Increased processivity, misincorporation, and nucleotide incorporation efficiency in Sulfolobus solfataricus Dpo4 thumb domain mutants

The present study aimed to increase the processivity of Sulfolobus solfataricus DNA polymerase Dpo4. Protein engineering and bioinformatics were used to compile a library of potential Dpo4 mutation sites. Ten potential mutants were identified and constructed. A primer extension assay was used to evaluate the processivity of Dpo4 mutants. Thumb (A181D) and finger (E63K) domain mutants showed a processivity of 20 and 19 nucleotides (nt), respectively. A little finger domain mutant (I248Y) exhibited a processivity of 17 nt, only 1 nt more than wild-type Dpo4. Furthermore, the A181D mutant showed lower fidelity and higher nucleotide incorporation efficiency (4.74 × 10^-4 s^-1 μM^-1) than E63K and I248Y mutants. When tasked with bypassing damage, the A181D mutant exhibited a 3.81-fold and 2.62-fold higher catalytic efficiency (k_cat/K_m) at incorporating dCTP and dATP, respectively, than wildtype Dpo4. It also showed a 55% and 91.5% higher catalytic efficiency when moving beyond the damaged 8-oxoG:C and 8-oxoG:A base pairs, respectively, compared to wild-type Dpo4. Protein engineering and bioinformatics methods can effectively increase the processivity and translesion synthesis ability of Dpo4.