CHARACTERIZATION OF THE TRYPTOPHAN FLUORESCENCE AND HYDRODYNAMIC PROPERTIES OF RAT DNA-POLYMERASE-BETA

We have examined the biophysical properties of DNA polymerase beta (be ta-pol) in solution. Time-resolved and steady-state fluorescence were used to investigate the microenvironment of the lone tryptophanyl resi due (Trp324), and a combination of sedimentation equilibrium, sediment ation velocity and fluorescence anisotropy decay measurements were use d to study the hgdrodynamic properties of the enzyme. Trp324 appears t o be exposed to water as judged by the tryptophan emission and steady- state and lifetime quenching experiments. The fluorescence is easily q uenched by a neutral quencher acrylamide (k(q) = 1.59x10(9)M(-1)s(-1)) , and by a negatively charged ionic quencher, I- (k(q) = 1.60x10(9) M( -1)s(-1)), but not by a positively charged ionic quencher, Cs+ (X(q) = 0.2x10(9) M(-1)s(-1)). The fluorescence lifetime of beta-pol is best described by the sum of two exponentials with a longer lifetime compon ent of 8.4 ns and a shorter lifetime component of 1.3 ns. Decay associ ated spectra (DAS) show emission maxima at 340 nm and at 345 nm for th e shorter lifetime and longer lifetime components, respectively, with corresponding centers of gravity at 347 nm and 348 nm. Sedimentation e quilibrium experiments show that the enzyme exists as a monomer at the KCl concentrations (>0.05 M) studied in the absence of divalent metal s. Zn2+ causes higher order aggregation, but no such aggregates are se en with Mg2+ and Mn2+. In the presence of 1 mM manganese, the average lifetime decreased approximately 10%, from 8.14 ns to 7.38 ns, with a concomitant increase of average rotational correlational time (phi) fr om 24 ns to 28 ns. The accessibility of the positively charged quenche r (Cs+) to tryptophan also decreases approximately 50%, indicating alt eration of the tryptophan microenvironment. By contrast, Mg2+ + causes minor changes in fluorescence properties. The hy drodynamic shape of the intact enzyme and its single-stranded (8 kDa) and double-stranded (31 kDa) DNA binding domains were further investigated by sedimentatio n velocity measurements. The value of SX(20.W)(0) for the intact enzym e is 2.97 S, and the calculated axial ratio is 5.0. In contrast to the 8 kDa domain. which has a less asymmetric shape with an axial ratio o f 2.3, the 3 1 kDa domain shows an elongated structure with an axial r atio of 5.5. These data suggest that the axial ratio of the intact enz yme may be the result of marked bending of the molecule at the flexibl e hinge region between the two domains.