The thumb subdomain of Pyrococcus furiosus DNA polymerase is responsible for deoxyuracil binding, hydrolysis and polymerization of nucleotides

Abstract

B-family DNA polymerases, which are found in eukaryotes, archaea, viruses, and some bacteria, participate in DNA replication and repair. Starting from the N-terminus of archaeal and bacterial B-family DNA polymerases, three domains include the N-terminal, exonuclease, and polymerase domains. The N-terminal domain of the archaeal B-family DNA polymerase has a conserved deoxyuracil-binding pocket for specially binding the deoxyuracil base on DNA. The exonuclease domain is responsible for removing the mismatched base pair. The polymerase domain is the core functional domain and takes a highly conserved structure composed of fingers, palm and thumb subdomains. Previous studies have demonstrated that the thumb subdomain mainly functions as a DNA-binding element and has coordination with the exonuclease domain and palm subdomain. To further elucidate the possible functions of the thumb subdomain of archaeal B-family DNA polymerases, the thumb subdomain of Pyrococcus furiosus DNA polymerase was mutated, and the effects on three activities were characterized. Our results demonstrate that the thumb subdomain participates in the three activities of archaeal B-family DNA polymerases as a common structural element. Both the N-terminal deoxyuracil-binding pocket and thumb subdomain are critical for deoxyuracil binding. Moreover, the thumb subdomain assists DNA polymerization and hydrolysis reactions, but it does not contribute to the fidelity of DNA polymerization.

Keywords: B-family DNA polymerase; Deoxyuracil-binding; Exonuclease; Polymerase; Thumb subdomain.