Polypeptides capable of interacting with the human...

Details Polypeptides capable of interacting with the human... Polypeptides capable of interacting with the human...

2001-06-25

2004-03-16

Hutson, Richard (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Isomerase

C435S183000, C435S252300, C435S320100, C435S325000, C536S023100, C536S023200, C536S023500, C530S350000, C530S358000

Reexamination Certificate

active

06706514

ABSTRACT:

The present invention relates to novel polypeptides capable of interacting with human topoisomerase III&agr; and to the nucleic acid sequences encoding these polypeptides. It also relates, in addition, to a method for identifying compounds capable of interacting with said polypeptides and to a method for identifying molecules capable of modulating the interaction of topoisomerase III&agr; with said polypeptides.
The replication of DNA is a complex mechanism which involves a large number of factors. DNA exists in the physiological state in a supercoiled form and access to the information which it contains requires substantial modification of the degree of coiling. Replication requires the suppression of the supercoils, the separation of the two strands of the DNA double helix and the maintaining of DNA in single-stranded form.
The modification of the degree of coiling is brought about in vivo by topoisomerases which are enzymes capable of modifying the DNA superstructures. It is possible to distinguish type I topoisomerases which cut only one of the two DNA strands and which eliminate the supercoils, and type II topoisomerases which act by cutting the two DNA strands and which are capable of eliminating or creating the supercoils. Eukaryotic topoisomerases are less well known than their prokaryotic homologs and their mechanism of action has still not yet been elucidated to date.
The separation of the two strands of a DNA duplex is catalyzed by a group of enzymes, called DNA helicases, which act in an ATP-dependent manner so as to produce the single-stranded DNA used as template for the DNA replication and transcription processes. Generally, the helicases bind to the single-stranded DNA or to the junctions between the single- and double-stranded DNA, and move in a single direction along the DNA in the double-stranded region, destroying the hydrogen bonds joining the two strands. All helicases exhibit a DNA-dependent ATPase (or. NTPase) activity which hydrolyzes the gamma phosphate of the ribonucleoside or deoxyribonucleoside 5′-triphosphate and provides the energy necessary for the reaction. The first helicase was discovered in
E. coli
in 1976. Since then, more than 60 helicases have been isolated in prokaryotes and eukaryotes. The role of human helicases has still not been elucidated in most cases, with the exception of HDHII (repair of the lesions induced by X-rays), HDHIV (assembly of preribosomes), ERCC2 and ERCC3, which are involved in repair through excision and cell viability. Little is known on the structure of these helicases. A large portion of the information available on the structures and functions of helicases has been obtained by comparative analysis of the amino acid sequences. In particular, conserved motifs have made it possible to group helicases into subfamilies based on the sequence homologies.
Human Topoisomerase III belongs to the family of type IA topoisomerases and therefore exhibits sequence homologies with
E. coli
topoisomerases I and III, yeast Topoisomerase III as well as reverse gyrase from archaebacteria. Human Topoisomerase III is now called Topoisomerase III&agr; so as to differentiate it from human topoisomerase III&bgr; which was recently discovered during the sequencing of the human immunoglobulin &lgr; gene locus (Kawasaki, K., Minoshima, S., Nakato, E., Shibuya, K., Shintani, A., Schmeits, J. L., Wang, J. and Shimizu, N. 1997, Genome Research 7: 250-261), and for which no functional activity has been shown. Yeast-expressed and unpurified topoisomerase III&agr; exhibits an activity of partial relaxation of a highly negatively supercoiled DNA (Hanai, R., Caron, P. R. and Wang, J. C. 1993. Proc. Natl. Acad. Sci. USA 93: 3653-3657).
Topoisomerase III&agr; is a protein of 976 amino acids and with a molecular weight of about 110 kDa. The gene encoding human Topoisomerase III&agr; is present in a single copy on chromosome 17p11.2-12 (Hanai, R., Caron, P. R. and Wang, J. C. 1996. Proc. Natl. Acad. Sci. USA 93: 3653-3657). A murine homolog of Topoisomerase III was recently cloned (Seki, T., Deki, M., Katada, T. and Enomoto, T. 1998. Biochim Biophys Acta 1396: 127-131).
Topoisomerase III&agr; exhibits a strong sequence homology with yeast Topoisomerase III, namely 44% sequence identity and 61% similarity. The homology which it exhibits with bacterial topoisomerases I and III is less strong, namely 24% identity and 44% similarity. However, Topoisomerase III&agr; resembles
E. coli
Topoisomerase I more than it resembles the other members of the group of type IA topoisomerases from the point of view of the organization of the protein into domains. Indeed, these two polypeptides contain a C-terminal domain which has no equivalent in
E. coli
or yeast Topoisomerase III. This C-terminal domain contains motifs with 4 cysteines (3 motifs for
E. coli
Topoisomerase I and 1.5 motif for human Topoisomerase III&agr;), as well as an extreme C-terminal domain for which a DNA-binding role has been demonstrated for
E. coli
Topoisomerase I.
The role of human topoisomerase III&agr; in the cell has not yet been identified.
Human Topoisomerase III&agr; appears to be essential, at least during embryogenesis, since the knock-out of the murine homolog of Topoisomerase III&agr; is lethal (Li, W. and Wang, J. C. 1998 Proc. Natl. Acad. Sci. USA 95: 1010-1013). The messenger RNAs for Topoisomerase III&agr; are present in numerous tissues (heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas) in the form of three transcripts of 7.2, 6 and 4 kilobases in size (Fritz, E., Elsea, S. H., Patel, P. I. and Meyn, M. S. 1997 Proc. Natl. Acad. Sci. USA 94: 4538-4542).
Moreover, it has been assumed that Topoisomerase III&agr; plays a role in maintaining the stability of the genome. Indeed, the cDNA CAT4.5, encoding a truncated human Topoisomerase III&agr; of 141 N-terminal amino acids, is capable of complementing the phenotype for hypersensitivity to ionizing radiation in AT (Ataxia-Telangectasia) cells exhibiting a mutation in the ATM gene (Fritz, E., Elsea, S. H., Patel, P. I. and Meyn, M. S. 1997 Proc. Natl. Acad. Sci. USA 94: 4538-4542).
In yeast, two independent studies have shown the existence of an interaction between the helicase SGS1 and yeast Topoisomerase III. On the one hand, the sgs1- mutants are suppressers of the top3- phenotype (slow growth, hyperrecombination) in the yeast
S. cerevisiae
(Gangloff, S., McDonald, J. P., Bendixen, C., Arthur, L. and Rothstein, R. 1994. Mol. Cell. Biol. 14: 8391-8398). On the other hand, it has been shown that the first 500 amino acids of SGS1 interact with yeast Topoisomerase III (Gangloff, S., McDonald, J. P., Bendixen, C., Arthur, L. and Rothstein, R. 1994. Mol. Cell. Biol. 14: 8391-8398, Lu, J., Mullen, J. R., Brill, S. J., Kleff, S., Romeo, A. M. and Sternglanz, R. 1996. Nature 383: 678-679). However, to date, no interaction between a helicase and human Topoisomerase III&agr; has been identified.
The identification of partners of human topoisomerase III&agr; therefore constitutes a major challenge for the understanding of the role of human topoisomerase III&agr;, and of its mechanism of action.
The present invention results from the demonstration of novel polypeptides capable of interacting with topoisomerase III&agr; (called hereinafter polypeptide partners of topoisomerase III&agr;). It also results from the discovery that these polypeptides show a strong homology with proteins which exhibit structural characteristics common to RNA helicases and for which no function had so far been described. The demonstration of this interaction and of these homologies designate these proteins as DNA helicase partners of topoisomerase III&agr;. The identification of these partners makes it possible to envisage numerous applications based on the combined action of these partner proteins and of topoisomerase III&agr;; these applications relate in particular to:
1) The destruction of the nucleosomal structure: to undergo some processes such as replication, transcription, repair or recombination, DNA should be accessib

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