Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-08-29
2002-04-02
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C530S350000, C536S023200, C536S023400
Reexamination Certificate
active
06365355
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the field of molecular biology, and more particularly, detection of DNA sequence variation, DNA mutations, DNA damage and DNA base pair mismatches. In particular, the invention relates to proteins including chimeric proteins capable of detecting DNA sequence variations, DNA mutations, damaged DNA or DNA with mismatched base pairs.
BACKGROUND OF THE INVENTION
Natural DNA sequence variation exists in identical genomic regions of DNA among individual members of a species. It is of interest to identify similarities and differences in such genomic regions of DNA because such information can help identify sequences involved in susceptibility to disease states as well as provide genetic information for characterization and analysis of genetic material.
When a cell undergoes reproduction, its DNA molecules are replicated and precise copies are passed on to its descendants. The linear base sequence of a DNA molecule is maintained during replication by complementary DNA base pairing. Occasionally, an incorrect base pairing does occur during DNA replication, which, after further replication of the new strand, results in a double-stranded DNA offspring with a sequence containing a heritable single base difference from that of the parent DNA molecule. Such heritable changes are called “genetic polymorphisms, genetic mutations,” “single base pair mutations,” “point mutations” or simply, “DNA mismatches”. In addition to random mutations during DNA replication, organisms are constantly bombarded by endogenous and exogenous genotoxic agents which injure or damage DNA. Such DNA damage or injury can result in the formation of DNA mismatches or DNA mutations such as insertions or deletions.
The consequences of natural DNA sequence variation, DNA mutations, DNA mismatches and DNA damage range from negligible to lethal, depending on the location and effect of the sequence change in relation to the genetic information encoded by the DNA. In some instances, natural DNA sequence variation, DNA mutations, DNA mismatches and DNA damage can lead to cancer and other diseases of which early detection is critical for treatment.
There is thus a tremendous need to be able to rapidly identify differences in DNA sequences among individuals. In addition there is a need to identify DNA mutations, DNA mismatches and DNA damage to provide for early detection of cancer and other diseases.
SUMMARY OF THE INVENTION
The present invention concerns the use of proteins that function biologically to recognize DNA mutations and their application in defined systems for detecting and mapping DNA mutations, DNA mismatches and DNA damage. The present invention provides methods for using such DNA mutation recognition proteins, alone or in combination with other proteins, for detecting DNA sequence variability, detecting and localizing DNA mutations and for comparing DNA sequences among individuals of a species.
In one embodiment, the present invention is directed to chimeric proteins where the chimeric protein includes a DNA mutation binding protein, a linker and a nuclease.
The DNA mutation binding proteins of the chimeric proteins of the invention will bind to genetic mutations, single base pair mutations, point mutations, DNA mismatches, DNA insertions, DNA deletions, DNA transversions, DNA transitions, frameshift mutations, damaged DNA, and other changes or alterations in a normal or wild type DNA sequence.
DNA mutation binding proteins which find use in the invention include human MutS homologue2 (hMSH2), xeroderma pigmentosum complementation group A (XPA), xeroderma pigmentosum C (XPC), xeroderma pigmentosum complementation group E (XPE),
Thermus thermophilus
Mut S (TthMutS), thymine DNA glycosylase (TDG),
Escherechia coli
Fpapy-DNA glycosylase,
Escherechia coli
endonuclease III,
Escherechia coli
exonuclease III,
Escherechia coli
endonuclease IV, T4 endonuclease,
Escherechia coli
uracil DNA glycosylase,
Escherachia coli
A/G-specific adenine DNA glycosylase (MutY),
Escherechia coli
Uvr A DNA mutation binding protein,
Escherechia coli
Uvr B DNA mutation binding protein and other DNA damage binding proteins.
The DNA mutation binding proteins of the invention include those proteins having amino acid sequences depicted in SEQ ID NO:1, 3, 7, 9, 11, 15, 19, 21, 23, 25, 29, 31, 39, 35, 37, 101 and 103. The DNA mutation binding proteins of the invention are encoded by DNA which have the nucleotide sequences depicted in SEQ ID NO:2, 4, 8, 10, 12, 16, 20, 22, 24, 26, 30, 32, 40, 36, 38, 102 and 104.
The nucleases of the chimeric proteins of the invention are proteins and peptides capable of cleaving or cutting DNA. Nucleases include the N-terminus of human excision repair cross-complementing rodent repair deficiency (XPF),
Serratia marcescens
nuclease (Nuc),
Escherechia coli
Fpapy-DNA glycosylase;
Escherechia coli
endonuclease III;
Escherechia coli
endonuclease IV; T4 endonuclease;
Escherechia coli
uracil DNA glycosylase;
Escherechia coli
A/G-specific adenine DNA glycosylase,
Escherechia coli
Uvr B nuclease,
Escherechia coli
Uvr C nuclease and other DNA nucleases.
The nucleases include those proteins having amino acids depicted in SEQ ID NO:5, 11, 13, 25, 31, 35, 37, 39, 103 and 105. The nucleases are encoded by DNA having the nucleotide sequences depicted in SEQ ID NO 6, 12, 14, 26, 31, 36, 38, 40, 104 and 106.
In one embodiment, the chimeric proteins of the invention are recombinant proteins having the formula A-L-B or B-L-A, wherein: A is a peptide having DNA mutation binding activity; L is a linker peptide; and B is a peptide having nuclease activity. The invention is further directed to DNA encoding the chimeric proteins of the invention. The DNA may be in a vector. Furthermore, the vector may be in a suitable host such as bacteria, yeast or fungi.
In another embodiment, the present invention is directed to an isolated and purified chimeric protein comprising a pair of proteins wherein the pair of proteins are selected from the group consisting of XPF and XPA, XPF and hMSH2, XPA and XPF, hMSH2 and XPF, Nuc and hMSH2, Nuc and XPA, MutS and XPF, XPF and MutS, Nuc and MutS, XPA and XPF, and Nuc and XPA, wherein XPF is human excision repair cross-complementing rodent repair deficiency, XPA is xeroderma pigmentosum complementation group A, hMSH2 is human MutS homologue2, Nuc is
Serratia marcescens
nuclease and TthMutS is
Thermus thermophilus
Mut S.
The linker peptide of the chimeric peptide of the invention generally consists of 8 amino acids rich in glycine and proline or other amino acids known to disrupt protein secondary structure. For example, the sequence GSGPSPGS (SEQ ID NO:17) finds use in the invention. However, in some circumstances the linker peptides will be as short as zero amino acids where the nuclease and DNA binding protein retain activity in the absence of a linker peptide. In other circumstances the peptide will have up to 5, 6, 7, 8 9 10, 11-15, 16-20 or 21-30 amino acids.
In another embodiment, the present invention is directed to an isolated and purified nucleic acid encoding a chimeric polypeptide comprising a DNA mutation binding protein and a nuclease. The nucleic acid may by in a nucleic acid construct. The nucleic acid construct may be operably associated with an expression control sequence functional in a microbial cell such as a bacterial cell.
In another embodiment, the present invention is directed to a recombinant bacterial cell comprising a nucleic acid encoding a chimeric polypeptide comprising a DNA mutation binding protein and a nuclease.
In another embodiment, the present invention is directed to an isolated and purified nucleic acid encoding a chimeric protein having the formula A-L-B or B-L-A, wherein:A is a peptide having DNA mutation binding activity; L is a linker peptide; and B is a peptide having nuclease activity.
In another embodiment, the present invention is directed to a method of detecting a DNA sequence variation, comprising: a) obtaining a polynucleotide; b) obtaining a chimeric protein wh
Horlick Kenneth R.
The Regents of the University of California
Thompson Alan H.
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