Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-01-14
2001-12-11
Myers, Carla J. (Department: 1655)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S005000, C435S006120, C435S091200, C435S194000, C435S320100, C435S325000, C435S419000, C435S252300, C536S023200
Reexamination Certificate
active
06329178
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the field of molecular biology. Specifically, the present invention relates to a DNA polymerase that is a mutant form of a naturally occurring DNA polymerase, in which one or more amino acids within the active site are altered.
BACKGROUND OF THE INVENTION
DNA polymerases are responsible for the replication and maintenance of the genome, a role that is central to accurately transmitting genetic information from generation to generation. DNA polymerases function in cells as the enzymes responsible for the synthesis of DNA. They polymerize deoxyribonucleoside triphosphates in the presence of a metal activator, such as Mg
2+
, in an order dictated by the DNA template or polynucleotide template that is copied. Even though the template dictates the order of nucleotide subunits that are linked together in the newly synthesized DNA, these enzymes also function to maintain the accuracy of this process. The contribution of DNA polymerases to the fidelity of DNA synthesis is mediated by two mechanisms. First, the geometry of the substrate binding site in DNA polymerases contributes to the selection of the complementary deoxynucleoside triphosphates. Mutations within the substrate binding site on the polymerase can alter the fidelity of DNA synthesis. Second, many DNA polymerases contain a proof-reading 3′-5′ exonuclease that preferentially and immediately excises non-complementary deoxynucleoside triphosphates if they are added during the course of synthesis. As a result, these enzymes copy DNA in vitro with a fidelity varying from 5×10
−4
(1 error per 2000 bases) to 10
−7
(1 error per 10
7
bases) (Fry and Loeb,
Animal Cell DNA Polymerases
), pp. 221, CRC Press, Inc., Boca Raton, Fla. (1986); Kunkel,
J. Biol. Chem.
267:18251-18254(1992)).
In vivo, DNA polymerases participate in a spectrum of DNA synthetic processes including DNA replication, DNA repair, recombination, and gene amplification (Korberg and Baker,
DNA Replication
, pp. 929, W. H. Freeman and Co., New York (1992)). During each DNA synthetic process, the DNA template is copied once or at most a few times to produce identical replicas. In vitro DNA replication, in contrast, can be repeated many times, for example, during polymerase chain reaction (Mullis, U.S. Pat. No. 4,683,202).
In the initial studies with polymerase chain reaction (PCR), the DNA polymerase was added at the start of each round of DNA replication (U.S. Pat. No. 4,683,202). Subsequently, it was determined that thermostable DNA polymerases could be obtained from bacteria that grow at elevated temperatures, and these enzymes need to be added only once (Gelfand, U.S. Pat. No. 4,889,818). At the elevated temperatures used during PCR, these enzymes would not denature. As a result, one can carry out repetitive cycles of polymerase chain reactions without adding fresh enzymes at the start of each synthetic addition process. DNA polymerases, particularly thermostable polymerases, are the key to a large number of techniques in recombinant DNA studies and in medical diagnosis of disease. For diagnostic applications in particular, a target nucleic acid sequence may be only a small portion of the DNA or RNA in question, so it may be difficult to detect the presence of a target nucleic acid sequence without PCR amplification. Due to the importance of DNA polymerases in biotechnology and medicine, it would be highly advantageous to generate DNA polymerase mutants having desired enzymatic properties such as altered fidelity and high activity.
Polymerases contain an active site architecture that specifically configures to an incorporates each of the four deoxynucleoside triphosphates while taking direction from templates with diverse nucleotide sequences. In addition, the active site tends to exclude altered nucleotides produced during cellular metabolism. The overall folding pattern of polymerases resembles the human right hand and contains three distinct subdomains of palm, fingers and thumb. (Beese et al.,
Science
260:352-355 (1993); Patel et al.,
Biochemistry
34:5351-5363 (1995); these two references are incorporated herein by reference. While the structure of the fingers and thumb subdomains vary greatly between polymerases that differ in size and in cellular functions, the catalytic palm subdomains are all superimposable. Motif A, which interacts with the incoming dNTP and stabilizes the transition state during chemical catalysis, is superimposable with a mean deviation of about one Å amongst mammalian pol &agr; and prokaryotic pol I family DNA polymerases (Wang, et al.,
Cell
89:1087-1099 (1997)). Motif A begins structurally at an antiparallel &bgr;-strand containing predominantly hydrophobic residues and continues to an &agr;-helix (FIG.
1
). The primary amino acid sequence of DNA polymerase active sites are exceptionally conserved. Motif A retains the sequence DYSQIELR in polymerases from organisms separated by many millions years of evolution including
Thermus aquaticus, Chlamydia trachomatis
, and
Escherichia coli
. Taken together, these results indicate polymerases function by similar catalytic mechanisms and that the active site of polymerases may be immutable in order to ensure the survival of organisms.
U.S. Pat. No. 5,939,292 is directed to a recombinant thermostable DNA polymerase that is a mutant form of a naturally occurring thermostable DNA polymerase, wherein said naturally occurring thermostable DNA polymerase has an amino acid sequence comprising amino acid sequence motif SerGlnIleGluLeuArgXaa (SEQ ID NO:1) wherein “Xaa” at position 7 of said sequence motif is a valine residue or an isoleucine residue; wherein said mutant form has been modified to contain an amino acid other than glutamic acid (Glu) at position 4 of said sequence motif; and wherein said mutant form possesses reduced discrimination against incorporation of an unconventional nucleotide in comparison to said naturally occurring thermostable DNA polymerase. In the '292 patent, the thermostable DNA polymerase mutant has an activity to incorporate ribonucleotides in vitro. The mutant has a single mutation in the active site, namely, the glutamic acid residue is altered. We believe that bacteria dependent on such a DNA polymerase mutant with a single mutation of altering glutamic acid residue in the active site is not able to survive in vivo because the mutant does not have enough activity for DNA replication. Our results suggest that bacteria depending on a DNA polymerase mutant which has a Glu615 residue substitution will only survive if the Glu is substituted by Asp and there is at least one additional substitution at other sites in motif A (FIG.
2
).
The present invention evaluates the degree of mutability of a polymerase active site in vivo. Our results counter the common paradigm that amino acid substitutions within the catalytic site lead to reduced stability and enzymatic activity. We find that the DNA polymerase active site is highly mutable and can accommodate many amino acid substitutions without affecting DNA polymerase activity significantly. The instant application shows that mutation on the catalytic site can produce highly active enzymes with altered substrate specificity. Mutant DNA polymerases may offer selective advantages such as ability to resist incorporation of chain terminating nucleotide analogs, increased catalytic activity, ability to copy through hairpin structures, increased processivity, and altered fidelity.
SUMMARY OF THE INVENTION
This invention is directed to a DNA polymerase that is a mutant form of a naturally occurring DNA polymerase, in which one or more amino acids in the active site is mutated. The DNA polymerase mutant of this invention is characterized by altered substrate specificity, altered fidelity or higher enzymatic activity in comparison with the naturally occurring DNA polymerase. A host cell dependent on the DNA polymerase mutant is able to survive and replicate repetitively. The invention also provides a method of pr
Loeb Lawrence A.
Patel Premal H.
Halluin Albert P.
Howrey Simon Arnold & White , LLP
Kung Viola T.
Myers Carla J.
Taylor Janell E.
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