Selective inhibition of DNA replicase assembly by a non-natural nucleotide: exploiting the structural diversity of ATP-binding sites

Abstract

DNA synthesis is catalyzed by an ensemble of proteins designated the replicase. The efficient assembly of this multiprotein complex is essential for the continuity of DNA replication and is mediated by clamp-loading accessory proteins that use ATP binding and hydrolysis to coordinate these events. As a consequence, the ability to selectively inhibit the activity of these accessory proteins provides a rational approach to regulate DNA synthesis. Toward this goal, we tested the ability of several non-natural nucleotides to inhibit ATP-dependent enzymes associated with DNA replicase assembly. Kinetic and biophysical studies identified 5-nitro-indolyl-2'-deoxyribose-5'-triphosphate as a unique non-natural nucleotide capable of selectively inhibiting the bacteriophage T4 clamp loader versus the homologous enzyme from Escherichia coli. Modeling studies highlight the structural diversity between the ATP-binding site of each enzyme and provide a mechanism accounting for the differences in potencies for various substituted indolyl-2'-deoxyribose-5'-triphosphates. An in vivo assay measuring plaque formation demonstrates the efficacy and selectivity of 5-nitro-indolyl-2'-deoxyribose as a cytostatic agent against T4 bacteriophage while leaving viability of the E. coli host unaffected. This strategy provides a novel approach to develop agents that selectively inhibit ATP-dependent enzymes that are required for efficient DNA replication.

Figure 1

Structures and electron density surface potentials of natural and non-natural nucleosides and nucleotides used in this study. For convenience, only the nucleobase portion is provided. (A) Comparison of the structures of adenine and 5-nitroindole (B) Structures of substituted indolyl deoxynucleotides. All models were constructed using Spartan ’04 software (www.wavefun.com). The electron density surface potentials of adenine and non-natural nucleobases were then generated. The most electronegative regions are in red, neutral charges are in green, and the most electropositive regions are in blue.

Figure 2

d5-NITP is not hydrolyzed by gp44/62 but instead acts as a competitive inhibitor. (A) Hydrolysis of various nucleotide substrates by gp44/62 quantified by colorimetric ATPase assay. Assay conditions are as described in methods section. The concentration of all nucleotide substrates was maintained at 500 µM (b) Dose-dependent inhibition of gp44/62 ATPase activity by d5-NITP (▲) and r5-NITP (■). K_i values are 4.8 ± 0.5 µM and 10.8 ± 0.7 µM, respectively. (c) Double reciprocal plot of rate versus ATP concentration at several fixed concentrations of d5-NITP. The following concentrations of d5-NITP were used: no inhibitor (■), 5 µM (▲), 25 µM (▼), and 50 µM (♦). The K_i value of d5-NITP was determined by fitting the data to the following rate equation: v = V_max[S]/K_m(1+[I]/K_i) + [S]. The inset shows a plot of the slope of each line ([ATP]/rate) as a function of d5-NITP concentration.

Figure 3

d5-NITP inhibits assembly of the bacteriophage T4 replicase. (A) Diagram of strand displacement assay used to monitor replicase assembly and function. DNA polymerase alone (I) can incorporate nucleotides up to the forked strand but is unable to extend beyond it. As such, the longest product possible is a 44-mer. DNA polymerase in the presence of accessory proteins (II) defines the replicase and is able to extend the primer beyond the forked strand up to the abasic site (SP) present at position 51 in the template. In this case, the longest product formed is a 50-mer. When an ATPase inhibitor is present (III), replicase assembly is prevented. As a result, extension beyond the forked strand is not observed and the longest product detected is 44-mer generated by DNA polymerase alone. (b) Representative denaturing gel electrophoretic images of DNA synthesis catalyzed by the bacteriophage T4 polymerase and replicase in the absence or presence of d5-NITP. DNA substrate alone (lane 1), T4 DNA polymerase (lane 2), T4 DNA replicase (polymerase and accessory proteins) (lane 4), T4 DNA polymerase with 100 µM d5-NITP (lane 3), and T4 DNA replicase with 100 µM d5-NITP. (lane 5) (C) Quantification of product formation using the strand displacement assay described above. (D) Fluorescence changes associated with opening of the bacteriophage T4 processivity factor by the clamp loader, gp44/62. In the presence of 1 mM ATP, gp44/62 opens the closed ring of the homotrimeric gp45 labeled with fluorescent probe to generate a rapid change in fluorescence. In the presence of 1 mM d5-NITP, a significantly smaller change in fluorescence is observed indicating that clamp opening does not occur upon binding of the non-natural nucleotide.

Figure 4

Molecular modeling of the active sites of gp44/62 and γ-complex. The active site of the bacteriophage T4 clamp loader, gp44/62, bound with (A) ATP or (B) d5-NITP. The active site of the E.coli clamp loader, γ-complex, bound with (C) ATP or (D) d5-NITP.

Figure 5

Inhibition of T4 plaque formation by d5-NI. (A) The addition of 100 µg/ml d5-NI reduces plaque formation. Arrows indicate plaques caused by the lysis of phage-infected E.coli. (B) Graphical quantification of plaque reduction by d5-NI (n=4, **p<0.01 vs others). (C) Effects of 100 µg/ml d5-NI (♦), d5-EyI (▲) and ampicillin (●) compared to a DMSO control treated normal growth curve of E. coli (■). (D) Comparing the ability of d5-NI, d5-EyI, d5-FI, and chloramphenicol to inhibit phage infectivity. (n=4, **p<0.01 vs others) (E) The addition of 100 µg/ml d5-NI reduces plaque formation more effectively in wild type E.coli (JA300) compared to the E. coli strain, KY895, that is deficient in deoxythymidine kinase activity (tdk-1). (n=3, *p<0.05).