Kinetic analysis by stopped-flow spectroscopy

Stopped-flow spectroscopy data were collected on an Applied Photophysics SX20 instrument under pseudo first-order conditions with the DNA in excess. Pol QM1 (1.25 μM) was preincubated with 2.5 μM DNA in a buffer containing 20 mM Tris-HCl (pH 8.0), 30 mM KCl, 200 μM EDTA, 250 μM TCEP. Nucleotides and Mg²⁺ (10 mM) were mixed rapidly with these preformed binary complexes of pol QM1. The fluorescence of endogenous TRP residues was then monitored at A₂₈₀ during the time course of the reaction. Fluorescence data were fit to mathematical models using an implementation of the nonlinear least-squares (NLLS) Marquardt-Levenberg algorithm in GNUplot. Variant polymerases were analyzed during cleavage and extension of ss14AG to orient the overall substrate processing reaction within the fluorescence trace. The relatively slower kinetics of the Y2387F variant assisted in aligning the end-trimming step with fluorescence quenching and the dNTP incorporation step with fluorescence enhancement (Fig S3A). Reactions with ds14AG/21T and dATP exhibited only increasing fluorescence and no apparent trough during the first phase, because end-trimming does not occur with this substrate. The products evolving while reacting the double-stranded substrate ds14AG/21T with dATP or dGTP (5 μM vs 250 μM) were resolved on a denaturing sequencing gel to correlate the second phase of increasing fluorescence with nucleotide incorporation, yielding the 15mer product (Fig S3B). When pol QM1 was mixed with dGTP and ds14AG/21T in the SX20 instrument, only flat fluorescence traces were obtained (data not shown), consistent with discrimination of the dGTP/dT mismatch apparent on the gel (Fig S3B).