A well conserved archaeal B-family polymerase functions as a mismatch and lesion extender

Abstract

B-family DNA polymerases (PolBs) of different groups are widespread in Archaea and different PolBs often coexist in the same organism. Many of these PolB enzymes remain to be investigated. One of the main groups that are poorly characterized is PolB2 whose members occur in many archaea but are predicted as an inactivated form of DNA polymerase. Herein, Sulfolobus islandicus DNA polymerase 2 (Dpo2), a PolB2 enzyme was expressed in its native host and purified. Characterization of the purified enzyme revealed that the polymerase harbors a robust nucleotide incorporation activity, but devoid of the 3’-5’ exonuclease activity. Enzyme kinetics analyses showed that Dpo2 replicates undamaged DNA templates with high fidelity, which is consistent with its inefficient nucleotide insertion activity opposite different DNA lesions. Strikingly, the polymerase is highly efficient in extending mismatches and mispaired primer termini once a nucleotide is placed opposite a damaged site. Together, these data suggested Dpo2 functions as a mismatch and lesion extender, representing a novel type of PolB that is primarily involved in DNA damage repair in Archaea. Insights were also gained into the functional adaptation of the motif C in the mismatch extension of the B-family DNA polymerases.

Enzymatic Switching Between Archaeal DNA Polymerases Facilitates Abasic Site Bypass

“…We have recently found that Sulfolobus Dpo2, an exo − B-family DNA polymerase, is very inefficient in nucleotide incorporation, but readily extends primer termini paired to the AP lesion (Supplementary Figure 2; Feng et al, 2021). A closer examination of replication products by Dpo2 on APcontaining templates revealed that the longest product is 1 nucleotide shorter than the template strand, indicative of the occurrence of a single nucleotide omission during the TLS extension (Supplementary Figure 2).…”

“…Sulfolobus strains were grown in SCV (0.2% sucrose, 0.2% casamino acids, 1% vitamin solution plus basic salts) or ACV media (0.2% D-arabinose, 0.2% casamino acids, 1% vitamin solution plus basic salts) at 78 • C as previously described (Peng et al, 2012). The Dpo2 (SiRe_0615) (Feng et al, 2020) and Dpo1 (SiRe_1451) expression strain (Feng et al, 2021) were constructed previously and the Dpo4 expression strain was constructed in this study.…”

“…The Dpo2 and Dpo1 DNA polymerases were purified previously as described (Feng et al, 2021). The Dpo4-encoding gene (SiRe_0236) was amplified from S. islandicus genomic DNA using primers indicated in Supplementary Table 1 and cloned into the expression vector, pSeSD (Peng et al, 2012), using Nde I and Sal I restriction sites.…”

“…In a previous work, we showed Dpo2 is very inefficient in nucleotide incorporation opposite the AP site under the tested condition (Feng et al, 2021). To gain a further insight into the properties of Dpo2, the same set of DNA substrates, which was used for testing Dpo4 AP insertion activity above, was employed in the assay.…”

“…Dpo3, another PolB that may also be involved in chromosome replication, also shows the 3 -5 exonuclease activity (Choi et al, 2011;Bauer et al, 2012). In contrast, Dpo2, the third PolB that mediates the targeted mutagenesis in Sulfolobuss islandicus (Feng et al, 2020), is devoid of the 3 -5 exonuclease activity (Feng et al, 2021). Nevertheless, it is still elusive whether these DNA polymerases could coordinate with each other to efficiently bypass DNA lesions, and how Dpo2, the only DNA damage-inducible DNA polymerase in the organism, could contribute to the DNA lesion bypass.…”