H-DNA and d(TC)n binding proteins
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AbstractRepetitive d(TC)n tracts have been shown in vitro to adopt an unusual DNA conformation, H-DNA. H-DNA is an intramolecular triplex favored to form in sequences with mirror symmetry and having either a homopurine and /or homopyrmidine strand bias. These d(TC)n tracts are prevalently found in eukaryotic genomes. Specifically, these sequences have been isolated in the regulatory regions of genes, sites of recombination and origins of replication. Many biological roles have been proposed and a putative role in transcription has been demonstrated. The adventitious multi-functional aspect of this sequence strongly suggests a more global role in the structure and function relationship, such as in the organization of chromatin and the nuclear matrix. If these sequences have biological functions in the cell, the question is whether these functions include the formation of triple stranded structures. Two d(TC)n·d(GA)n bearing plasmids, pTClS and pTC32 have been constructed to examine the relationships between superhelical density, protonation and insert size in the context of H-DNA formation. The former plasmid contains a synthetic d(TC)is insert and the latter plasmid contains a synthetic d(TCh2 insert. Two dimensional topoisomer electrophoretic analysis is a useful and powerful techique for examining thermodynamically stable transitions. From these results, thermodynamic parameters were calculated. The formation of H-DNA for d(TC)n sequences is favored under acidic conditions and is length dependent. The pattern of Sl nuclease activity for pTC15 was consistent with H-DNA and for pTC32 was not consistent with a single or predominant conformer of H-DNA but a mixed population of conformers. A natural sequence with a polypyrmidine strand bias derived from mouse was characterized to form H-DNA in vitro. The cloned insert contains a (CT)io insert flanked by (T ,C) rich tracts. The conditions for H-DNA formation of this mouse-derived sequence is similar to the conditions of the synthetic insert cloned within pTC15. A protein-DNA interaction with the sequence d(TC)0 was examined using nuclear protein extracts. A cluster of proteins was discovered to bind to the singlestranded oligonucleotide d(TC)i6 but not its complementary strand, d(GA)l6 nor the duplex sequence d(TCh6·d(GAh6- These single-stranded d(TC)n binding proteins are found among several mammalian species examined and have been sized as 55.5 and 57 kD doublets using southwestern analysis. The existence of this protein is incompatible with postulated in vivo existence of the d(TC)n type triplexes in eukaryotic cells.
Bibliography: p. 133-143.
CitationYee, H. A. (1991). H-DNA and d(TC)n binding proteins (Unpublished master's thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/19749
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