Thermostable Pyrococcus furiosus DNA ligase catalyzes the synthesis of (di)nucleoside polyphosphates

Abstract

DNA ligase from the hyperthermophilic marine archaeon Pyrococcus furiosus (Pfu DNA ligase) synthesizes adenosine 5 -tetraphosphate (p4A) and dinucleoside polyphosphates by displacement of the adenosine 5 -monophosphate (AMP) from the Pfu DNA ligase-AMP (E-AMP) complex with tripolyphosphate (P 3), nucleoside triphosphates (NTP), or nucleoside diphosphates (NDP). The experiments were performed in the presence of 1-2 μM [α- 32P]ATP and millimolar concentrations of NTP or NDP. Relative rates of synthesis (%) of the following adenosine(5 )tetraphospho(5 )nucleosides (Ap4N) were observed: Ap4guanosine (Ap4G) (from GTP, 100);Ap4deoxythymidine (Ap4dT) (from dTTP, 95); Ap4xanthosine (Ap4X) (from XTP, 94); Ap 4deoxycytidine (Ap4dC) (from dCTP, 64); Ap 4cytidine (Ap4C) (from CTP, 60); Ap 4deoxyguanosine (Ap4dG) (from dGTP, 58); Ap 4uridine (Ap4U) (from UTP, <3). The relative rate of synthesis (%) of adenosine(5 )triphospho(5 )nucleosides (Ap3N) were: Ap3guanosine (Ap3G) (from GDP, 100); Ap 3xanthosine (Ap3X) (from XDP, 110); Ap3cytidine (Ap3C) (from CDP, 42); Ap3adenosine (Ap3A) (from ADP, <1). In general, the rate of synthesis of Ap4N was double that of the corresponding Ap3N. The enzyme presented optimum activity at a pH value of 7.2-7.5, in the presence of 4 mM Mg2+, and at 70°C.The apparent Km values for ATP and GTP in the synthesis of Ap4G were about 0.001 and 0.4 mM, respectively, lower values than those described for other DNA or RNA ligases. Pfu DNA ligase is used in the ligase chain reaction (LCR) and some of the reactions here reported [in particular the synthesis of Ap4adenosine (Ap4A)] could take place during the course of that reaction. © Springer-Verlag 2001.