Thermolabile uracil-DNA-glycosylas, process for its...

Details Thermolabile uracil-DNA-glycosylas, process for its... Thermolabile uracil-DNA-glycosylas, process for its...

1998-08-06

2001-02-13

Prats, Francisco (Department: 1651)

Chemistry: molecular biology and microbiology

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

Hydrolase

C435S195000, C435S091210, C435S091200, C435S091100

Reexamination Certificate

active

06187575

ABSTRACT:

The invention concerns a thermolabile (heat labile) enzyme with uracil-DNA-glycosylase activity, a process for isolating the enzyme from gram-positive microorganisms and an improved method for the detection or removal of uracil from DNA containing uracil in particular from DNA fragments that are obtained after specific amplification (e.g. PCR).
Uracil-DNA-glycosylases (UNG; EC 3.2.2.3) are wide-spread, highly conserved and extremely specific DNA repair enzymes. Their biological function is to specifically remove the base uracil from DNA. Uracil can form in DNA by the spontaneous deamination of cytosine or by the misincorporation of dUTP during DNA synthesis. The deamination of cytosine leads to promutagenic U:G mismatches which, if not corrected, lead to transition mutations in the next cycle of DNA synthesis (Lindahl, T. (1993) Nature 362, 709-715).
UNGs are used especially within the framework of PCR technology to decontaminate PCR mixtures. The so-called carry-over contamination of PCR mixtures by amplified target-DNA can lead to false-positive results. Carry-over contamination can be kept under control by incorporating dUTP into all PCR products (whereby dTTP is replaced by dUTP) and treating ready-mixed PCR reactions with UNG followed by thermal inactivation of UNG. In this process UNG cleaves uracil from all DNAs containing uracil but has no effect on natural (i.e. target) DNA. The a basic sites which are formed block the replication of DNA by DNA polymerases. This carry-over prevention technology prevents PCR products from resulting PCRs from causing false-positive results by contamination (Longo et al. (1990) Gene 93, 125-128). UNG from
E. coli
is usually used for this method (WO 92/0181, EP 0 415 755). The corresponding use of UNGs for isothermal amplification is also described (EP 0 624 643).
Most of the UNGs known today have an adequately high specificity for the efficient cleavage of uracil from single-stranded and double-stranded DNA and can thus in principle be used to optimize specific amplification methods. In contrast the UNGs do not show any activity towards other “normal” DNA bases or towards uracil in RNA.
A series of UNGs isolated from prokaryotic and eukaryotic organisms as well as some of viral origin have been described. Microbial UNGs are in particular known from
E. coli
(T. Lindahl, PNAS 71 (9), 3649-3653 (1974); Lindahl et al., J. Biol. Chem. 252 (10), 3286-3294 (1977)),
Bacillus subtilis
(Cone et al., Biochemistry 16 (14), 3194-3201 (1977)),
Bacillus stearothermophilus
(Kaboev et al., FEBS Letters 132 (2), 337-340 (1981)),
Thermothrix thiopara
(Kaboev et al., J. Bacteriology 164 (1), 421-424 (1985)) and
Micrococcus luteus
(Leblanc et al., J. Biol. Chem. 257 (7), 3477-3483 (1982). In addition a UNG from humans (Krokan et al., Nucl. Acid Res. 9 (11), 2599-2613 (1981) and some UNGs of viral origin have been described. Moreover the structural basis for the specificity and catalysis of UNG has recently been elucidated (Savva et al., Nature 373, 487-493; Mol et al., Cell 80, 869-878 (1995)).
However, most UNGs do not meet the requirements for use as a carry-over prevention method for amplification methods such as e.g. PCR due to their inadequate degree of purity and other properties especially their thermolability that is too low. Thus even after drastic heat treatment such as for example 10 minutes, 95° C. and subsequent PCR a residual activity of UNG is still detected (Thornton et al., Bio Techniques 13 (2), 180-183 (1992)). The residual activity of UNG i.e. the continued degradation of PCR products containing uracil is usually prevented by further incubating the corresponding mixtures after the PCR reaction at high temperatures of about 70° to 72° C. Moreover it was observed that storage of the PCR mixture/PCR product even at ca 4° C. often leads to a further degradation of the PCR product. Therefore much lower temperatures such as ca. −20° C. are recommended for the storage and/or the inhibition of the residual activity of UNG by the addition of chloroform or phenol. In addition the search for more suitable heat labile mutants has not yet been successful (Duncan et al., J. Bacteriology 134, (3), 1039-1045 (1978); WO 92/01814).
Thus the activity of the UNGs that are presently available cannot be completely switched off or only by using additional measures which additionally complicate the entire process.
Therefore the object of the present invention was to provide an enzyme with uracil-DNA-glycosylase activity which enables the difficulties known from the state of the art in removing uracil from DNA to be largely solved or avoided.
The object is achieved by a thermolabile enzyme with uracil-DNA-glycosylase activity which is obtainable from gram-positive microorganisms with a degree of purity of at least 95% (SDS-gel) and by a half-life of less than 5 minutes at 40° C. and of approximately or less than 2 minutes at 45° C. In addition to Arthrobacter microorganisms of the genus Micrococcus come especially into consideration. It has proven to be particularly advantageous when the microorganism DSM 10239 (BMTU 3346) is used as the enzyme source. DSM 10239 is deposited at the “Deutsche Sammlung für Mikroorganismen und Zellkulturen GmbH”, Mascheroder Weg 1b, D-38124 Braunschweig.
The enzyme according to the invention is usually purified below ca. 10° C., advantageously at ca. 4° C. Firstly the cells are disrupted by measures known to a person skilled in the art; this is preferably carried out mechanically by means of a high pressure press or a homogenizer. Subsequently the DNA components are separated e.g. by a Polymin precipitation. For the further purification the supernatant is firstly subjected to a hydroxyapatite chromatography (e.g. hydroxyapatite Ultrogel) which is followed by an anion exchange chromatography (preferably on Q-Sepharose ff high load) and a hydrophobic interaction chromatography. The latter can for example take place on phenyl Sepharose ff.
Details of the purification of the enzyme are as follows:
A certain amount of cells are suspended in the form of their dry weight in a frozen state with a low concentrated substance which buffers well in the pH range of ca. 7.2 to 8.0 such as e.g. phosphate buffer containing an SH reagent. Subsequently the cells are disrupted by incubation with lysozyme; usually 30 minutes at ca. 4° C. are adequate for this. The actual cell disruption is carried out mechanically for example by means of a high pressure press or a homogenizer. Usually a degree of lysis of ca. 30% is achieved.
In order to separate nucleic acid components these are precipitated under non-denaturing conditions. A step-wise precipitation with a dilute Polymin solution has proven to be particularly suitable in this case. After a short incubation phase and centrifugation, the supernatant is advantageously dialysed against the buffer solution which had been used for suspending the biomass. It has turned out that the dialysis is usually completed after ca. 16 hours. The dialysate is separated over a hydroxyapatite Ultrogel column. In every case the appropriate chromatography material is firstly equilibrated with the solution in which the fraction which is to be separated is also present. The fraction containing the enzyme is eluted with a linear gradient of ca. 10 mM to 1 M buffer solution e.g. a phosphate buffer at ca. pH 7.5. The combined fractions are dialysed against a solution buffering at ca. pH 8.0. Tris/HCl and also triethanolamine, N-methyldi-ethanolamine or other organic or inorganic buffers with a buffer capacity between pH 7.8 to 8.4 are suitable as buffers in this case. The combined dialysate is applied to an anion exchanger column equilibrated with this dialysate buffer such as for example Q-Sepharose ff high load and eluted with a linear gradient of increasing concentrations of sodium chloride. The combined eluate fractions are admixed with ammonium sulfate (final concentration: 1.3 M) and applied to a hydrophobic column material. In this case phenyl Sepharose ff has proven to be particularly suitable as the c

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