Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1999-03-03
2004-03-30
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S006120, C435S018000, C435S440000, C536S023200
Reexamination Certificate
active
06713294
ABSTRACT:
This invention relates to new DNA-glycosylases, in particular new cytosine-, thymine- and uracil-DNA glycosylases, and their use for mutagenesis, for NA modification and cell killing.
Damage to DNA arises continually throughout the cell cycle and must be recognised and repaired prior to the next round of replication to maintain the genomic integrity of the cell. DNA base damage can be recognised and excised by the ATP-dependent nucleoside excision repair systems or by base excision repair systems exemplified by the DNA glycosylases.
DNA glycosylases are enzymes that occur normally in cells. They release bases from DNA by cleaving the bond between deoxyribose and the base in DNA. Naturally occurring glycosylases remove damaged or incorrectly placed bases. This base excision repair pathway is the major cellular defence mechanism against spontaneous DNA damage.
DNA glycosylases which have been identified are directed to specific bases or modified bases. An example of a DNA glycosylase which recognizes an unmodified base is uracil DNA glycosylase (UDG), which specifically recognises uracil in DNA and initiates base excision repair by hydrolysing the N-C1′ glycosylic bond linking the uracil base to the deoxyribose sugar. This creates an abasic site that is removed by a 5′-acting apurinic/apyrimidic (AP) endonuclease and a deoxyribophosphodiesterase, leaving a gap which is filled by DNA polymerase and closed by DNA ligase.
The activity of UDG serves to remove uracil which arises in DNA as a result of incorporation of dUMP instead of dTMP during replication or from the spontaneous deamination of cytosine. Deamination of cytosine to uracil creates a premutagenic U:G mismatch that, unless repaired, will cause a GC→AT transition mutation.
In vivo, UDGs specifically recognise and remove uracil from within DNA and cleave the glycosylic bond to initiate the uracil excision pathway. In vitro, UDG's can recognise and remove uracil from both single stranded DNA (ssDNA) and double-stranded DNA (dsDNA) substrates.
UDGs are ubiquitous enzymes and have been isolated from a number of sources. Amino acid sequencing reveals that the enzymes are conserved throughout evolution with greater than about 55% amino acid identity between human and bacterial proteins. A cDNA for human UDG has been cloned and the corresponding gene has been named UNG (Olsen et al. (1989) EMBO J., 8: 3121-3125).
The crystal structures of the human enzyme (Mol et al., (1995) Cell, 80: 869-878) and the herpes simplex virus enzymes (Sava et al. (1995) Nature, 373: 487-493) have recently been determined and reveal that uracil binds in a rigid pocket at the base of the DNA binding groove of human UDG. The absolute specificity of the enzyme for uracil over the structurally related DNA bases thymine and cytosine is conferred by shape complementary, as well as main chain and side chain hydrogen bonds.
Although UDG's do not have activity against other bases as a result of the afore-mentioned specific spatial and charge characteristics of the active site, other glycosylases with different activities have been identified, which may or may not be restricted to single substrates.
A naturally-occurring thymine-DNA glycosylase has been identified which in addition to releasing thymine also releases uracil (Nedderman & Jiricny (1993) J. Biol. Chem., 268: 21218-21114; Nedderman & Jiricny (1994) J. Proc. Natl. Acad. Sci. U.S.A., 91: 1642-1646). This thymine-DNA glycosylase however has activity in respect of only certain substrates and has an absolute requirement for a mismatched U or T opposite of a G in a double-stranded substrate and will not recognise T or U from T(U): A matches or a single-stranded substrate. DNA glycosylases which recognize and release unmodified bases other than uracil and thymine (in certain substrates, as mentioned above) have not been identified.
A DNA glycosylase recognizing unmodified cytosine has not been reported, although a 5-hydroxymethylcytosine-DNA glycosylase activity was detected in mammalian cells (Cannon et al. (1988) Biochem. Biophys. Res. Comm., 151: 1173-1179). The sequences of the afore-mentioned thymine and 5-hydroxymethylcytosine DNA glycosylases have not yet been reported and it is unknown whether their active site may be structurally related to UDG.
It has now surprisingly been found that the substitution of certain of the UDG amino acids has a profound effect on the substrate specificity of the glycosylase. In particular, the replacement of Asn204 by Asp204 results in the production of a mutant enzyme which has acquired cytosine-DNA glycosylase (CDG) activity, while retaining some UDG-activity. Alternatively, replacing Tyr147 with Ala147 allows for binding of thymine, resulting in an enzyme that has acquired thymine-DNA glycosylase (TDG) activity.
These new DNA glycosylases are not product-inhibited by added uracil, in contrast to UDG and other UDG-mutants. Compared with the efficiency of wild type UDG in removal of uracil, the activity of the new DNA glycosylases that remove normal pyrimidines in DNA is low, but distinct and easily detectable. However, it should be noted that the very high turnover of UDG appears to be unique among DNA glycosylases and turnover numbers of other DNA glycosylases may be as low, or even lower than those of the engineered glycosylases CDG and TDG. This may result from the narrow substrate specificity of UDG.
Furthermore, an additional new UDG has been identified. The complete sequence of the UNG gene was recently published (Haug et al., 1996, Genomics, 36, p408-416). As mentioned previously, cDNA to this UNG gene has been identified by Olsen et al., 1989, supra (hereinafter referred to as UNG1 cDNA and the expressed protein referred to as UNG1). Other workers have described the location, gene structure and recombinant expression of UNG1 (Slupphaug et al., 1993, Nucl. Acids Res., Vol. 21, No. 11, p. 2579-2584; Haug et al., 1994, FEBS Letters, 353, p. 180-184; Slupphaug et al., 1995, Biochemistry, 34, p. 128-138, respectively). It has now surprisingly been found that alternative splicing of the genomic DNA (UNG) with an exon located 5′ of exon 1 which was not previously recognized results in a new distinct cDNA with an open reading frame of 313 amino acids. The new UNG CDNA is referred to hereinafter as UNG2 cDNA, and the product which it encodes, UNG2. The latter protein has a predicted size of 36 kDa.
UNG2 differs from the previously known form (UNG1, ORF 304 amino acid residues) in the 44 amino acids of the N-terminal presequence, which is not necessary for catalytic activity. The rest of the presequence and the catalytic domain, altogether 269 amino acids, are identical. The alternative presequence in UNG2 arises by splicing of a previously unrecognized exon (exon 1A) into a consensus splice site after codon 35 in exon 1B (previously designated exon 1). The UNG1 presequence starts at codon 1 in exon 1B and thus has 35 amino acids not present in UNG2. Coupled transcription/translation in rabbit reticulocyte lysates demonstrated that both proteins are catalytically active. Similar forms of UNG1 and UNG2 are expressed in mouse which has an identical organization of the homologous gene. Furthermore, the presequence of a putative Xiphophorus UNG2 protein predicted from the gene structure is homologous to mammalian UNG2, but much shorter, suggesting a very high degree of conservation from fish to man.
The invention therefore provides a DNA glycosylase capable of releasing cytosine bases from single stranded (ss) DNA and/or double stranded (ds) DNA or thymine bases from both single stranded (ss) DNA and double stranded (ds) DNA or from single stranded (ss) DNA or uracil bases from single stranded (ss) DNA and/or double stranded (ds) DNA, wherein said uracil-DNA glycosylase is encoded by a nucleic acid molecule comprising the sequence (SEQUENCE I.D. No 1):
1
CACAGCCACA GCCAGGGCTA GCCTCGCCGG TTCCCGGGTG GCGCGCGTTC GCTGCCTCCT
61
CAGCTCCAGG ATGATCGGCC AGAAGACGCT CTACTCCTTT TTCTCCCCCA GCCCCGCCAG
121
GAAGCGACAC GCCCCCAGCC CCGAGCCGGC CGTCCAGGG
Kavli Bodil
Krokan Hans E.
Mol Clifford D.
Slupphaug Geir
Tainer John A.
Achutamurthy Ponnathapu
Pak Yong
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