Maize Ku70 orthologue and uses thereof

Details Maize Ku70 orthologue and uses thereof Maize Ku70 orthologue and uses thereof

1999-08-30

2001-01-30

Fox, David T. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

C800S286000, C800S306000, C800S314000, C800S321000, C800S320000, C800S320100, C800S320200, C800S320300, C800S322000, C435S069100, C435S069200, C435S468000, C435S419000, C435S430000, C435S431000, C435S320100, C536S023100, C536S023200, C536S023600, C536S024100, C536S024500

Reexamination Certificate

active

06180850

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 (1, 2). Left unrepaired, 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 (1,2). The first pathway involves a series of very specific biochemical reactions catalyzed by a complex of cellular proteins (3). Due to the large number of proteins involved in this complex, it is referred to as a ‘recombinosome’ (4). This pathway is the dominant mode of DSB repair in lower eukaryotes such as yeast (2).
The non-homologous end-joining pathway is the major route of DSB repair in higher eukaryotes (1,2). 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] (5,6). 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 (5,6). While PADPRP is a single polypeptide of ~115 kDa (7), DNA-PK exists as a complex of two subunits (8,9). 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 (9). 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 (8,9).
Although enzymology of DNA-PK
cs
has been investigated extensively (8), its biological function was identified only recently (10). 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, Tellp, Tor1p, Tor2p, FRAP, Rad3p, Mec1p and Mei4l (10). 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 (10). Although several members of this subfamily have been cloned from animals, no known information on plant DNA-PK
cs
is available in the literature.
The non-catalytic subunit of DNA-PK appear to be identical to previously well characterized mammalian Ku proteins (8). The Ku complex, also a heterodimer of 70 kDa and 86 kDa proteins, was shown to be a nuclear DNA-binding autoantigen (12,13). Patients diagnosed of a variety of autoimmune diseases have been known to develop antibodies to Ku proteins (14). 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 (15). 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 (15). Recently, Yoo and Dynan have also demonstrated RNA binding activity of the Ku protein (16).
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 (16). Boulton & Jackson have shown that the yeast Ku70 potentiates illegitimate DNA DSB repair and serves as a barrier to error-prone DNA repair pathways (17). Studies with mutant rodent cell lines have clearly shown that Ku proteins are required for the V (D) J DNA recombination (18) and immunoglobulin isotype switching (19). Components of DNA-PK are also involved in the non-homologous end-joining pathway in telomeric length maintenance and telomere silencing (20) as well as telomere integrity (21). 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 (22). A role for Ku protein in modulation of heat shock response (23), and hyperthermic radiosensitization (24) has also been advocated. 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 orthologues have been cloned from human (25, 26), mouse (27),
Drosophila melanogaster
(28),
Rhipicephalus appendiculatus
(29) and
Caenorhabditis elegans
(30). Interestingly, Ku orthologues have also been reported in
Saccharomyces cerevisiae
(31-33).
Control of homologous recombination or non-homologous end joining by modulating Ku provides the means to modulate the efficiency which heterologous nucleic acids are incorporated into the genomes of a target plant cell. Control of these processes has important implications in the creation of novel recombinantly engineered crops such as maize. The present invention provides this and other advantages.
SUMMARY OF THE INVENTION
Generally, it is the object of the present invention to provide nucleic acids and proteins relating to the maize Ku70 orthologue. It is an object of the present invention to provide: 1) antigenic fragments of the proteins of the present invention; 2) transgenic plants comprising the nucleic acids of the present invention; 3) methods for modulating, in a transgenic plant, the expression of the nucleic acids of the present invention.
Therefore, in one aspect, the present invention relates to an isolated nucleic acid comprising a member selected from the group consisting of (a) a polynucleotide having a specified sequence identity to a polynucleotide encoding a polypeptide of the present invention, wherein the polypeptide when presented as an immunogen elicits the production of an antibody which is specifically reactive to the polypeptide; (b) a polynucleotide which is complementary to the polynucleotide of (a); and (c) a polynucleotide comprising a specified number of contiguous nucleotides from a polynucleotide of (a) or (b). The isolated nucleic acid can be DNA.
In another aspect, the present invention relates to recombinant expression cassettes, comprising a nucleic acid as described, supra, operably linked to a promoter. In some embodiments, the nucleic acid is operably linked in antisense orientation to the promoter.
In another aspect, the present invention is directed to a host cell transfected with the recombinant expression cassette as described, supra. In some embodiments, the host cell is a sorghum (
Sorghum bicolor
) or maize (
Zea mays
) cell.
In a further aspect, the present invention relates to an isolated protein comprising a polypeptide having a specified number of contiguous amino acids encoded by the isolated nucleic acid referred to, supra.
In another aspect, the present invention relates to an isolated nucleic acid comprising a polynucleotide of specified length which selectively hybridizes under stringent conditions to a nucleic acid of the present invention, or a complement thereof. In some embodiments, the isolated nucleic acid is operably linked to a promot

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