RESUMO
Human RECQL4 protein was expressed in insect cells using a baculovirus protein expression system and it was purified to near homogeneity. The protein sedimented at a position between catalase (230 kDa) and ferritin (440 kDa) in glycerol gradient centrifugation, suggesting that it forms homo-multimers. Activity to displace annealed 17-mer oligonucleotide in the presence of ATP was co-sedimented with hRECQL4 protein. In ion-exchange chromatography, both DNA helicase activity and single-stranded DNA-dependent ATPase activity were co-eluted with hRECQL4 protein. The requirements of ATP and Mg for the helicase activity were different from those for the ATPase activity. The data suggest that the helicase migrates on single-stranded DNA in a 3'-5' direction. These results suggest that the hRECQL4 protein exhibits DNA helicase activity.
Assuntos
DNA Helicases/metabolismo , RecQ Helicases/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/isolamento & purificação , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/efeitos da radiação , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/efeitos da radiação , Baculoviridae/genética , Centrifugação com Gradiente de Concentração , Cromatografia por Troca Iônica , Clonagem Molecular , DNA Helicases/genética , DNA Helicases/isolamento & purificação , DNA Helicases/efeitos da radiação , DNA Polimerase I/metabolismo , DNA Polimerase I/efeitos da radiação , DNA Complementar , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Magnésio/metabolismo , Polidesoxirribonucleotídeos/metabolismo , Ligação Proteica , RecQ Helicases/genética , RecQ Helicases/isolamento & purificação , RecQ Helicases/efeitos da radiação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/efeitos da radiação , Raios UltravioletaRESUMO
Two human melanoma cell lines (one radiosensitive, HT144 and one radioresistant, SK Mel-3) and one normal human fibroblast (AG1522) were evaluated for thermal radiosensitization and the thermal enhancement ratios (TERs) were calculated. These were compared with residual polymerase activity to determine if this activity could be used to predict TERs. In all three cell lines, there was a good correlation between TER and residual polymerase alpha or beta activity. Polymerase beta was more sensitive than polymerase alpha as an indicator for TER. There were small cell line-dependent differences (not related to radiosensitivity) among the correlation curves, indicating that for each cell/tumor-type polymerase activity, vs. TER may have to be calibrated.