A Unique B-Family DNA Polymerase Facilitating Error-Prone DNA Damage Tolerance in Crenarchaeota

Abstract

Sulfolobus islandicus codes for four DNA polymerases: three are of the B-family (Dpo1, Dpo2, and Dpo3), and one is of the Y-family (Dpo4). Western analysis revealed that among the four polymerases, only Dpo2 exhibited DNA damage-inducible expression. To investigate how these DNA polymerases could contribute to DNA damage tolerance in S. islandicus, we conducted genetic analysis of their encoding genes in this archaeon. Plasmid-borne gene expression revealed that Dpo2 increases cell survival upon DNA damage at the expense of mutagenesis. Gene deletion studies showed although dpo1 is essential, the remaining three genes are dispensable. Furthermore, although Dpo4 functions in housekeeping translesion DNA synthesis (TLS), Dpo2, a B-family DNA polymerase once predicted to be inactive, functions as a damage-inducible TLS enzyme solely responsible for targeted mutagenesis, facilitating GC to AT/TA conversions in the process. Together, our data indicate that Dpo2 is the main DNA polymerase responsible for DNA damage tolerance and is the primary source of targeted mutagenesis. Given that crenarchaea encoding a Dpo2 also have a low-GC composition genome, the Dpo2-dependent DNA repair pathway may be conserved in this archaeal lineage.

PolB1 Is Sufficient for DNA Replication and Repair Under Normal Growth Conditions in the Extremely Thermophilic Crenarchaeon Sulfolobus acidocaldarius

“…On the other hand, Lundgren and Bernander (2007) suggested that the PolB1 of S. acidocaldarius performs both leading- and lagging-strand syntheses based on the temporal patterns of transcription of all four polymerases with respect to the cell-division cycle. More recently, biochemical properties of the B-family polymerases of Sulfolobus and related genera have been investigated, and argue that the PolB1 enzymes play this replicative role ( Choi et al, 2011 ; Bauer et al, 2012 ; Peng et al, 2016 ; Yan et al, 2017 ), and this conclusion is supported by functional genetic criteria in S. acidocaldarius (this study) and closely related crenarchaea ( Feng et al, 2020 ). The recent evidence that Sulfodiicoccus acidiphilus , a member of the order Sulfolobales lacks PolB3 ( Sakai and Kurosawa, 2019 ) also strongly supports a replicative role of PolB1.…”

“…A contribution of TLS in the survival of bulky lesions has been suggested by in vitro studies in which S. solfataricus Dpo4 bypassed benzyl adducts, whereas the PolB2 and PolB3 enzymes of S. solfataricus did not ( Choi et al, 2011 ; see Table 1 ), and in which Dpo4 replicated past cisplatin lesions ( Boudsocq et al, 2001 ). The PolB2 polymerases are distinct from the other DNAPs of Sulfolobales in being induced by UV ( Fröls et al, 2007 ; Götz et al, 2007 ; Feng et al, 2020 ). In addition, phenotypic analyses indicate that the PolB2 of a strain related to S. solfataricus increases survival of UV-C, cisplatin, and NQNO ( Feng et al, 2020 ).…”

“…Previous in vivo , in vitro , and bioinformatic studies suggest that PolB1 is the main replicative DNAP in these archaea ( Choi et al, 2011 ; Makarova et al, 2014 ). The biochemical evidence that PolB2 and Dbh may be specialized for DNA repair, TLS, or both ( Boudsocq et al, 2004 ; Choi et al, 2011 ) has been supported by phenotypic analysis of dbh ( polY ) mutants of S. acidocaldarius ( Sakofsky et al, 2012 ; Jain et al, 2020 ), and dpo2 ( polB2 ) mutants of a related genus ( Feng et al, 2020 ). Although the PolB3 class of polymerases has the widest distribution among archaea ( Cooper, 2018 ), its functional significance remains unclear.…”

“…In addition, PolB2 can bypass uracil and methylate bases, and PolB3 can bypass cyclobutane thymine dimers (Table 1), and the expression level of polB2 has been found to increase after UV irradiation (Fröls et al, 2007;Götz et al, 2007;Feng et al, 2020). A PolB1 is found in all members of the TACK (Thaumarchaota, Aigarchaota, Crenarchaeota, and Korarchaeota) superphylum of Archaea (Makarova et al, 2014;Cooper, 2018).…”

“…Similarly, PolB2 and PolB3 has been reported to bypass some DNA damage (i.e., hypoxanthine and 8-oxoG) in vitro ( Choi et al, 2011 ; Table 1 ). In addition, PolB2 can bypass uracil and methylate bases, and PolB3 can bypass cyclobutane thymine dimers ( Table 1 ), and the expression level of polB2 has been found to increase after UV irradiation ( Fröls et al, 2007 ; Götz et al, 2007 ; Feng et al, 2020 ).…”