Mnemonic aspects of Escherichia coli DNA polymerase I : Interaction with one template influences the next interaction with another template

• Published in: Journal of molecular biology Vol. 189; no. 3; pp. 435 - 448
• Main Authors: Papanicolaou, Catherine, Lecomte, Philippe, Ninio, Jacques
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Ltd 05.06.1986; Elsevier
• Subjects: Base Composition; Biological and medical sciences; Deoxyadenine Nucleotides - metabolism; Deoxycytidine Monophosphate - metabolism; Deoxyguanine Nucleotides - metabolism; DNA Polymerase I - metabolism; DNA Replication; Escherichia coli - enzymology; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Kinetics; Models, Biological; Molecular and cellular biology; Molecular genetics; Poly dA-dT - metabolism; Replication; Templates, Genetic; Thymine Nucleotides - metabolism; DNA polymerase 1; DNA; Escherichia coli; Bacteria; Molecular interaction; Replication; Mechanism of action; Escherichieae; Enterobacteriaceae
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/0022-2836(86)90315-3

When Escherichia coli DNA polymerase I (Pol I) replicates a homopolymer, the excision/polymerization (exo/pol) ratio varies with enzyme and initiator concentration. The study of this effect in the case of poly(dA) · oligo(dT) replication led us to propose a mnemonic model for Pol I, in which the 3′ to 5′ excision activity warms up when the enzyme is actively polymerizing, and cools down when it dissociates from the template. The model predicts that the exo/pol ratio must increase with processivity length and initiator concentration and decrease with enzyme concentration. It predicts also that contact of the enzyme with one template alters its excision efficiency towards another template. The exo/pol ratio and processivities of Pol I and its Klenow fragment were studied on four templates: poly(dA) · (dT) 10, poly(dT) · (dA) 10, poly(dC) · (dG) 10 and poly-(dI) · (dC) 10. We show that the Klenow fragment is usually much less processive than Pol I and when this is the case it has a much lower exo/pol ratio. At equal processivity. the exo/pol ratios are nearly equal. Furthermore, many factors that influence processivity length (e.g. manganese versus magnesium, inorganic pyrophosphate, ionic strength) influence the exo/pol ratio in the same direction. The study of deaminated poly(dC) replication, where we followed incorporation and excision of both G and A residues, allowed us to assign the origin of the dNMP variations to changes in the 3′ to 5′ proof-reading activity of Pol I. Similarly, the lower dNMP turnover of the Klenow fragment observed with deaminated poly(dC) was specifically assigned to a decreased 3′ to 5′ exonuclease activity. The exo/pol ratio generally increased with initiator and decreased with enzyme concentration, in agreement with the model, except for poly(dI) · oligo(dC), where it decreased with initiator concentration. However, by terminating chain elongation with dideoxy CTP, we showed directly that, even in this system, excision is relatively inefficient at the beginning of synthesis. Interaction of Pol I with poly(dA) · (dT) or with poly(dC) · (dG) modifies its exo/pol characteristics in the replication of poly(dI) · (dC) and poly(dA) · (dT), respectively. The Klenow enzyme is not sensitive to such influences and this correlates with its reduced processivity on the influencing templates. Our results reveal the existence of differences between Pol I and its Klenow fragment that are more profound than has been thought previously. They reveal also that the exo/pol ratio, and hence error rates, are subject to multiple influences, and in particular they suggest that mutational hot-spots may occur in vivo whenever processive replication is interrupted.