RNA template-dependent 5 ' nuclease activity of Thermus aquaticus and Thermus thermophilus DNA polymerases

DNA replication and repair require a specific mechanism to join the 3'- and 5'-ends of two strands to maintain DNA continuity. In order to understand the details of this process, we studied the activity of the 5' nucleases wi th substrates containing an RNA template strand. By comparing the eubacteri al and archaeal 5' nucleases, we show that the polymerase domain of the eub acterial enzymes is critical for the activity of the 5' nuclease domain on RNA containing substrates. Analysis of the activity of chimeric enzymes bet ween the DNA polymerases from Thermus aquaticus (TaqPol) and Thermus thermo philus (TthPol) reveals two regions, in the "thumb" and in the "palm" subdo mains, critical for RNA-dependent 5' nuclease activity. There are two criti cal amino acids in those regions that are responsible for the high activity of TthPol on RNA containing substrates. Mutating glycine 418 and glutamic acid 507 of TaqPol to lysine and glutamine, respectively, increases its RNA -dependent 5' nuclease activity 4-10-fold, Furthermore, the RNA-dependent D NA polymerase activity is controlled by a completely different region of Ta qPol and TthPol, and mutations in this region do not affect the 5' nuclease activity. The results presented here suggest a novel substrate binding mod e of the eubacterial DNA polymerase enzymes, called a 5' nuclease mode, tha t is distinct from the polymerizing and editing modes described previously. The application of the enzymes with improved RNA-dependent 5' nuclease act ivity for RNA detection using the invasive signal amplification assay is di scussed.