Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-04-15
2001-02-06
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
Reexamination Certificate
active
06183969
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a powerful assay for detecting and characterizing chromosomal rearrangements in eukaryotic cells. Using this assay, environmental factors such as drugs, genetic factors such as specific gene mutations, as well as the presence or absence of specific DNA segments can all be examined for their role in promoting chromosome instability via nonhomologous recombination.
BACKGROUND OF THE INVENTION
Cancer is a genetic disorder in which a series of mutations subvert the normal developmental program of a cell and allow it to proliferate without constraint. Accumulation of deleterious mutations appear to represent the basis of cancer progression (Kinzler, K. W. and Vogelstein, B.
Nature
1997 386:761-763). While these mutations can take many forms, the most characteristic form of genetic change in cancer cells is karyotypic instability with aneuploidy and chromosomal rearrangements, particularly balanced translocations. By joining together previously unlinked chromosomal arms, balanced translocations can result in the creation of hybrid genes with altered expression patterns for potential oncogenes or tumor suppressor genes. Karyotypic detection of translocations has been very useful for cancer researchers. Clinically, the presence or absence of specific translocations has therapeutic and prognostic implications. More fundamentally, genes identified at the translocation breakpoints are strong candidates for involvement in malignant transformation (Sanchez-Garcia, I.
Annu. Rev. Genet.
1997 31:429-453). These translocations serve as markers of the malignant state and can be either the cause or the consequence of the transformed state. For example, the Philadelphia chromosome is a specific t(9;22)(q34;q11) translocation that fuses the B-cell antigen receptor gene BCR and the ABL oncogene (De Klein et al.
Nature
1982 300: 765-767). This fusion is thought to represent the crucial event in the development of chronic granulocytic leukemia. However, this translocation can also appear later in the course of multiple forms of leukemia. In general, it appears that all hematologic malignancies originate from such “dangerous liaisons” between unlinked chromosomal segments. Solid tumors as well may have characteristic translocations, suggesting that the development of an unstable chromosomal state increases the likelihood of translocations which in turn increase the likelihood of tumor progression (Rabbitts, T. H.
Nature
1994 372:143-149; Sanchez-Garcia, I.
Annu. Rev. Genet.
1997 31:429-453).
While the identification and analysis of the genes present at specific translocation breakpoints has become an area of great research interest, causes of these translocations are still poorly understood. Clearly an understanding of possible causes, however, is important for cancer prevention, for identification of at risk individuals, and for developing potential targets of drug intervention.
Translocations are believed to arise by recombination, a descriptive term given to any process whereby double-stranded DNAs are broken and rejoined in ways that alter the linkage relationship of the genes near the breaks. At least three different recombination pathways that operate in cells which can cause translocations have been proposed.
First, during homology-dependent or homologous recombination, identical sequences on nonhomologous chromosomes are believed to crossover, resulting in new linkages of nonhomologous chromosome arms. This model is supported by the fact that repetitive sequences in the human genome such as Alu elements or retrotransposons such as LINE elements are occasionally observed at translocation breakpoints (Kato et al.
Gene
1991 97:239-244). Studies from a number of model organisms, particularly
Saccharomyces cerevisiae
, indicate that the presence of a double strand break (DSB) greatly stimulates the process of homologous recombination, via strand invasion of a linear single-stranded end into complementary double-stranded sequences elsewhere in the genome (Petes et al. (1991) Recombination in yeast. “The Molecular and Cellular Biology of the yeast Saccharomyces”. Broach, J. R., Pringle, J. R. and Jones, E. W., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Model systems for homology-directed transpositions have been developed in yeast and in mammalian cells which take advantage of the stimulatory effect of an induced DSB (Fasullo et al.
Mutat Res.
1994 314:121-133; Haber, J. E. and Leung, W.
Proc. Natl. Acad. Sci. USA
1996 93:13949-13954; and Richardson et al.
Genes
&
Dev.
1998 12:3831-3842). However, it appears that homologous recombination is at best a minor pathway for translocation-formation in human cancers.
Site-specific recombination requiring a site-specific recombinase, as well as specific DNA recognition sequences, have also been proposed. In humans, B and T cell precursors go through a site-specific recombinational process known as V(D)J rejoining that is essential to their maturation. V(D)J rejoining assembles antigen receptor variable genes by making DSBs at specific recombination signal sequences and then rejoining non-contiguous intrachromosomal segments. Many reciprocal translocations associated with lymphoid malignancies involve a V(D)J cleavage-rejoining site at the breakpoint (Rabbitts, T. H.
Nature
1994 372:143-149). This has led to the concept that these translocations result from aberrant nonhomologous rejoining events during this normally site-specific process. The V(D)J cleavage site represents one break. Potential sources of the second break include both physiological (e.g. transcription, replication, repair) and environmental (e.g. x-rays, free radicals). In these cases, the V(D)J break might be joined to another simultaneous break. It was recently demonstrated that the V(D)J recombinase can function as a transposase, capable of actively inserting cleaved DNA ends into random targets (Hiom et al.
Cell
1998 94:463-470). Similar experimental systems using other site-specific recombinases have also been designed that can generate recombinase-dependent translocations in the presence of a recombinase and appropriate recognition sequences on separate nonhomologous chromosomes (Golic, K. G. and Lindquist, S.
Cell
1989 59:499-509; Sauer, B.
J. Mol. Biol.
1992 223:911-928; and Van Deursen et al.
Proc. Natl. Acad. Sci. USA
1995 92:7376-7380).
Nonhomologous recombination is an inherently imprecise form of recombination that appears to be the major pathway for DSB repair in human cells (Meuth, M. (1989) Illegitimate recombination in mammalian cells. “Mobile DNA”. Berg, D. E. and Howe, M. M., American Society for Microbiology, Washington, D.C.; and Roth, D. and Wilson, J. (1988) Illegitimate recombination in mammalian cells. “Genetic Recombination”. Kucherlapati, R. and Smith, G. R., American Society for Microbiology, Washington, D.C.), although it represents a minor pathway in
Saccharomyces cerevisiae
(Haber, J. E.
Bioessays
1995 17:609-620). In this form of recombination, no special sequences are present at the break sites. Instead variable length deletions or rearrangements have been observed at break sites. The recombination joints normally involve at most, a few (<5) overlapping bases. Based on analysis of breakpoints, it is believed that most cancer-causing chromosomal translocations occur by nonhomologous end-joining of simultaneous DSBs that are present on separate nonhomologous chromosomes. This type of recombination has been studied in mammalian systems by analyzing sites of integration and excision of DNA viruses and transfected linear marker DNA, as well as by determining the genetic components required for the normal recombinational repair of V(D)J site-specific cleavage events (Roth et al.
Current Biol.
1995 5:496-499). Further information has come from studies in
Saccharomyces cerevisiae
. The data so far suggests that there may be multiple end-joining pathways which utilize (amongst other proteins) Rad50, Mre11, Xrs2, Ku70, Ku80, the DNA-dependent protein kin
Ketter James
Law Offices of Jane Massey Licata
Rutgers The State University of New Jersey
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