Figure 7.

Pol β catalyzed nucleotide insertion is reduced in the 601 NCP resulting in a lack of processivity. (A) NCP processive substrate design. Schematic depicting positions of 1-nt gaps and labeling strategy. Oligonucleotide B is 5΄ labeled with a ³²P phosphate. Site one is located 15 nts and site two 32 nts from the 5΄ end. Reactions were performed as described in Figure 2A. (B) Crystal structure of the NCP containing 601 sequence (PDB: 3LZ0). The positions of the 1-nt gaps are shown as red spheres. (C) Bar graphs representing the fraction of processive Pol β molecules in DNA and NCP reactions at 60 mM and 150 mM ionic strength. The F_p values at 60 mM ionic strength are 0.46 ± 0.05 and 0.1 ± 0.03 for DNA and NCP, respectively. The F_p values at 150 mM ionic strength are 0.12 ± 0.03 and 0.08 ± 0.05 for DNA and NCP, respectively. The error bars report the mean and standard deviation from at least two measurements performed with two independent NCP preparations. (D) Bar graph representing the k_cat (for total product formation) for DNA and NCP at 60 and 150 mM ionic strength. The k_cat values at 60 mM ionic strength are 0.38 ± 0.07 s⁻¹ and 0.02 ± 0.002 s⁻¹ for DNA and NCP, respectively. The k_cat values at 150 mM ionic strength are 0.41 ± 0.09 s⁻¹ and 0.02 ± 0.002 s⁻¹ for DNA and NCP, respectively. The error bars report the mean and standard deviation from at least two measurements performed with two independent NCP preparations. (E) Single-turnover time-courses for Pol β catalyzed nucleotide insertion at site two. Reaction conditions include 2 μM Pol β with 50 nM DNA (closed) and NCP (open), 150 mM ionic strength, 50 μM dGTP, and standard reaction conditions. The non-zero y-intercept for the NCP time-course is proposed to be a result of the NCP preparation containing a fraction of free DNA or a population of NCP that reacts before the major phase. The data are fit to a single-exponential equation yielding k_obs values of 3.0 ± 0.2 s⁻¹ and 0.006 ± 0.001 s⁻¹ for DNA and NCP, respectively.