Table 2.

Kinetic parameters for nucleotide insertion reactions catalyzed by hPolη

ReactionInsertionSiteadNTP addedKm (μM)bVmax(%/min)bVmax/Km fincc
Standing startOpposite an A by hPolη5′----CAGdTTP23.6 ± 1.35.6 ± 0.150.24ND
----GTCACT
Opposite an A by hPolη + PCNA, RFC, and RPA5′----CAGdGTP70 ± 121.1 ± 0.050.0165 × 10−3
----GTCACTdATP83 ± 282.3 ± 0.20.0278.5 × 10−3
dTTP2.7 ± 0.38.5 ± 0.33.151
dCTP102 ± 190.98 ± 0.080.00963 × 10−3
Running startOpposite a C by hPolη5′----CAGdGTP14.3 ± 1.56.6 ± 0.450.46ND
----GTCACT
Opposite a C by hPolη + PCNA, RFC, and RPA5′----CAGdGTP1 ± 0.097.6 ± 0.437.61
----GTCACTdATP82 ± 162.5 ± 0.410.033.9 × 10−3
dTTP238 ± 512.8 ± 0.30.0121.5 × 10−3
dCTP380 ± 950.78 ± 0.450.0022.7 × 10−4
  • a Sequences represent a portion of the substrate, as also shown in Fig. 4. Boldfacing indicates the template base which directed nucleotide insertion.

  • b Values are reported as means and standard deviations.

  • c f inc, frequency of nucleotide misincorporation. ND, not determined.