Ku80 homologue and uses thereof

Details Ku80 homologue and uses thereof Ku80 homologue and uses thereof

2002-06-03

2004-11-23

Andres, Janet (Department: 1647)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

C435S069100, C435S320100, C435S471000, C435S183000

Reexamination Certificate

active

06822077

ABSTRACT:

TECHNICAL FIELD
The present invention relates generally to plant molecular biology. More specifically, it relates to nucleic acids and methods for modulating their expression in plants.
BACKGROUND OF THE INVENTION
Cellular DNA undergoes double strand breakage during the course of many physiological events as well as in response to a variety of environmental insults (Friedburg, E., Walker, G. and Siede, W.,
DNA Repair and Mutagenesis,
ASM Press, Washington, D.C., 1995; Nickollof, J. and Hoekstra, M.,
DNA Damage and Repair,
Humana Press, Totowa, N.J., 1998). Left un-repaired, such double strand breaks (DSBs) lead to mutations that may prove lethal to the organism. Therefore, these DSBs are repaired promptly via two independent pathways: i) homologous recombination ii) non-homologous end joining (Friedburg, E., Walker, G. and Siede, W.,
DNA Repair and Mutagenesis,
ASM Press, Washington D.C., 1995; Nickollof, J. and Hoekstra, M.,
DNA Damage and Repair,
Humana Press, Totowa, N.J., 1998). The first pathway involves a series of very specific biochemical reactions catalyzed by a complex of cellular proteins (Shinohara and Ogawa,
Trends in Biochem. Sci.
237:387-391, 1995). Due to the large number of proteins involved in this complex, it is referred to as a ‘recombinosome’ (Hays et al.,
Proc. Natl. Acad. Sci. USA
92:6925-6929, 1995). This pathway is the dominant mode of DSB repair in lower eukaryotes such as yeast (Nickollof, J. and Hoekstra, M.,
DNA Damage and Repair,
Humana Press, Totowa, N.J., 1998).
The non-homologous end-joining pathway is the major route of DSB repair in higher eukaryotes (Friedburg, E., Walker, G. and Siede, W.,
DNA Repair and Mutagenesis,
ASM Press, Washington, D.C., 1995; Nickollof, J. and Hoekstra, M.,
DNA Damage and Repair,
Humana Press, Totowa, N.J., 1998). This pathway is also catalyzed by a group of cellular proteins. This group contains, in addition to hitherto unidentified factors, some well-characterized enzymes such as DNA ligases, Poly(ADP-Ribose) Polymerase [PADPRP], and DNA -dependent Protein Kinase [DNA-PK] (Lindahl et al.,
Trends in Biochem. Sci.
237: 405-411, 1995; Jackson & Jeggo,
Trends in Biochem. Sci.
237:412-415, 1995). These enzymes have been studied in detail using lower as well as higher vertebrate systems including mammals. Both PADPRP and DNA-PK have been shown to be activated by DNA ends. Moreover, these two enzymes also bind DNA ends (Lindahl et al.,
Trends in Biochem. Sci.
237:405-411, 1995; Jackson & Jeggo,
Trends in Biochem. Sci.
237:412-415, 1995). While PADPRP is a single polypeptide of ~115 kDa, DNA-PK exists as a complex of two subunits (Shah et al.,
Anal. Biochem.
227:1-13, 1995; Dvir et al.,
Proc. Natl. Acad. Sci. USA
89:11920-11924, 1992; Anderson et al.,
Crit. Rev. Eukaryot. Gene Express.
4:283-314, 1992). The catalytic subunit [DNA-PK
cs
] is composed of a single polypeptide of ~450 kDa. It is a serine-threonine type of protein kinase that phosphorylates a variety of nuclear enzymes, transcription factors and oncogenes (Anderson et al.,
Crit. Rev. Eukaryot. Gene Express.
4:283-314, 1992). However, DNA-PK
cs
by itself does not bind DNA. The non-catalytic subunit of DNA-PK is a heterodimer composed of 70 kDa and 86 kDa proteins. The non-catalytic subunit acts as a regulator of DNA-PK
cs
by virtue of its' ability to bind to DNA ends, thereby recruiting the catalytic subunit to the site of DSBs (Dvir et al.,
Proc. Natl. Acad. Sci. USA
89:11920-11924, 1992; Anderson et al.,
Crit. Rev. Eukaryot. Gene Express.
4:283-314, 1992).
Although the enzymology of DNA-PK
cs
has been investigated extensively, its biological function was identified only recently (Dvir et al.,
Proc. Natl. Acad. Sci. USA
89:11920-11924, 1992 ;Jeggo,
Mutation Res.
384:1-14, 1997). Availability of the full-length cDNA sequence of mammalian DNA-PK
cs
allowed identification of this protein as a member of the phosphotidyl inositol 3-kinase (PI kinase) gene family. While most members of this family are lipid kinases, a small number of proteins forming a subfamily specifically phosphorylate proteins. Members of this subfamily are known as PI-K related kinases and include the ATM protein, Tel1p, Tor1p, Tor2p, FRAP, Rad3p, Mec1p and Mei41 (Jeggo,
Mutation Res.
384: 1-14, 1997). In addition to their structural and biochemical similarities, members of this subfamily also appear to share a common biological function. They are all involved in repair of DNA that is damaged in response to a variety of genetic, physiological or environmental events (Jeggo,
Mutation Res.
384:1-14, 1997). Although several members of this subfamily have been cloned from animals, no information on plant DNA-PK
cs
is available in the literature.
The non-catalytic subunit of DNA-PK consists of two proteins of ~70 kDa and 86 kDa (Dvir et al.,
Proc. Natl. Acad. Sci. USA
89:11920-11924, 1992; Gotlib and Jackson,
Cell
72:131-142, 1993). These two proteins appear to be identical to previously well-characterized mammalian Ku proteins (Dvir et al.,
Proc. Natl. Acad. Sci. USA
89:11920-11924, 1992). The Ku complex, also a heterodimer of 70 kDa and 86 kDa proteins, was shown to be a nuclear DNA-binding autoantigen (Mimori et al.,
J. Clin. Invest.
68:611-620, 1981; Mimori et al.,
J.Biol. Chem.
261:2274-2278, 1986). Patients diagnosed with a variety of autoimmune diseases have been known to develop antibodies to Ku proteins (Yaneva & Arnettt,
Clin. Exp. Immunol.
76:366-372, 1989). Further biochemical analysis has established that Ku binds with strong affinity to DNA ends, stem-loop structures, DNA bubbles, or transitions between double stranded DNA and two single strands (Chu,
J. Biol. Chem.
272:24097-24100, 1997). Subsequent to binding to the ends, Ku molecules can translocate along the DNA, such that three or more molecules can bind to the linear DNA fragment. Both components of Ku have a DNA dependent ATPase activity and an ATP dependent helicase activity (Chu,
J. Biol. Chem.
272: 24097-24100, 1997). Recently, Yoo and Dynan have also demonstrated RNA binding activity of the Ku protein (Yoo & Dynan,
Biochemistry
37:1336-1343, 1998).
Recent genetic studies using rodent cell lines defective in DNA strand break repair have provided the important link between Ku protein, DNA-PK and DSB repairs during DNA replication, repair and recombination (Yoo & Dynan,
Biochemistry
37:1336-1343, 1998). Boulton & Jackson have shown that the yeast Ku70 potentiates illegitimate DNA DSB repair and serves as a barrier to error-prone DNA repair pathways (Boulton & Jackson,
EMBO J.
15:5093-5103, 1996). Studies with mutant rodent cell lines have clearly shown that Ku proteins are required for the V (D) J DNA recombination and immunoglobulin isotype switching (Roth et al.,
Current Biol.
5: 96-498, 1995; Casellas et al.,
EMBO J.
17:2404-2411, 1998). Components of DNA-PK are also involved in the non-homologous end-joining pathway in telomeric length maintenance and telomere silencing as well as telomere integrity (Boulton & Jackson,
EMBO J.
17:1819-1828, 1998; Polotnianka et al.,
Current Biol.
8:831-834, 1998). Ramsden & Gellert have recently observed that Ku protein stimulates DNA end joining by mammalian DNA ligases and proposed a direct role for Ku in DSB repair (Ramsden & Gellert,
EMBO J.
17:609-614, 1998). A role for Ku protein in modulation of heat shock response and hyperthermic radiosensitization has also been advocated (Yang et al.,
Mol. Cell. Biol.
16: 3799-3806, 1996; Burgman et al.,
Cancer Res.
57: 2847-2850, 1997). As discussed above, recent studies have established the role of DNA-PK components in various cellular processes involving DSB. During the course of these investigations, Ku homologues have been cloned from human, mouse,
Drosophila melanogaster, Rhipicephalus appendiculatus
and
Caenorhabditis elegans
(Reeves & Sthoeger,
J. Biol. Chem.
264:5047-5052, 1989; Chan et al.,
J. Biol. Chem.
264: 3651-3654, 1989; Porges et al.,
J. Immunol.
145:222-4228, 1990; Jacoby et al.,
J. Biol. Chem.
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