Proficient Replication of the Yeast Genome by a Viral DNA Polymerase

Abstract

DNA replication in eukaryotic cells requires minimally three B-family DNA polymerases: Pol ␣, Pol ␦, and Pol ⑀. Pol ␦ replicates and matures Okazaki fragments on the lagging strand of the replication fork. Saccharomyces cerevisiae Pol ␦ is a threesubunit enzyme (Pol3-Pol31-Pol32). A small C-terminal domain of the catalytic subunit Pol3 carries both iron-sulfur cluster and zinc-binding motifs, which mediate interactions with Pol31, and processive replication with the replication clamp proliferating cell nuclear antigen (PCNA), respectively. We show that the entire N-terminal domain of Pol3, containing polymerase and proofreading activities, could be effectively replaced by those from bacteriophage RB69, and could carry out chromosomal DNA replication in yeast with remarkable high fidelity, provided that adaptive mutations in the replication clamp PCNA were introduced. This result is consistent with the model that all essential interactions for DNA replication in yeast are mediated through the small C-terminal domain of Pol3. The chimeric polymerase carries out processive replication with PCNA in vitro; however, in yeast, it requires an increased involvement of the mutagenic translesion DNA polymerase during DNA replication.Replication of genomic DNA during each cell cycle requires the action of replicative DNA polymerases. To ensure faithful transmission of genomic information from the parent to the daughter cells, these polymerases must work efficiently and with very high fidelity (1). The eukaryotic replicative DNA polymerases are members of the B-family polymerases, which are classified as such according to the structure of their catalytic domains (2-5). Three B-family DNA polymerases participate in DNA replication. The current model states that Pol ⑀ 2 replicates the leading strand of the replication fork, whereas Pol ␣-primase initiates Okazaki fragments on the lagging strand that are elongated and matured by Pol ␦ (6). This simple "division of labor" model is still a matter of debate (7-9). Furthermore, under certain conditions, such as those of replication restart following DNA recombination, Pol ␦ carries out substantial DNA synthesis of both strands (10). The fourth B-family enzyme, Pol , is required for translesion synthesis in response to DNA damage, which results in the bulk of damageinduced mutagenesis in eukaryotes (11). Pol also participates in replication past structural blocks when normal replication forks stall (12).B-family DNA polymerases are ubiquitous; they are found in eukaryotes, bacteria, archaea, and in both bacterial and eukaryotic DNA-based viruses (13). All B-family enzymes contain three conserved domains: a structural N-terminal domain (NTD), a 3Ј-5Ј exonuclease domain, and the polymerase domain containing the palm, finger, and thumb sub-domains. The NTD is highly conserved, but a specific function for this domain has been assigned only to some archaeal enzymes, in which the NTD recognizes template uracil residues and inhibits continued replication by the DNA polymerase (14, 15). The...

Increased MAD2L2 expression predicts poor clinical outcome in Colon Adenocarcinoma

“…When DNA damage occurs, POL3 is normally required in normal cells to complete repair. In contrast to normal cells, cancer cells generally have a penchant for relying on error-repair bypasses, especially the TLS repair bypass, and this pathway is commonly performed by the error-prone and more efficient POLζ, of which MAD2L2 is a subunit (Stodola et al, 2016). We hypothesize that the passive high expression of MAD2L2 in COAD contributes to genomic instability and mutational load tolerance in cancer cells.…”

Engineering Ca2+-Dependent DNA Polymerase Activity

“…coli failed multiple attempts (Figure S27 for one example), perhaps due to host interactions. Recent works showed that the yeast genome could be replicated replacing Pol3 with bacteriophage polymerase RB69 . If Pol3 possesses an interchangeable function with a native E.…”

Yeast DNA polymerase ζ maintains consistent activity and mutagenicity across a wide range of physiological dNTP concentrations

“…Various defects in the catalytic and accessory subunits of yeast replicative DNA polymerases impede the progression of the replication fork and cause DRIM ( 45 , 48 – 50 , 52 , 54 , 56 ). Among these, the pol3-Y708A mutation has been used most commonly for the mechanistic studies of DRIM ( 45 – 47 ) because of its rather strong mutator phenotype that is almost entirely Polζ-dependent.…”

“…While originally discovered as a response to mutations in the replicative DNA polymerases α, δ and ε ( 45 , 48 – 49 ), DRIM can also be promoted by defects in non-catalytic replisome components ( 45 , 50 – 54 ) or replication-coupled chromatin remodelling ( 55 ), as well as by exposure of wild-type cells to the replication inhibitor hydroxyurea (HU) ( 47 ). Most recently, DRIM has been observed in yeast strains, in which replication deficiency was caused by a replacement of the catalytic domain of Polδ with that of bacteriophage RB69 DNA polymerase ( 56 ), providing further evidence that the recruitment of Polζ is a general response to replication impediment. Like DNA damage-induced mutagenesis, the DRIM phenotype is completely dependent on monoubiquitylation of proliferating cell nuclear antigen (PCNA) by Rad6/Rad18 ( 45 ), suggesting that the recruitment of Polζ-Rev1 to undamaged DNA is regulated similarly to the DNA damage response.…”