Higher order coiling or supercoiling exists in the DNA of all organisms as well as some viruses. The long term goal of this proposal is to understand the structure of supercoiled DNA and the molecular mechanisms by which supercoiling affects biological function. Natural supercoiling can lead to formation of open (unwound or unrepaired) regions in DNA. Such regions are frequently associated with altered DNA secondary structures, eg. cruciforms and Z-DNA. We will continue to use mung bean nuclease, a single-strand-specific endonuclease, to probe for altered secondary structures under solution conditions where DNA can function in vitro. In prokaryotic and eukaryotic DNAs, the enzyme often cleaves DNA sequences involved in regulation of transcription and DNA replication. Potential cruciforms and Z-DNA structures are recognized as is a dA+dT-rich sequence with a novel conformation. The goal of the proposal is to examine the nature and biological significance of altered secondary structures in supercoiled DNA. To achieve this goal we will continue to study plasmid pBR322 DNA and simian virus 40 DNA, and we will (1) examine the effects of point mutations on detection of altered secondary structure, (2) examine the effects of point mutations on gene expression in vivo and transcription in vivo and in vitro, (3) determine the requirements for recognition of a new DNA conformation detected in certain dA-dT-rich sequences. Point mutations will be introduced by either oligonucleotide directed mutagenesis or by gap repair mutagenesis at mung bean nuclease nicks. Recombinant DNA technology and prokaryotic and eukaryotic expression vectors will be utilized to assess gene expression in vivo. The level and accurate initiation of RNA transcripts will be determined by annealing mRNA to labeled complementary DNA and analyzing the DNA protected from single-strand-specific nuclease digestion on DNA sequencing gels.
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