Broad specificity DNA damage endonuclease

Details Broad specificity DNA damage endonuclease Broad specificity DNA damage endonuclease

1999-06-08

2002-04-09

Achutamurthy, Ponnathapu (Department: 1652)

Drug, bio-affecting and body treating compositions

Enzyme or coenzyme containing

Hydrolases

C435S196000

Reexamination Certificate

active

06368594

ABSTRACT:

BACKGROUND OF THE INVENTION
The field of the present invention is the area of DNA repair enzymes. In particular, the invention concerns the identification of stable ultraviolet DNA endonuclease polypeptide fragments, their nucleotide sequences and recombinant host cells and methods for producing them and for using them in DNA repair processes.
Cellular exposure to ultraviolet radiation (UV) results in numerous detrimental effects including cell death, mutation and neoplastic transformation. Studies indicate that some of these deleterious effects are due to the formation of two major classes of bipyrimidine DNA photoproducts, cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts (6-4 PPs). (Friedberg et al. [1995] in
DNA Repair and Mutagenesis
, pp. 24-31, Am. Soc. Microbiol., Washington, D.C.).
Organisms have evolved several different pathways for removing CPDs and 6-4 PPs from cellular DNA (Friedberg et al. [1995] supra; Brash et al. [1991
] Proc. Natl. Acad. Sci. U.S.A.
8810124-10128). These pathways include direct reversal and various excision repair pathways which can be highly specific or nonspecific for CPDs and 6-4 PPs. For example, DNA photolyases specific for either CPDs or 6-4 PPs have been found in a variety of species and restore the photoproduct bases back to their original undamaged states (Rubert, C. S. [1975
] Basic Life Sci.
5A:73-87; Kim et al. [1994
] J. Biol. Chem.
269:8535-8540; Sancar, G. B. [1990
] Mutat. Res.
236:147-160). Excision repair has been traditionally divided into either base excision repair (BER) or nucleotide excision repair (NER) pathways, which are mediated by separate sets of proteins but which both are comprised of DNA incision, lesion removal, gap-filling and ligation reactions (Sancar, A. [1994
] Science
266:1954-19560; Sancar, A. and Tang, M. S. [1993
] Photochem. Photobiol.
57:905-921). BER N-glycosylase/AP lyases specific for CPDs cleave the N-glycosidic bond of the CPD 5′ pyrimidine and then cleave the phosphodiester backbone at the abasic site via a &bgr;-lyase mechanism, and have been found in several species including T4 phage-infected
Escherichia coli, Micrococcus luteus
, and
Saccharomyces cerevisiae
(Nakabeppu, Y. et al. [1982
] J. Biol. Chem.
257:2556-2562; Grafstrom, R. H. et al. [1982
] J. Biol. Chem.
257:13465-13474; Hamilton, K. K. et al. [1992
] Nature
356:725-728). NER is a widely distributed, lesion non-specific repair pathway which orchestrates DNA damage removal via a dual incision reaction upstream and downstream from the damage site, releasing an oligonucleotide containing the damage and subsequent gap filling and ligation reactions (Sancar and Tang [1993] supra).
Recently, an alternative excision repair pathway initiated by a direct acting nuclease which recognizes and cleaves DNA containing CPDs or 6-4 PPs immediately 5′ to the photoproduct site has been described (Bowman, K. K. et al. [1994
] Nucleic. Acids Res.
22:3026-3032; Freyer, G. A. et al. [1995
] Mol. Cell. Biol.
15:4572-4577; Doetsch, P. W. [1995
] Trends Biochem. Sci.
20:384-386; Davey, S. et al. [1997
] Nucleic Acids Res.
25:1002-1008; Yajima, H. et al. [1995
] EMBO J.
14:2393-2399; Yonemasu, R. et al. [1997
] Nucleic Acids Res.
25:1553-1558; Takao, M. et al. [1996
] Nucleic Acids Res.
24:1267-1271). The initiating enzyme has been termed UV damage endonuclease (UVDE, now termed Uve1p). Homologs of UVDE have been found in
Schizosaccharomyces pombe, Neurospora crassa
and
Bacillus subtilis
(Yajima et al. [1995] supra; Yonemasu et al. [1997] supra; Takao et al. [1996] supra). The Uve1p homologs from these three species have been cloned, sequenced and confer increased UV resistance when introduced into UV-sensitive strains of
E. coli, S. cerevisiae
, and human cells (Yajima et al. [1995] supra; Takao et al, [1996] supra). In
S. pombe
Uve1p is encoded by the uve1+ gene. However, because of the apparently unstable nature of partially purified full-length and some truncated UVDE derivatives, UVDE enzymes have been relatively poorly characterized and are of limited use (Takao et al. [1996] supra).
Because of the increasing and widespread incidence of skin cancers throughout the world and due to the reported inherent instability of various types of partially purified full-length and truncated UVDE derivatives, there is a long felt need for the isolation and purification of stable UVDE products, especially for use in skin care and medicinal formulations.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide purified stable UVDE (Uve1p), polypeptide fragments which retain high levels of activity, particularly those from the
Schizosaccharomyces pombe
enzyme. In a specific embodiment, the polypeptide fragment is &Dgr;228-UVDE, which contains a 228 amino-acid deletion of the N-terminal region of the
S. pombe
uve1+ gene product; a second specific embodiment is the fusion protein GST-&Dgr;228-UVDE. The DNA sequence encoding GST-full-length UVDE from
S. pombe
is given in SEQ ID NO:1. The deduced amino acid sequence of full-length GST-UVDE is given in SEQ ID NO:2. The DNA sequence encoding &Dgr;228-UVDE is given in SEQ ID NO:3. The deduced amino acid sequence of &Dgr;228-UVDE is given in SEQ ID NO:4. The DNA coding sequence and deduced amino acid sequence for GST-&Dgr;228-UVDE are given in SEQ ID NO:5 and SEQ ID NO: 6, respectively. Also encompassed within the present invention are truncated UVDE proteins wherein the truncation is from about position 100 to about position 250 with reference to SEQ ID NO:2, and wherein the truncated proteins are stable in substantially pure form.
Also within the scope of the present invention are nucleic acid molecules encoding such polypeptide fragments and recombinant cells, tissues and animals containing such nucleic acids or polypeptide fragments, antibodies to the polypeptide fragments, assays utilizing the polypeptide fragments, pharmaceutical and/or cosmetic preparations containing the polypeptide fragments and methods relating to all of the foregoing.
A specifically exemplified embodiment of the invention is an isolated, enriched, or purified nucleic acid molecule encoding &Dgr;228-UVDE. Another exemplified embodiment is an isolated, enriched or purified nucleic acid molecule encoding GST-&Dgr;228-UVDE.
In a specifically exemplified embodiment, the isolated nucleic acid comprises, consists essentially of, or consists of a nucleic acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:5.
In another embodiment, the invention encompasses a recombinant cell containing a nucleic acid molecule encoding &Dgr;228-UVDE or GST-&Dgr;228-UVDE. The recombinant nucleic acid may contain a sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, a synonymous coding sequence or a functional derivative of SEQ ID NO:3 or SEQ ID NO:5. In such cells, the &Dgr;228-UVDE coding sequence is generally expressed under the control of heterologous regulatory elements including a heterologous promoter that is not normally coupled transcriptionally to the coding sequence for the UVDE polypeptide in its native state.
In yet another aspect, the invention relates to a nucleic acid vector comprising a nucleotide sequence encoding &Dgr;228-UVDE or GST-&Dgr;228-UVDE and transcription and translation control sequences effective to initiate transcription and subsequent protein synthesis in a host cell. Where a GST full length or truncated derivative is expressed, the GST portion is desirably removed (after affinity purification) by protease cleavage, for example using thrombin.
It is yet another aspect of the invention to provide a method for isolating, enriching or purifying the polypeptide termed &Dgr;228-UVDE.
In yet another aspect, the invention features an antibody (e.g., a monoclonal or polyclonal antibody) having specific binding affinity t

Affiliated with

Also associated with

Rating

Broad specificity DNA damage endonuclease does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Broad specificity DNA damage endonuclease, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Broad specificity DNA damage endonuclease will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2923102