Recombinant helix modification recognition proteins and uses...

Details Recombinant helix modification recognition proteins and uses... Recombinant helix modification recognition proteins and uses...

1995-11-28

2001-05-15

Arthur, Lisa B. (Department: 1655)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S320100, C435S325000, C435S069700, C435S252300, C536S023400, C536S023740, C530S350000, C530S371000

Reexamination Certificate

active

06232095

ABSTRACT:

BACKGROUND OF THE INVENTION
This invention relates to proteins that bind nucleotide mismatches, DNAs encoding such proteins, and uses thereof, particularly in mismatch detection assays.
Mutation detection techniques provide powerful tools for the prediction and diagnosis of disease arising from one or more changes in a nucleotide sequence. Such changes generally occur in coding regions and result in protein products that are either inactive or are altered in their level or type of activity. Less commonly, mutations resulting in disease states occur in nucleotide sequences that do not encode a protein product; for example, mutations in repetitive DNA have been shown to be associated with such diseases as human fragile-X syndrome, spinal and bulbar muscular dystrophy, and myotonic dystrophy.
To detect mismatches at the nucleic acid level, a number of screens have been developed, many employing a technique commonly referred to as heteroduplex analysis. This technique involves the formation of a duplex between one strand of a control nucleic acid (typically, of wild-type sequence) and one strand of a test nucleic acid (for example, one suspected of including a mutation). The presence of a mismatch in the duplex is then revealed by any of a number of standard approaches, including RNAse A digestion, chemical cleavage, or PCR-based or primer extension-based techniques (reviewed in Cotton,
Curr. Opinion in Biotech
. 3: 24, 1992).
Another means by which mismatches may be identified in a heteroduplex molecule is by the use of mismatch-specific proteins. These proteins are capable of recognizing and either binding or cleaving at or near a site of mismatch. One particular class of mismatch-specific binding proteins is the “Mut” series of bacterial polypeptides. An exemplary member of this class, the MutS protein, has been reported to bind heteroduplex DNA at sites of certain base pair mismatches and at loops resulting from deletions or insertions of up to four bases in length (Parker et al.,
Proc. Natl. Acad. Sci. USA
89: 1730-1734, 1992; Modrich et al., U.S. Pat. No. 5,459,039, 1995).
Given the role played by mismatch-specific proteins in mutation detection assays, the isolation and characterization of additional proteins having the capacity to identify mutant sites in nucleic acid substrates is of great value. These proteins find use in assays for the diagnosis and prognosis of diseases such as cancer, and are also useful for perinatal screening for inherited diseases, differential diagnosis of diseases not readily detectable by conventional tests (for example, Marfan's syndrome), and the detection of genetic alleles (for example, for genetic mapping, tissue matching, or identification purposes).
SUMMARY OF THE INVENTION
In general, the invention features a substantially pure recombinant Hmp polypeptide. Preferably, this polypeptide includes an amino acid sequence substantially identical to the sequence shown in
FIG. 2
(SEQ ID NO: 2), for example, includes the exact amino acid sequence shown in
FIG. 2
(SEQ ID NO: 2); is derived from a fungus (for example, Ustilago); or is fused to a second polypeptide (for example, bacteriophage T4 endonuclease VII).
In a related aspect, the invention also features substantially pure DNA that includes a sequence encoding a recombinant Hmp polypeptide. Preferably, this DNA encodes an amino acid sequence substantially identical to the amino acid sequence shown in
FIG. 2
(SEQ ID NO: 2); includes a DNA sequence substantially identical to the DNA sequence shown in
FIG. 2
(SEQ ID NO: 3); or includes the exact DNA sequence shown in
FIG. 2
(SEQ ID NO: 3).
In other related aspects, the invention features substantially pure DNA that includes a sequence encoding an Hmp fusion polypeptide; vectors and cells that include any of the substantially pure DNAs of the invention; substantially pure antibody that specifically binds an Hmp polypeptide (for example, an Hmp1 polypeptide); and a method of producing a recombinant Hmp polypeptide that involves providing a cell transformed with DNA encoding an Hmp polypeptide positioned for expression in the cell, culturing the transformed cell under conditions for expressing the DNA, and isolating the recombinant Hmp polypeptide (for example, the recombinant Hmp1 polypeptide). Also included in the invention is an Hmp polypeptide produced by expression of the substantially pure DNAs of the invention.
In yet another related aspect, the invention features a kit for the detection of a mismatch in a test nucleic acid sequence that includes a substantially pure Hmp polypeptide. In preferred embodiments, the Hmp polypeptide is purified from
Ustilago maydis
; the Hmp polypeptide is recombinant; the Hmp polypeptide is an Hmp1 polypeptide; the Hmp1 polypeptide is purified from
Ustilago maydis
; the Hmp1 polypeptide is recombinant; and the Hmp polypeptide is fused to a second polypeptide.
In a final related aspect, the invention features a method for detecting at least one mismatch in a test nucleic acid which hybridizes to a control nucleic acid, the method involving: a) providing a single-stranded control nucleic acid; b) annealing the single-stranded control nucleic acid to a single-stranded test nucleic acid to form a hybridized duplex between the nucleic acids; c) contacting the duplex with an Hmp polypeptide capable of recognizing at least one base pair mismatch in the duplex, under conditions allowing the Hmp polypeptide to bind the duplex at or near the site of mismatch; and d) detecting the binding as an indication of the presence of at least one mismatch in the test nucleic acid.
In preferred embodiments, the mismatch is a mutation; either or both of the test nucleic acid and the control nucleic acid is amplified; the Hmp polypeptide is derived from a eukaryotic organism; the Hmp polypeptide is purified from
Ustilago maydis
; the Hmp polypeptide is recombinant; the Hmp polypeptide is an Hmp1 polypeptide; the Hmpl polypeptide is purified from
Ustilago maydis
; the Hmp1 polypeptide is recombinant; and the Hmp polypeptide is fused to a second polypeptide.
By “Hmp” is meant any polypeptide within the class of
h
elix
m
odification recognition
p
roteins. Such polypeptides are capable of binding cruciform DNA, DNA duplexes exhibiting one or more mismatched base pairs (for example, substitutions, insertions, or deletions), or otherwise distorted helical DNA. In particular, an Hmp polypeptide is capable of binding, under standard solution conditions, to a sequence that includes a C/C mismatch, in preference to binding an otherwise identical sequence lacking a mismatch. Hmp polypeptides are further characterized by their capacity to bind all possible base pair mismatches (i.e., A/A, T/T, C/C, GIG, A/C, A/G, C/T, and T/G mismatches), and to do so in preference to binding homoduplex DNA (i.e., DNA lacking mismatched nucleotides). By “HMP” is meant a gene encoding a member of the Hmp class of polypeptides (as defined above).
By “Hmpl” is meant one member of the Hmp class of polypeptides having the amino acid sequence shown in
FIG. 2
(SEQ ID NO: 2) or a sequence that is substantially identical to that amino acid sequence. An Hmp1 polypeptide is encoded by an “HMP1” nucleic acid sequence.
By “polypeptide” or “protein” is meant any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).
By “substantially identical” is meant a polypeptide exhibiting at least 60%, preferably 75%, more preferably 90%, and most preferably 95% identity to a reference amino acid sequence or is meant a nucleic acid sequence exhibiting at least 50%, preferably 70%, more preferably 85%, and most preferably 90% identity to a reference nucleic acid sequence. For polypeptides, the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids. For nucleic acids, the length of comparison sequences will generally be at least 30 nucleotides, preferably at least 60 n

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