Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-05-11
2004-06-22
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S004000, C435S007310, C530S300000, C530S327000
Reexamination Certificate
active
06753151
ABSTRACT:
The present invention relates to assays, screening methods, peptides, mimetics, and methods of use based on the discovery and characterisation of an interaction between Ku70 and Ku80 and DNA-PK
cs
. More particularly, aspects of the invention are based around peptide fragments of Ku70 and Ku80. The invention relates to numerous cellular processes which are of interest in therapeutic contexts.
Ku is a protein that is found in a wide range of organisms, ranging from
Saccharomyces cerevisiae
to man (Dynan and Yoo, 1998). It is expressed in all human tissues examined. Ku comprises two tightly-associated subunits of about 69 kDa and about 83 kDa. These are termed Ku70 and Ku80 (or Ku86), respectively. Although some information has been obtained regarding the regions of the Ku polypeptides that interact with one another (Cary et al., 1998; Jin and Weaver, 1997; Koike et al., 1998; Osipovich et al., 1997; Wang et al., 1998a; Wang et al., 1998b; Wu and Lieber, 1996), little is known about the precise sites of interaction and the molecular mechanism underlying it.
The most highly characterised function of Ku at the biochemical level is its ability to bind avidly to certain disruptions of the DNA double helix in a sequence independent fashion. The most well studied example of such a disruption is the DNA double-strand break (DSB; Blier et al., 1993; Devries et al., 1989; Mimori and Hardin, 1986). Other discontinuities that are recognised by Ku include single-strand breaks in the sugar-phosphate backbone of double-stranded DNA (dsDNA), and DNA single-strand to double-strand transitions, such as those that occur in hairpin loops or single-stranded gaps in a dsDNA molecule (Blier et al., 1993; Falzon et al., 1993). Once bound to a dsDNA end, Ku can move to internal positions in the DNA in an ATP-independent fashion (Paillard and Strauss, 1991; Devries et al., 1989). Ku has also been reported to be capable of sequence-specific DNA interactions (Giffin et al., 1996): for a review see (Dynan and Yoo, 1998). It has also been reported that Ku70 and possibly Ku80 are capable of interacting with DNA in the absence of their heterodimerisation partner (Chou et al., 1992; Wang et al., 1994). It has also been demonstrated that Ku heterodimers bound to DNA are able to specifically associate with one another (Cary et al., 1997).
When complexed with DNA, Ku can interact with an approximately 460 kDa polypeptide, the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). DNA-PKcs is a member of the PI 3-kinase-like (PIKL) protein kinase family (Hartley et al., 1995) and, together with Ku and DNA, forms a catalytically active DNA-PK complex with Ser/Thr kinase activity (Dvir et al., 1992; Gottlieb and Jackson, 1993; Suwa et al., 1994).
Little is currently known about how Ku interacts with DNA-PKcs, although the fact that it is apparently unable to bind DNA-PKcs in the absence of DNA (Suwa et al., 1994) suggests that DNA binding by Ku induces a conformational change that permits the DNA-PKcs interaction. In particular, previous studies have provided little information about the regions of the Ku heterodimer which are involved in the DNA-PK
cs
interaction.
It has been shown that, under certain conditions, DNA-PKcs can bind to dsDNA ends and become activated in the absence of Ku (Hammarsten and Chu, 1998; Yaneva et al., 1997). Thus, whilst allosteric activation of DNA-PKcs by Ku might occur, it appears that direct interactions between DNA-PKcs and DNA can be sufficient to activate the kinase in vitro. This suggests that contacts between DNA-PKcs and DNA play an important role in DNA-PK activation, even in the presence of Ku.
A breakthrough in the understanding of DNA-PKcs/Ku function came with the discovery that defects in these proteins are associated with a subset of mutant mammalian cell lines that are defective in DNA DSB rejoining, and are profoundly sensitive to ionising radiation and other agents that generate DNA DSBs as their principal lethal lesion (Jackson, S. P., et al (1995) TIBS 20, 412-415; Critchlow, S. E., et al (1998) TIBS 23, 394-398). Indeed, the mutant phenotypes of these cells are corrected by the introduction of the appropriate Ku or DNA-PKcs expression vector, and recent work using extracts of mammalian or
Xenopus laevis
cells has provided evidence for a direct involvement of Ku and DNA-PKcs in DNA DSB rejoining (Baumann and West, 1998; Labhart, P., (1999) Mol. Cell. Biol. 19, 2585-2593). Furthermore, DNA-PK catalytic activity has been implicated at an early stage of DNA DSB repair in Xenopus cell-free extracts (Gu et al., 1996; Gu et al., 1998) and for radiation-induced DNA repair in cultured human cells (Okayasu, R., et al (1998) Radiat. Res. 149, 440-445). Coupled with the fact that Ku displays a very high affinity for dsDNA ends in vitro, these data suggest that DNA-PK functions directly in the recognition and resolution of radiation-induced DNA DSBs in vivo.
Cells deficient in DNA-PKcs, Ku80, or Ku70 are also severely impaired in V(D)J recombination, a site-specific genomic rearrangement process that takes place in the developing vertebrate immune system to help generate the vast antigen recognition capacity of antibody and T-cell receptor molecules (Jackson, S. P., et al (1995) TIBS 20, 412-415; Critchlow, S. E., et al (1998) TIBS 23, 394-398). This process requires the production of DNA DSBs between the recombining gene segments by the RAG1/RAG2 proteins (Jackson, S. P., et al (1995) TIBS 20, 412-415; Critchlow, S. E., et al (1998) TIBS 23, 394-398) and the subsequent rejoining of the DNA ends via DNA-PK-dependent mechanisms. For a single DNA rearrangement between two coding segments (V, D, or J regions) to occur, a join between the two coding sequences (known as the coding join) and one between the two non-coding signal ends (the signal join) are made. Interestingly, Ku is essential for both types of join, whereas DNA-PKcs appears to be required only for coding joins and plays a non-essential and variable role in the generation of signal joins (Bogue et al., 1998). This suggests that, at least for the repair of a sub-set of DNA DSBs, Ku is able to function in the absence of DNA-PKcs.
Consistent with the Ku-associated DNA DSB repair pathway being highly conserved throughout eukaryotic evolution, Ku is found in
S. cerevisiae
and is essential for repair of DNA DSBs by the pathway of non homologous end-joining (Boulton and Jackson, 1996b; Siede et al., 1996 and see Critchlow and Jackson, 1998 for review). Perhaps surprisingly there is no clear orthologue of DNA-PKcs encoded by the fully-sequenced
S. cerevisiae
genome. Thus, in yeast, Ku carries out DNA-repair functions independently of DNA-PK. Although it is possible that the functions of mammalian DNA-PKcs are assumed by other members of the PIKL protein kinase familly, such as Mec1p and/or Tel1p, there is no evidence to suggest that these interact physically or genetically with Ku.
Interestingly,
S. cerevisiae
Ku has also been shown to play important roles in telomere length maintenance, and in the transcriptional silencing of genes placed close to telomeric DNA (Boulton and Jackson, 1996a; Boulton and Jackson, 1998 Porter et al., 1996).
The present inventors have investigated interactions between Ku70 and Ku80, and between the two Ku subunits and DNA-PKcs. The data presented herein lead to the conclusion that the two Ku subunits are structurally and functionally related to each other, and appear to associate via a pseudo-homodimerisation mechanism. Furthermore, the work demonstrates that the extreme C-terminus of Ku80 plays an important role in the interaction between Ku and DNA-PKcs. These results provide for modulation of the structure and physiological functions of DNA-PKcs and Ku, for instance by means of peptides corresponding to conserved regions in Ku70 or Ku80 and/or regions of interaction between Ku70 and Ku80 and/or other molecules such as DNA-PKcs, and allow for postulation of a model for the evolution of the DNA-PK complex.
Based on the experimental work and discussion herein the invention is further
Gell David A.
Jackson Stephen P.
Ketter James
Kudos Pharmaceuticals Limited
Lambertson David
Nixon & Vanderhye P.C.
LandOfFree
Interactions of Ku polypeptides and applications thereof does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Interactions of Ku polypeptides and applications thereof, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Interactions of Ku polypeptides and applications thereof will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3338742