Interaction of DNA with the Klenow fragment of DNA polymerase I studied by time-resolved fluorescence spectroscopy

摘要: The interaction of a fluorescent duplex DNA oligomer with the Klenow fragment polymerase I from Escherichia coli has been studied in solution by using time-resolved fluorescence spectroscopy. An aminonaphthalenesulfonate (dansyl) probe was linked propyl chain to C5-modified uridine base located at specific site primer strand oligomer. bound tightly (KD = 7.9 nM), and probe's position within DNA-protein complex varied stepwise elongation upon addition appropriate deoxynucleoside triphosphates. decay total intensity polarization anisotropy were measured picosecond laser time-correlated single photon counting system. lifetimes, correlation time for internal rotation, angular range rotation according complex. These results showed that five or six bases upstream 3' terminus contact protein this region there differences degree solvent accessibility closeness contact. Further, minor binding mode identified, on basis heterogeneity environment observed when positioned seven terminus, which resulted distinctive "dip rise" decay. Experiments an epoxy-terminated site-directed mutant established labeled active (major form) spatially distinct 3'----5' exonuclease (minor form). abundance each these modes estimated under conditions analyzing dansyl probe. About 12% site. This method should be useful investigating editing mechanism important enzyme.