The presence of inverted repeat (palindromic) sequences in DNA is ubiquitous. Whether at the DNA or RNA level, their importance has been established for termination of transcription and for attenuation in prokaryotes (Rosenberg and Court in (1979) Annu. Rev. Genet. 13, 319-353). In addition, inverted repeats have been found at the origin of replication of many mammalian viruses including SV40, polyoma, BK and herpes simplex I viruses, as well as at the replication origin of several prokaryotes. It has recently been shown by Zannis-Hadjopoulos et al. (1984) J. Mol. Biol. 179, 577-586 that such sequences may also be important in the initiation of mammalian DNA replication, since it has been found that origin-rich monkey DNA segments are enriched for inverted repeats.
Inverted repeat sequences have the potential of forming cruciform symmetrical (stem-loop or hairpin) structures through intrastrand nucleotide pairing. Although the existence of cruciforms in vivo has not been directly demonstrated, their existence in vitro has. It has long been postulated that cruciforms may form transiently as regulatory signals, e.g. to act as the attachment site within DNA replication origins for proteins involved in replication (Hand, R. (1978) Cell 15, 317-325, and Mizuuchi et al. (1982) J. Mol. Biol. 156, 229-243). In support of this, Collins et al., (1982) Biochem. 21, 2414-3419 found that, while there were no Sl nuclease-sensitive sites in nonproliferating cells, the number of Sl sites increased when cells moved from Go to Gl and peaked at the onset of S phase. Lilley ((1980) Proc. Nat. Acad. Sci. U.S.A. 77, 6468-6472) and Panayotatos et al. ((1981) Nature 289, 466-470) have demonstrated that cruciform structures were sensitive to single-stranded specific nucleases like Sl by virtue of their single-strandedness at the tip of the hairpin.
Although double-stranded DNA is poorly immunogenic, monoclonal antibodies have been raised against such alternative DNA structures as Z-DNA and carcinogen-modified DNA.
Consequently, the usefulness of these antibodies for studying alternative DNA structures suggested that the preparation of a monoclonal antibody for the detection of cruciform DNA structures would be highly desirable.
As mentioned before, it has long been postulated that cruciforms may form transiently in vivo, to act as special regulatory signals on the DNA, for the initiation of DNA replication and serve as the attachment sites for the initiator proteins.
The ability to form a stem-loop (cruciform structure) is known to be essential for function of the origin of replication on the plasmid ColE1, for example, where mutations that disrupt folding result in replication failure. In this case, a multiple stem-loop structure is necessary for the association of a primer-precursor RNA with the DNA template prior to generation of the primer by RNase H cleavage; control of the confirmation of the primer precursor controls plasmid replication. The ability to assume this configuration is conserved among different ColE1 type origins despite considerable divergence in their primary structure. The potential for secondary structural interactions is also a characteristic of the regions surrounding the light (L) and heavy (H) strand replication origins (O.sub.L and O.sub.H) of mitochondrial DNA.
In the case of L strand initiation of DNA synthesis, the stem-loop structure found at the origin is essential for initiation in vitro in a human mitochondrial system.
Although the cruciform conformation is energetically unfavoured in a relaxed DNA molecule, negative supercoiling favors it; studies using single strand specific nucleases and electron microscopy have provided evidence for the existence of cruciforms in a supercoil DNA. Furthermore, it was recently reported that a dominant factor in the kinetics of cruciform extrusion is the DNA sequence that flanks the inverted repeat, with the sequence of the inverted repeat itself having little or no influence. The flanking sequence with the dominant influence on cruciform kinetics is very AT-rich, acts in cis independent of polarity, and it can have an effect over distances of at least 100 bp.
Despite the evidence provided by the nuclease S1 digestion experiments that argues in favor of the existence of cruciforms, their existence in vivo has not been directly demonstrated, although the presence of a native cruciform structure has recently been reported in E. Coli.
The obtention of anti-cruciform monoclonal antibodies that could enhance DNA replication in cell systems containing cruciforms acting as promoters in DNA replication would also be desirable.
USPTO Backgrounds
USPTO Backgrounds