Synthetic Activity of Sso DNA Polymerase Y1, an Archaeal DinB-like DNA Polymerase, Is Stimulated by Processivity Factors Proliferating Cell Nuclear Antigen and Replication Factor C

Abstract

DNA replication efficiency is dictated by DNA polymerases (pol) and their associated proteins. The recent discovery of DNA polymerase Y family (DinB/UmuC/ RAD30/REV1 superfamily) raises a question of whether the DNA polymerase activities are modified by accessory proteins such as proliferating cell nuclear antigen (PCNA). In fact, the activity of DNA pol IV (DinB) of Escherichia coli is enhanced upon interaction with the ␤ subunit, the processivity factor of DNA pol III. Here, we report the activity of Sso DNA pol Y1 encoded by the dbh gene of the archaeon Sulfolobus solfataricus is greatly enhanced by the presence of PCNA and replication factor C (RFC). Sso pol Y1 per se was a distributive enzyme but a substantial increase in the processivity was observed on poly(dA)-oligo(dT) in the presence of PCNA (039p or 048p) and RFC. The length of the synthesized DNA product reached at least 200 nucleotides. Sso pol Y1 displayed a higher affinity for DNA compared with pol IV of E. coli, suggesting that the two DNA polymerases have distinct reason(s) to require the processivity factors for efficient DNA synthesis. The abilities of pol Y1 and pol IV to bypass DNA lesions and their sensitive sites to protease are also discussed.The recently identified novel family of DNA polymerases (pol) 1 (DinB/UmuC/Rev1/Rad30 superfamily or family Y) comprises proteins from different species including Bacteria, Eukarya, and Archaea (1, 2). Members of this superfamily can bypass lesions on template DNA, such as ultraviolet light photoproducts, and mismatched primer/template DNA (3-7). Thus, the new DNA polymerases seem to function by assisting the conventional DNA replicases to cope with faulty DNA templates by taking over the DNA synthesis at aberrant sites (for recent reviews, see Refs. 1 and 8 -12). Common features of family Y DNA polymerases include the lack of 3Ј-exonuclease proofreading activity and synthesis of DNA with low fidelity in a distributive fashion. The human DNA pol has been shown recently to possess 5Ј-deoxyribose phosphate lyase activity, suggesting a role in base excision repair (13). It has been hypothesized that the very low processivity of these polymerases prevents excessive introduction of mutations because of extended error-prone DNA synthesis. This has been, however, questioned recently when DNA polymerase IV of Escherichia coli (pol IV) (7) was found to synthesize tracks of more than 300 nucleotides upon interaction with the ␤ subunit of pol III (14). The ␤ subunit and its eukaryotic counterpart, i.e. PCNA, play essential roles in processive chromosomal replication by forming a sliding platform that mediates the interaction of DNA polymerases with DNA (15). In addition, the sliding clamps also interact with a variety of proteins other than DNA polymerases involved in DNA processing and even in cell cycle control (16). It is therefore of general interest to elucidate the relationships between various members of Y family DNA polymerases and the corresponding sliding clamps.Archaea, the third domain of life, is t...

<i>Escherichia coli</i> DinB inhibits replication fork progression without significantly inducing the SOS response

“…DinB may inhibit RecA activation on SSB-coated ssDNA because it can bind ssDNA (Grúz et al, 2001;Furukohri et al, 2008). However, the affinity of DinB for ssDNA is approximately 2400-fold lower than that of SSB (Grúz et al, 2001). Even the highest amount of overproduced DinB in this report (about 80 μM; Fig.…”

“…The alternative explanation of the results is that ssDNA may be present but unavailable for RecA activation. DinB may inhibit RecA activation on SSB-coated ssDNA because it can bind ssDNA (Grúz et al, 2001;Furukohri et al, 2008). However, the affinity of DinB for ssDNA is approximately 2400-fold lower than that of SSB (Grúz et al, 2001).…”

Investigating the Role of the Little Finger Domain of Y-family DNA Polymerases in Low Fidelity Synthesis and Translesion Replication

“…In addition, the lesion bypass properties of Dpo4 are somewhat like those of the eukaryotic translesion polymerases in that Dpo4 can bypass thymine-thymine cyclobutane pyrimidine dimers (CPDs) (15,18,19) and abasic sites (15,20). In contrast, Dbh is a much more distributive polymerase when replicating undamaged DNA, is unable to incorporate a base opposite a CPD, and bypasses an abasic site with very low efficiency (16,17,21).…”

“…Dpo4 (DNA polymerase IV) was identified in the genome of S. solfataricus P2 through BLAST searches (13) of the complete P2 genome (14), using the dbh gene as a search query (15). Overall, the Dbh and Dpo4 proteins share 54% identity, yet the two polymerases exhibit different enzymatic properties (15)(16)(17). Dpo4 is thermostable and exhibits robust polymerase activity.…”

Revealing the essentiality of multiple archaeal pcna genes using a mutant propagation assay based on an improved knockout method

“…A similar protein sequence motif is also present in diverse archaeal PCNAinteracting proteins from different archaeal species. The identified archaeal PCNA-interacting proteins include DNA replication and repair polymerases (Cann et al, 1999;Daimon et al, 2002;Dionne et al, 2003;Grú z et al, 2001), DNA ligases (Dionne et al, 2003;Kiyonari et al, 2006;Mayanagi et al, 2009), nucleases, helicases and DNA glycosylases (Dionne & Bell, 2005;Kiyonari et al, 2008;Yang et al, 2002). Structural analyses of several PCNAenzyme complexes have yielded considerable insights into the functions of PCNA as a sliding clamp or docking platform (Doré et al, 2006;Matsumiya et al, 2001;Miyata et al, 2005;Xing et al, 2009).…”