Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2001-05-23
2004-04-20
Prouty, Rebecca (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S232000, C435S455000, C424S094610, C530S387300, C530S388210
Reexamination Certificate
active
06723548
ABSTRACT:
BACKGROUND
DNA damage caused by ultraviolet (UV) light can lead to mutations, carcinogenesis, and cell death (Ananthaswamy and Pierceall,
Photochem. Photobiol.,
52, 1119-1136 (1990), Ziegler et al.,
Photochem. Photobiol.,
63, 432-435 (1996)). UV-induced DNA damage occurs frequently in DNA as the bases of nucleic acids absorb light in a range coincident with that of natural sunlight, making the bases susceptible to photochemically induced alterations (Patrick, In:
Photochemistry and Photobiology of Nucleic Acids
, Wang (ed.) Vol. II, Academic Press, New York, pp. 1-32 (1976)).
Ultraviolet (UV) light is the principle cause of basal and squamous cell carcinomas and possibly melanomas. UV light can also lead to mutations and cell death (Ananthaswamy and Pierceall,
Photochem. Photobiol.,
52, 1119-1136 (1990), Ziegler et al.,
Photochem. Photobiol.,
63, 432-435 (1996)). The vast majority of nonmelanoma skin cancers occur on portions of the body that are chronically exposed to sun. Additionally, molecular analyses of DNA sequences of oncogenes in skin tumor cells often reveals a signature tandem UV-induced mutations of CC to TT (Wikonkal and Brash,
J. Investig. Dermatol. Symp. Proc.,
4, 6-10 (1999)). This tandem mutation is strongly indicative of cis-syn cyclobutane pyrimidine dimers and (6-4) photoproducts, two types of photoproducts produced by exposure of DNA to sunlight.
Following exposure to UV light, humans undergo a temporary, reversible immunosuppression (Kripke
Cancer Res.,
54, 6102-6105 (1994), Ullrich et al,
J. Investig. Dermatol Symp Proc.,
4, 65-69 (1999)). Recent data suggest that the molecular trigger for this signal transduction cascade is the persistence of the damaged DNA itself (Nishigori et al.,
Proc. Natl. Acad. Sci., U.S.A.,
93, 10354-10359 (1996), and Wolf et al.,
J. Invest. Dermatol.,
104, 287-92 (1995)).
The human DNA repair system that is responsible for the removal of these DNA lesions is the nucleotide excision repair (NER) pathway, which removes a patch of damaged DNA by incising the damage-containing DNA strand both 5′ and 3′ to the damage (Cleaver,
J. Dermatol Sci.,
23, 1-11 (2000), and Sarasin,
Mutat. Res.,
428, 5-10 (1999)). Polymerases and helicases act in conjunction to remove the patch and resynthesize new, undamaged DNA. A DNA ligase then completes repair by sealing the remaining break (reviewed in Benhamou and Sarasin,
Mutat. Res.,
462, 149-158 (2000)).
In contrast to the NER pathway, human cells have the capacity to avoid the consequences of replicating damaged DNA by moving the damaged strand through homologous recombination opposite an undamaged DNA. This mechanism does not remove damage, but gives the cell additional time to excise the lesion without being forced to replicate potentially mutagenic DNA.
Human cells also have an additional pathway for removing many types of DNA lesions, including cis-syn cyclobutane pyrimidine dimers, that arise from UV light, oxidative stress, alkylation damage and deamination, among others. This pathway is termed the base excision repair (BER) system, and although it removes many lesions, in humans there are no enzymes that initiate repair at sites of UV induced damage. The first step in this pathway involves the recognition and removal of the damaged base by a class of enzymes called glycosylases. These enzymes break the glycosyl bond and a subset of these enzymes also possesses the ability to incise the phosphodiester backbone through a lyase reaction. Downstream of these reactions, the pathway requires the activities of an abasic (AP) site endonuclease, DNA polymerase(s) and DNA ligase. Thus in humans, the pathway is intact and robust, but concerning the repair of UV-induced damage, the first enzyme is missing.
Glycosylases exist that can initiate repair at sites of UV induced damage. The T4-pdg enzyme (also referred to as endonuclease V), produced by the denV gene of the bacteriophage T4, catalyzes the rate limiting, first step in the removal of UV-induced DNA damage, namely, single strand incision of DNA at the site of damage. Other glycosylases having the ability to repair DNA damage have also been identified, and include the
Micrococcus luteus
ultraviolet N-glycosylase/AP lyase and the
Paramecium bursaria chlorella
Virus-1 PBCV-1 pyrimidine dimer-specific glycosylase.
SUMMARY OF THE INVENTION
The present invention represents an advance in the art of repairing DNA lesions that result from, for instance, UV light, oxidative stress, alkylation damage and/or deamination. The introduction to human cells of a glycosylase having the appropriate initiating repair activity would result in cells possessing a fully functional BER pathway. The implications of this would be a faster, more efficient repair of potentially mutagenic and carcinogenic damage. Another benefit would be that this enhanced rate of repair would help to prevent immunosuppression caused by DNA damage. T4-pdg, the glycosylase/AP lyase that can initiate repair at sites of UV induced damage, has been delivered to human cells to increase the repair of damaged DNA; however, the enzyme has not been targeted to the cellular organelles containing the DNA to be repaired, i.e., the nucleus and the mitochondria of a cell. In the present invention, amino acid sequences that promote intracellular nuclear and mitochondrial targeting have been added to enzymes that initiate repair in the BER system.
The present invention provides a polypeptide having pyrimidine glycosylase activity, preferably, pyrimidine glycosylase/AP lyase activity. The polypeptide includes a targeting sequence, preferably an exogenous target sequence. The invention includes a composition that contains the polypeptide and a pharmaceutically acceptable carrier.
In some aspects of the present invention, the polypeptide includes an amino acid sequence of SEQ ID NO:41, SEQ ID NO:42, or SEQ ID NO:43 and a targeting sequence, preferably an exogenous targeting sequence. In other aspects of the present invention, the polypeptide includes an amino acid sequence having pyrimidine glycosylase/AP lyase activity and having at least about 15% identity with an amino acid sequence of SEQ ID NO:41, SEQ ID NO:42, or SEQ ID NO:43, and a targeting sequence, preferably an exogenous targeting sequence.
The present invention is further directed to a polynucleotide that includes a coding sequence encoding a polypeptide having pyrimidine glycosylase activity, preferably pyrimidine glycosylase/AP lyase activity. The polypeptide includes a targeting sequence, preferably, an exogenous targeting sequence.
In some aspects of the present invention, the polynucleotide includes a coding sequence encoding a polypeptide having pyrimidine glycosylase/AP lyase activity and a targeting sequence, preferably, an exogenous coding sequence. The polynucleotide includes a nucleotide sequence of SEQ ID NO:44, SEQ ID NO:45, or SEQ ID NO:46. In other aspects of the present invention, the polynucleotide includes a coding sequence encoding a polypeptide having pyrimidine glycosylase/AP lyase activity and including a targeting sequence, preferably, an exogenous coding sequence. The polynucleotide includes a nucleotide sequence having at least about 10% identity with a nucleotide sequence of SEQ ID NO:44, SEQ ID NO:45, or SEQ ID NO:46.
The present invention provides a method for increasing the repair rate of damaged bases in a cell. The method includes introducing to a cell exposed to or at risk of exposure to an agent that damages DNA a composition that includes an amount of a polypeptide effective to increase the repair rate of damaged DNA in the cell compared to a cell that does not include the polypeptide. The polypeptide has pyrimidine glycosylase activity, preferably pyrimidine glycosylase/AP lyase activity, and includes a targeting sequence, preferably, an exogenous targeting sequence.
Also provided is a method for treating mutagenesis in a subject. The method includes introducing to a subject exposed to or at risk of exposure to an agent that damages DNA a composition that includes an effec
Lloyd R. Stephen
McCullough Amanda K.
Nguyen Khoa
Board of Regents , The University of Texas System
Mueting Raasch & Gebhardt, P.A.
Prouty Rebecca
Walicka Malgorzata A.
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