Regulatory Elements in ColE1 DNA Replication in Escherichia coli. Mutants of Saccharomyces cerevisiae DNA Polymerase I Resistant to Nucleotide Analogs: dNTP Binding Site Definition

Citation

Ma, Doreen (1989) Regulatory Elements in ColE1 DNA Replication in Escherichia coli. Mutants of Saccharomyces cerevisiae DNA Polymerase I Resistant to Nucleotide Analogs: dNTP Binding Site Definition. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/5b4k-wx16. https://resolver.caltech.edu/CaltechETD:etd-05222007-091103

Abstract

Control of Co1E1-type plasmid DNA replication in Escherichia coli was investigated. The initiation of DNA replication in Co1E 1-type plasmids is regulated by two trans-acting negative control elements: RNA I and the rop protein. RNA I is a transcript 108 nucleotides long made off the L-strand (lagging strand) of the plasmid and is complementary to the 5' portion of the preprimer, RNA II. The direct base-pairing interaction between the two RNA species precludes the formation of an RNA II:template DNA hybrid, which is processed by RNase H at the origin of replication to create the 3'-OH end of the mature primer. Another trans-acting regulatory element is a 63 amino acid plasmid-encoded protein, the rop gene product. By providing the rop gene product in trans on a compatible plasmid, suppression of the runaway replication phenotype of pJN75 was observed. Utilizing this property of the regulatory mechanism, we proceeded to select pJN75 derivatives that are insensitive to rop-mediated suppression. These mutant plasmids were designated pJN75nsr for non-suppressible by rop. Sequence analyses of 7 nsr, showed disruptions of base-pairing within the stem of loop structure III of RNA II and loop structure III' of RNA I, implying that rop mediates its action via the region comprising loop structures III and III'. We were also interested in the presence of a dnaA protein binding site about 90bp downstream of the ori-pBR322. We investigated the role of dnaA protein in Co1E1-type DNA replication by purifying the dnaA protein to homogeneity from an overproducing strain and examining its effect on various mutant DNA templates in an in vitro E. coli DNA replication extract developed in the Campbell lab. We found that the combination of dnaA protein binding at the dnaA consensus sequence can substitute for the lack of primosome assembly site (pas) on the H-strand (leading strand), which is postulated to be the point of transition between DNA polymerase I and polymerase III-dependent DNA synthesis. In the absence of the H-strand pas, dnaA protein may direct other essential proteins to form a replication complex at the dnaA site, functionally acting as proteins i, n, n' and n" at pas.

In an effort to identify and characterize the nucleotide and/or pyrophosphate binding site(s) of yeast DNA polymerise I, we have attempted to isolate pol1 mutants that are resistant to nucleotide/pyrophosphate analogs. We successfully constructed a Saccharomyces cerevisiae strain that depends on exogenous thymidine for survival and also contains a temperature-sensitive DNA polymerase I allele (pol1-17). Using this strain, 167-poll-17, we screened for pol1 mutants that are resistant to nucleotide/pyrophosphate analogs, e.g., AraT or PAA, which are normally impermeable to the cell wall of yeast. Our strategy was to introduce a plasmid containing a mutagenized pol1 gene into 167-poll-17, which contains a ts DNA polymerase. By screening for survivors at 37°C (non-permissive temperature of pol1-17 allele) on plates containing 1 mM AraT and 40 mM PAA, we hoped to isolate DNA polymerase I mutants on the transformed plasmid. To our disappointment, we were unable to isolate such DNA pol I mutants. We offer two explanations for the failure of our strategy: 1) the presence of another essential polymerase, CDC2 gene product and 2) perturbation of pyrimidine nucleotide pool. Finally, we propose to conduct site-directed mutagenesis of DNA polymerase I at putative dNTP/PP_i binding domains and to analyze the mutant polymerases in vitro for resistance to dNTP/PP_i analogs. Site-directed mutagenesis experiments are now in progress, and we are hopeful that these mutants will provide structural and functional information regarding the nucleotide/pyrophosphate binding site(s) of yeast DNA polymerase I.

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