DNA adenine methyltransferases and uses thereof

Details DNA adenine methyltransferases and uses thereof DNA adenine methyltransferases and uses thereof

1999-07-19

2002-07-02

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Transferase other than ribonuclease

C536S023200

Reexamination Certificate

active

06413751

ABSTRACT:

BACKGROUND OF THE INVENTION
A. Field of the Invention
This invention pertains to the field of microbiology and to the treatment of conditions caused by microbes. In particular, this invention pertains to the isolation, sequencing, and detection of a DNA adenine methyltransferase gene from a variety of micro-organisms.
B. Related Art
Most organisms modify their genomic DNA by the methylation of specific nucleotide bases. DNA methylation is critical to gene regulation and repair of mutational lesions (for recent reviews see Jost and Saluz,
DNA Methylation, Molecular Biology and Biological Significance.
Birhauser Verlag, Basel, Switzerland (1993); Palmer and Marinus,
Gene
143:1-12 (1994)).
DNA methylation is catalyzed by a class of enzymes of varying substrate specificity called DNA methyltransferase enzymes. A DNA methyltransferase from the bacterium
Caulobacter crescentus,
cell cycle regulated methyltransferase (“CcrM” refers to the protein and “ccrM” denotes the gene), methylates the adenine residue in the recognition sequence GANTC (Zweiger et al.,
J. Mol. Biol.
235: 472-485, 1994; N denotes any nucleotide). CcrM is unusual, as it is not part of a restriction modification system, and is the only known prokaryotic DNA methyltransferase shown to be essential for viability (Stephens et al.,
Proc. Natl. Acad. Sci.
93:1210-1214, 1996) outside of a restriction modification system (i.e., a coexpressed methylase and restriction enzyme which recognize a same nucleotide sequence).
The CcrM protein, and therefore its DNA methylation activity, is present only at the predivisional stage of the cell cycle (Zweiger et al.,
J. Mol. Biol.
235: 472-485, 1994; Stephens et al.,
Proc. Natl. Acad. Sci.
93:1210-1214, 1996). This is controlled in two ways; the ccrM gene is transcribed only in the predivisional cell (Stephens et al.,
J. Bacteriol.
177:1662-1669, 1995) and the CcrM protein is highly unstable and is completely degraded by the time of cell division in a Lon protease dependent process (Wright et al.,
Genes and Development
10:1532-1542, 1996).
SUMMARY OF THE INVENTION
The present invention comprises the isolation and sequence of a number of methyltransferase-encoding nucleic acids and their gene products, including the methyltransferase gene from
Rhizobium meliloti, Brucella abortus, Agrobacterium tumefaciens,
and
Helicobacter pylori.
These novel DNA methyltransferases are potential targets for new antimicrobial agents. Under the assay conditions provided herein, these enzymes exhibit a novel property called processivity.
In one series of embodiments, the invention comprises an isolated nucleic acid that encodes a
Rhizobium meliloti
DNA methyltransferase, including a nucleic acid having SEQ ID NO:1; cells that contain and express such nucleic acids; and isolated DNA adenine methyltransferases encoded by such a nucleic acid (e.g., SEQ ID NO: 2).
In another series of embodiments, the invention comprises an isolated nucleic acid that encodes a
Brucella abortus
DNA methyltransferase (e.g., SEQ ID NO:4), particularly a nucleic acid having SEQ ID NO:3; cells that contain and express such nucleic acids, and isolated DNA adenine methyltransferases encoded by such nucleic acid.
In another series of embodiments, the invention comprises an isolated nucleic acid (e.g., SEQ ID NO: 5) that encodes a partial sequence of
Agrobacterium tumefaciens
DNA methyltransferase (e.g., SEQ ID NO: 6).
In another series of embodiments, the invention comprises an isolated nucleic acid (e.g., SEQ ID NO: 7) that encodes a
Helicobacter pylori
DNA methyltransferase (e.g., SEQ ID NO: 8); cells that contain and express such nucleic acids, and isolated DNA adenine methyltransferases encoded by such nucleic acid.
The ccrM genes for
Rhizobium meliloti, Agrobacterium tumefaciens
and
Brucella abortus
exhibit homology to Caulobacter ccrM. It is highly likely that the ccrM homologs are a new DNA methyltransferase family which is not part of a restriction modification system.
Both Caulobacter and Rhizobium ccrM are essential for viability. Neither gene can be disrupted from the chromosome unless a copy is provided in trans on a plasmid (Stephens et al.,
Proc. Nat'l. Acad. Sci.
93:1210-1214, 1996; this application). The overexpression of both Rhizobium and Caulobacter ccrM results in defects in cell morphology and cell division, demonstrating the importance of DNA methylation in these two bacteria. Hemimethylated DNA could be detected in both Rhizobium and Caulobacter. In the case of Caulobacter this is due to the cell cycle regulation of ccrM.
In another embodiment, this invention provides for vectors incorporating any of the above-described nucleic acids. The vectors preferably include the above-described nucleic acid operably linked to (under the control of) a promoter, either constitutive or inducible. The vector can also include an initiation and a termination codon.
In another embodiment, this invention provides for cells that contain the above-mentioned nucleic acids and cells that express the above-mentioned nucleic acids that encode adenine methyltransferases. For example, host cells may be transfected with a nucleic acid of SEQ ID NO: 1, 3, 5, or 7.
In addition to providing for host cells stably transfested with nucleic acids encoding adenine methyltransferases, this invention also uses these transfected host cells to detect compounds that are capable of inhibiting adenine methyltransferase.
The invention further provides for nucleic acid probes that are capable of selectively hybridizing to a nucleic acid encoding an adenine methyltransferase. For example, the nucleic acid probe can be the nucleic acid of SEQ ID NO: 1, 3, 5, or 7. These probes can be used to measure or detect nucleic acids encoding adenine methyltransferases. The probes are incubated with a biological sample to form a hybrid of the probe with complementary nucleic acid sequences present in the sample. The extent of hybridization of the probe to these complementary nucleic acid sequences is then determined.
In another embodiment, this invention provides for antibodies to the methyltransferases encoded by the above-mentioned nucleic acids. Particularly preferred antibodies specifically bind a polypeptide comprising at least 10, more preferably at least 20, 40, 50, and most preferably at least 100, 200, and even 300 contiguous amino acids, or even the full length polypeptide encoded by a nucleic acid selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7; wherein said polypeptide elicits the production of an antiserum or antibody which specifically binds to a polypeptide selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ. ID NO: 6, or SEQ ID NO: 8, wherein the antiserum or antibody preferably does not cross-react with the
C. crescentus
adenine methyltransferase. The antibody can be polyclonal or monoclonal. The antibody can also be humanized or human.
This invention also provides for cells (e.g., recombinant cells such as hybridomas or triomas) which synthesize any of the above-described antibodies.
This invention also provides for kits for the detection and/or quantification of the above-mentioned nucleic acids. The kit can include a container containing one or more of any of the above identified nucleic acids, amplification primers, and antibodies with or without labels, free, or bound to a solid support as described herein. The kits can also include instructions for the use of one or more of these reagents in any of the assays described herein.
This invention further provides for methods and assays for identification and screening for novel antibiotics that target the methyltransferases of this invention. Such assays include those for screening for inhibitors of DNA methyltransferase activity that comprises: i. contacting in an aqueous reaction mixture a nucleic acid encoding a DNA methyltransferase wherein said methyltransferase has a molecular weight of about 30-45 kilodaltons and binds to a polyclonal antibody that specifically binds to a polypeptide

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