10.
18. A kit according to claim 17 which further comprises a control including at least one mismatch binding pair of oligonucleotides.
19. A kit according to claims 17 or 18 comprising at least one matched complementary binding pair of oligonucleotides.
20. The use of a polypeptide according to any one of claims 1-10 for detection of mismatched complementary oligonucleotide pairs or of mismatches in double-stranded nucleic acid fragments or in double-stranded PCR products.
21. Means for regulating the activity of a 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.
22. Means according to claim 21 wherein said regulation 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.
PCT/GB1996/002595 1995-10-24 1996-10-23 Dna repair WO1997015657A1 (en)

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WO2001073079A3 (en) * 2000-03-28 2002-05-16 Univ California Chimeric proteins for detection and quantitation of dna mutations, dna sequence variations, dna damage and dna mismatches
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US6582914B1 (en) 2000-10-26 2003-06-24 Genencor International, Inc. Method for generating a library of oligonucleotides comprising a controlled distribution of mutations

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