Effect of dGTP concentration on human and CHO telomerase.
Human telomerase produces a long ladder of six-base repeat additions to a primer, while CHO telomerase primarily adds only one or two repeat additions to a primer. Under the standard assay conditions, the concentration of dGTP is very low, so we investigated the effects of increasing dGTP concentration on human and CHO telomerase activities. Increasing dGTP concentration over a range of 1.5-50 microM caused the human telomerase to produce longer primer extension products until products were so large that no ladder pattern was apparent. Increasing dGTP concentration resulted in CHO telomerase producing one to eight repeat additions, though still not as many repeats as produced by human telomerase even under low dGTP conditions. CHO telomerase produced a six-base ladder pattern comparable to human telomerase only after raising the dGTP concentration to 500 microM under conditions in which the dATP concentration was low. Primer challenge experiments showed the human telomerase exhibited approximately 100% processivity at both low and high concentrations of dGTP, and thus increasing dGTP concentration appeared to affect only the extension rate. In contrast, CHO telomerase exhibited low processivity under low concentrations of dGTP and increased processivity at higher dGTP concentrations. One explanation for the low processivity of CHO was found in CHO telomerase's inability to extend the GGTTAG permuted primer under nonprocessive conditions, while able to extend the other five permuted primers. Competition studies of different permuted primers indicated that the GGTTAG primer cannot interact with the nonprocessive CHO telomerase. A model is proposed for explaining the nonprocessive behavior of CHO telomerase.
Maine
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IP
Department of Cancer Biology, Department of Experimental Therapeutics, The Institute for Drug Development, The Cancer Therapy & Research Center, San Antonio, Texas 78229, USA.
Chen
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SF
Windle
B
B
D000818:Animals; D016466:CHO Cells / Q000201:enzymology*; D006224:Cricetinae; D017931:DNA Primers / Q000378:metabolism; D003848:Deoxyguanine Nucleotides / Q000737:chemistry* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D011499:Protein Processing, Post-Translational; D012323:RNA Processing, Post-Transcriptional; D019098:Telomerase / Q000737:chemistry* / Q000378:metabolism*; D013997:Time Factors
D017931:DNA Primers; D003848:Deoxyguanine Nucleotides; C029603:deoxyguanosine triphosphate; D019098:Telomerase