This report demonstrates that the beta sliding clamp of E. coli binds two different DNA polymerases at the same time. One is the high-fidelity Pol III chromosomal replicase and the other is Pol IV, a low-fidelity lesion bypass Y family polymerase. Further, polymerase switching on the primed template junction is regulated in a fashion that limits the action of the low-fidelity Pol IV. Under conditions that cause Pol III to stall on DNA, Pol IV takes control of the primed template. After the stall is relieved, Pol III rapidly regains control of the primed template junction from Pol IV and retains it while it is moving, becoming resistant to further Pol IV takeover events. These polymerase dynamics within the beta toolbelt complex restrict the action of the error-prone Pol IV to only the area on DNA where it is required.

Sliding clamps are ring-shaped proteins that tether DNA polymerases to DNA, which enables the rapid and processive synthesis of both leading and lagging strands at the replication fork. The clamp-loading machinery must repeatedly load sliding-clamp factors onto primed sites at the replication fork. Recent structural and biochemical analyses provide unique insights into how these clamp-loading ATPase machines function to load clamps onto the DNA. Moreover, these studies highlight the evolutionary conservation of the clamp-loading process in the three domains of life.

Anionic Arabidopsis thaliana peroxidase ATP A2 was expressed in Escherichia coli and used as a model for the 95% identical commercially available horseradish peroxidase HRP A2. The crystal structure of ATP A2 at 1.45 A resolution at 100 K showed a water molecule only 2.1 A from heme iron [Ostergaard, L., et al. (2000) Plant Mol. Biol. 44, 231-243], whereas spectroscopic studies of HRP A2 in solution at room temperature [Feis, A., et al. (1998) J. Raman Spectrosc. 29, 933-938] showed five-coordinated heme iron, which is common in peroxidases. Presented here, the X-ray crystallographic, single-crystal, and solution resonance Raman studies at room temperature confirmed that the sixth coordination position of heme iron of ATP A2 is essentially vacant. Furthermore, electronic absorption and resonance Raman spectroscopy showed that the heme environments of recombinant ATP A2 and glycosylated plant HRP A2 are indistinguishable at neutral and alkaline pH, from room temperature to 12 K, and are highly flexible compared with other plant peroxidases. Ostergaard et al. (2000) also demonstrated that ATP A2 expression and lignin formation coincide in Arabidopsis tissues, and docking of lignin precursors into the substrate binding site of ATP A2 predicted that coniferyl and p-coumaryl alcohols were good substrates. In contrast, the additional methoxy group of the sinapyl moiety gave rise to steric hindrance, not only in A2 type peroxidases but also in all peroxidases. We confirm these predictions for ATP A2, HRP A2, and HRP C. The specific activity of ATP A2 was lower than that of HRP A2 (pH 4-8), although a steady-state study at pH 5 demonstrated very little difference in their rate constants for reaction with H2O2 (k1 = 1.0 microM(-1) x s(-1). The oxidation of coniferyl alcohol, ferulic, p-coumaric, and sinapic acids by HRP A2, and ATP A2, however, gave modest but significantly different k3 rate constants of 8.7 +/- 0.3, 4.0 +/- 0.2, 0.70 +/- 0.03, and 0.04 +/- 0.2 microM(-1) x s(-1) for HRP A2, respectively, and 4.6 +/- 0.2, 2.3 +/- 0.1, 0.25 +/- 0.01, and 0.01 +/- 0.004 microM(-1) x s(-1) for ATP A2, respectively. The structural origin of the differential reactivity is discussed in relation to glycosylation and amino acid substitutions. The results are of general importance to the use of homologous models and structure determination at low temperatures.