• Lafer, Eileen M (PI)

Project: Research project

Project Details


Experiments-aimed at elucidating the biological role of Z-DNA have
taken two forms. Natural sequences which can form Z-DNA have been
identified and proteins have been purified which bind better to Z-
DNA than B-DNA in vitro. However, the sequences have not been
shown to form Z-DNA in vivo, nor have the proteins been shown to
bind Z-DNA in vivo. We recently purified three proteins from coli
which bind specifically to Z-DNA in vitro, prepared monoclonal
antibodies against them, and cloned their genes. For the first
time reagents are available that make possible molecular analyses
of ,genes encoding Z-DNA binding proteins and extensive biochemical
studies of these proteins. The goal of these studies is to
elucidate the biological role of Z-DNA in E. coli by determining
the function of these Z-DNA binding proteins. We will sequence
their genes and locate them on the E. coli map, and analyze the
migration of the proteins on 2-D PACE. Comparison of this
information with available data banks will reveal if data on these
proteins or genes has been collected. Using allelic replacement
and constructs overexpressing or producing antisense mRNA for these
genes we will generate null mutants or conditional lethals and
strains which overexpress these genes ;or produce reduced levels
of these proteins. We will examine the mutants for defects in
processes executed at the DNA level: replication, recombination,
repair, supercoiling, and transcription. If we obtain conditional
lethals we will clone mutants in other genes which suppress the
original mutant phenotype. Using highly purified protein from
strains overexpressing these genes we will carry out an extensive
study of the interaction of these proteins with Z-DNA. We will
precisely quantitate the specificity of these proteins for Z-DNA.
Chemical modification experiments will reveal details, at the base
pair level, of the interaction. We will identify the in vivo
binding sites of these proteins by immunoprecipitating UV cross-
linked protein-DNA complexes from cell extracts and analyzing the
precipitated DNA by cloning and sequencing. Purified proteins will
also be assayed for NTPase, nuclease, topoisomerase, and
recombination activities. Since so little is known about what Z-
DNA does in a cell, we believe that E. coli, which is amenable to
genetic analysis and is one of the simplest and best characterized
laboratory organisms, is an ideal system in which to begin studying
the function of Z-DNA. These studies will have far reaching
implications since they will provide clues to the function of Z-
DNA in organisms other than E. coli.
Effective start/end date4/1/873/31/91


  • National Institutes of Health


  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)


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