An archaeal family-B DNA polymerase variant able to replicate past DNA damage: occurrence of replicative and translesion synthesis polymerases within the B family

Abstract

A mutant of the high fidelity family-B DNA polymerase from the archaeon Thermococcus gorgonarius (Tgo-Pol), able to replicate past DNA lesions, is described. Gain of function requires replacement of the three amino acid loop region in the fingers domain of Tgo-Pol with a longer version, found naturally in eukaryotic Pol ζ (a family-B translesion synthesis polymerase). Inactivation of the 3′–5′ proof-reading exonuclease activity is also necessary. The resulting Tgo-Pol Z1 variant is proficient at initiating replication from base mismatches and can read through damaged bases, such as abasic sites and thymine photo-dimers. Tgo-Pol Z1 is also proficient at extending from primers that terminate opposite aberrant bases. The fidelity of Tgo-Pol Z1 is reduced, with a marked tendency to make changes at G:C base pairs. Together, these results suggest that the loop region of the fingers domain may play a critical role in determining whether a family-B enzyme falls into the accurate genome-replicating category or is an error-prone translesion synthesis polymerase. Tgo-Pol Z1 may also be useful for amplification of damaged DNA.

Human PrimPol is a highly error-prone polymerase regulated by single-stranded DNA binding proteins

“…From raw mutation frequencies, an absolute error rate (number of mistakes made per base incorporated) was calculated as previously described ( 23 ). The Klenow fragment Taq-Pol A and Tgo-Pol B exo− presented error rates of 3.6 × 10 −5 and 1.6 × 10 −5 , respectively (Table 1 ), agreeing with the previously reported fidelities of these enzymes ( 23 , 37 ). Analysis of human PrimPol revealed an error-prone phenotype with a calculated error rate of 1 × 10 −4 , essentially an order of magnitude lower than the exonuclease-deficient variants of S. cerevesiae replicative DNA polymerases δ, ϵ and the TLS specialized DNA polymerase ζ ( 38 – 40 ).…”

Genomics insights into ecotype formation of ammonia‐oxidizing archaea in the deep ocean

“…). Remarkably, the gamma and delta AOA genomes lack the genes encoding the two subunits of DNA polymerase B (PolB), which is critical for the repair of deaminated adenine and cytosine bases (Jozwiakowski et al ., ; Abellon‐Ruiz et al ., ). There was no tag gene encoding a 3‐methyladenine DNA glycosylase in these gamma and delta genomes.…”

The Roles of Family B and D DNA Polymerases in Thermococcus Species 9°N Okazaki Fragment Maturation

“…The increased UV sensitivity of Tko strains lacking polB suggests an important role for polB in DNA nucleotide excision repair ( 5 ). In addition, Escherichia coli polB has been shown to be important for replication restart after replisome stalling at lesions and archaeal polB uracil recognition may be a mechanism to recruit DNA repair factors ( 17 , 18 ). Together, these data suggests that polB gap filling and strand displacement synthesis in vivo could play multiple roles in Okazaki fragment maturation, DNA repair, recombination, and replication restart.…”