Molecular Design of Inhibitors of in Vitro oriC DNA Replication Based on the Potential to Block the ATP Binding of DnaA Protein

Abstract

DnaA protein, the initiation factor for chromosomal DNA replication in Escherichia coli, is activated by binding to ATP. We earlier reported that 3-acetoxy-2,2 -bi-1H-indol inhibited the ATP binding to DnaA protein (Sasaki, S., Mizushima, T., Hashimoto, T., Maeda, M., and Sekimizu, K. (1994) Bioorg. Med. Chem. Lett. 4, 1771-1774). In the present study, derivatives of 3-acetoxy-2,2 -bi-1H-indol with different lengths of aliphatic chains at the 3-O position were synthesized, and their potential to inhibit the ATP binding to DnaA protein was examined. Elongation of the aliphatic chain resulted in inhibition of the ATP binding to DnaA protein at lower concentrations. Among the derivatives, 3-[N-(11-carboxyundecyl)] carbamoylmethoxy-2,2 -bi-1H-indol (structure 7 (3-CUCM-BI)) exhibited the most potent inhibition with an IC 50 value of 7 M. The mode of the inhibition was competitive. We further demonstrated that structure 7 (3-CUCM-BI) inhibited DNA replication of the oriC plasmid in a system reconstituted from purified proteins. This inhibition was specific for the initiation of DNA replication rather than for the elongation. The inhibition was overcome by preincubation of DnaA protein with ATP. Furthermore, structure 7 (3-CUCM-BI) showed little inhibition on DNA synthesis in the ABC primosome system. We propose that structure 7 (3-CUCM-BI) functions in the in vitro oriC DNA replication by inhibiting the ATP binding to DnaA protein.Replication of chromosomal DNA in Escherichia coli is regulated at the step of initiation. DnaA protein is the initiation factor for chromosomal DNA replication (1-3); thus, DnaA protein has been considered to play an important role in regulating DNA replication. DnaA protein has a high affinity for ATP (K d ϭ 0.03 M) and for ADP (K d ϭ 0.1 M) (4). In the oriC DNA replication system reconstituted from purified proteins, the ATP binding form of DnaA protein is active in DNA replication, whereas the ADP binding form is inactive (4). These results suggest that the ATP binding to DnaA protein activates the protein; however, the possibilities that the ADP binding to the protein inhibits the activity of DnaA protein in the initiation of oriC DNA replication and that the ATP binding to the protein is not essential for the process would need to be excluded. Studies on DnaAcos protein, which loses the affinity for ATP and ADP but is active in the initiation of oriC DNA replication in vitro, imply this notion (5, 6). To better understand the requirement of the ATP binding for the initiation of oriC DNA replication, development of specific inhibitors for the ATP binding to DnaA protein and examination of their effects on oriC DNA replication in vitro are important. The availability of such inhibitors would be good tools to study the biological relevance of the ATP binding to DnaA protein.We reported that 3-acetoxy-2,2Ј-bi-1H-indol inhibited the ATP binding of DnaA protein (7). This indol is the first known synthetic organic compound to inhibit the ATP binding of DnaA protein. However, concentration of...

Kinetics of ATP Binding to the Origin Recognition Complex of Saccharomyces cerevisiae

“…We also tried to detect the ATP hydrolysis by a filter binding assay. However, because the complex of ORC with ADP was very unstable, 2 we could not detect the ATP hydrolysis on ORC by a filer binding assay.…”

“…In Escherichia coli, DnaA, the initiator of chromosomal DNA replication, has a high affinity for both ATP and ADP (1). The ATP-DnaA complex is active for DNA replication, but the ADP-DnaA complex and nucleotide-free DnaA are inactive (1)(2)(3). DnaA has intrinsic ATPase activity, and ATP bound to DnaA can be hydrolyzed to ADP (1).…”

Alteration in the contents of unsaturated fatty acids in dnaA mutants of Escherichia coli

“…DnaA protein has a high affinity for ATP and ADP; the ATP-binding form is active in the oriC replication system in vitro, whereas the ADPbinding form is inactive (Sekimizu et al, 1987). Synthesized organic compounds that were designed to block the ATP binding to DnaA protein specifically inhibited oriC DNA replication in vitro (Mizushima et al, 1996a). We have shown recently that induction of an artificially constructed mutant DnaA protein, which has decreased ATPase activity, led to overinitiation of DNA replication in cells, resulting in a dominant lethal phenotype .…”

Suppression of temperature-sensitivity of a dnaA46 mutant by excessive DNA supercoiling

“…DnaA protein has high affinities for ATP and ADP, and the ATP-binding form is the active form for DNA replication [2]. Recent genetic and biochemical studies strongly suggest that adenine-nucleotide binding to DnaA protein regulates the initiation of chromosomal DNA replication in E. coli cells [3][4][5][6][7][8][9][10]. However, some other mechanisms should be investigated in order to understand completely the regulation of DNA replication.…”