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Yeast DNA Polymerase Zeta (␨)Is Essential for Error-Free Replication Past Thymine Glycol

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Yeast DNA Polymerase Zeta (␨)Is Essential for Error-Free Replication Past Thymine Glycol Downloaded from genesdev.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press Yeast DNA polymerase zeta (␨)is essential for error-free replication past thymine glycol Robert E. Johnson, Sung-Lim Yu, Satya Prakash, and Louise Prakash1 Sealy Center for Molecular Science, University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1061, USA DNA polymerase zeta (Pol␨) promotes the mutagenic bypass of DNA lesions in eukaryotes. Genetic studies in Saccharomyces cerevisiae have indicated that relative to the contribution of other pathways, Pol␨ makes only a modest contribution to lesion bypass. Intriguingly, however, disruption of the REV3 gene, which encodes the catalytic subunit of Pol␨, causes early embryonic lethality in mice. Here, we present genetic and biochemical evidence for the requirement of yeast Pol␨ for predominantly error-free replication past thymine glycol (Tg), a DNA lesion formed frequently by free radical attack. These results raise the possibility that, as in yeast, in higher eukaryotes also, Pol␨ makes a major contribution to the replicative bypass of Tgs as well as other lesions that block synthesis by replicative DNA polymerases. Such a preeminent role of Pol␨ in lesion bypass would ensure that rapid cell divisions continue unabated during early embryonic development, thereby minimizing the generation of DNA strand breaks, chromosome aberrations, and the ensuing apoptotic response. [Keywords: DNApolymerase ␨; thymine glycol; translesion DNAsynthesis; Pol ␨ as an extender; error-free translesion DNAsynthesis by Pol ␨; yeast] Received October 4, 2002; revised version accepted October 31, 2002. Genetic studies in the yeast Saccharomyces cerevisiae et al. 2000b; Washington et al. 2000). Genetic studies in have indicated the requirement of Rad6–Rad18-depen- yeast have additionally indicated a role for Pol␩ in the dent processes in promoting replication of damaged error-free bypass of cyclobutane pyrimidine dimers DNA. For example, the replicative bypass of UV-induced (CPDs) formed at TC and CC sites (Yu et al. 2001). DNAlesions is carried out by three different Rad6– Pol␨ differs from Pol␩ in its inability to replicate Rad18-controlled pathways (Torres-Ramos et al. 2002) in through CPDs, and that is because Pol␨ is very ineffi- which Rad5, an SWI/SNF DNA-dependent ATPase cient at inserting nucleotides opposite the 3ЈTofaT–T (Johnson et al. 1992, 1994), plays an important role in dimer. Pol␨, however, promotes the mutagenic bypass of promoting error-free replication through UV-induced DNAlesions by extending from nucleotides inserted op- DNAlesions. The other two pathways utilize the posite the lesion by another DNApolymerase (Johnson RAD30-encoded DNApolymerase eta (Pol␩; Johnson et et al. 2000a). For example, Pol␨ efficiently extends from al. 1999), and the REV3, REV7-encoded DNApolymerase a G inserted opposite the 3ЈT of either a T–T dimer or a zeta (Pol␨; Nelson et al. 1996), which primarily function (6–4) T–T photoproduct (Johnson et al. 2000a, 2001), and in the error-free and mutagenic modes, respectively, of it efficiently extends from an Aopposite an abasic site translesion DNAsynthesis (TLS) through UV lesions. (Haracska et al. 2001a). However, as inferred from ge- The mechanism by which Rad5 affects lesion bypass is netic studies in yeast, Pol␨ plays a relatively minor role not known; however, it has been suggested to promote a in the bypass of UV lesions or of abasic sites. Thus, a copy choice-type of DNAsynthesis (Torres-Ramos et al. rev3⌬ mutant displays only a modest increase in UV 2002). Pol␩ is very efficient at replicating through a cis- sensitivity in yeast (Johnson et al. 1992), and the discon- syn thymine–thymine (T–T) dimer, and steady-state ki- tinuities that are formed in DNAstrands synthesized netic studies have shown that it replicates through the from UV-damaged templates become fully repaired on two Ts of this DNAlesion with the same efficiency and subsequent incubation in a strain lacking Pol␨ (Torres- accuracy as it replicates through undamaged Ts (Johnson Ramos et al. 2002). In addition, introduction of the rev3⌬ mutation into the yeast apn1⌬ apn2⌬ strain, defective in the removal of abasic sites, causes no significant increase 1Corresponding author. in sensitivity to the alkylating agent methyl methane E-MAIL [email protected]; FAX (409) 747-8608. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ sulfonate (Johnson et al. 1998). gad.1048303. In the absence of any extraneous DNAdamage, the GENES & DEVELOPMENT 17:77–87 © 2003 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/03 $5.00; www.genesdev.org 77 Downloaded from genesdev.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press Johnson et al. rev3⌬ mutation in yeast confers no obvious adverse ef- out the subsequent extension reaction. Pol␨, on the other fect on cell viability or growth. Disruption of the REV3 hand, is able to replicate through this DNAlesion, and it gene in mice, however, causes early embryonic lethality does so by inserting an Aopposite it and by efficiently (Bemark et al. 2000; Esposito et al. 2000; Wittschieben et extending from the inserted nucleotide. Although Pol␩ al. 2000). This observation raised the possibility that can replicate through the Tg lesion, it is quite inefficient Pol␨ plays a more critical role in TLS through DNAle- at both the insertion and extension steps. Importantly, sions than had been envisaged based on the genetic stud- and quite unexpectedly, we found that of the three Rad6– ies carried out in yeast thus far. Rad18-dependent lesion bypass pathways, only the Pol␨ Free radical-induced DNAdamage makes a substantial pathway is necessary for Tg bypass, whereas the Pol␩- contribution to the spontaneous mutational burden, and and Rad5-dependent pathways are dispensable. More- consequently, it has been implicated in carcinogenesis over, Pol␨ predominantly promotes the error-free bypass and aging (Ames et al. 1993). Hydroxyl radicals, the prin- of this DNAlesion. These findings indicate that rather cipal DNA-damaging species formed from aerobic respi- than merely playing a minor role in the mutagenic by- ration or from exposure to chemical oxidizing agents or pass of DNAlesions, for certain DNAlesions, Pol ␨ pro- ionizing radiation, interact with the thymine or cytosine vides the primary if not the sole means of their bypass. residues, principally at the 5,6 double bond, leading to Consequently, efficient error-free TLS by Pol␨ could be the conversion of thymine to thymine glycol (5,6-dihy- crucial for maintaining the speed of rapid cell divisions roxy-5,6-dihydrothymine; Tg; Fig. 1A) and of cytosine to during early embryonic development, thereby account- cytosine glycol (Cg). Tg is a major stable product of thy- ing for the indispensability of this polymerase in higher mine modification in vitro and in vivo (Teoule et al. eukaryotes. 1974; Cathcart et al. 1984), whereas Cg is unstable and deaminates to uracil glycol (Ug; Wagner et al. 1992). Cg can also lead to the formation of two other stable prod- ucts, 5-hydroxycytosine (5-OHC; Dizdaroglu and Simic Results 1984; Teoule 1987; Wagner et al. 1992), and 5-hydroxy- Replication ofDNA containing a Tg by yeast DNA uracil (5-OHU; Wagner et al. 1992). polymerases ␦, ␨, and ␩ The oxidized pyrimidines are removed in yeast by two DNAglycosylases, Ntg1 and Ntg2 (Senturker et al. To examine the effect of a Tg on the DNAsynthetic 1998), that are related to Escherichia coli endonuclease activity of yeast DNApolymerases ␦, ␨, and ␩, we incor- III (Wallace 1997), which also functions in the removal of porated a site-specific Tg into a 76-nucleotide (nt) tem- these lesions. Nucleotide excision repair (NER) is a ver- plate, and annealed it to a 40-nt 5Ј 32P-labeled primer. satile repair system that functions in the removal of a DNAsynthesis was assayed in a running start reaction, diversity of DNAlesions, including UV-induced CPDs which allows for the synthesis of 5 nucleotides before and (6–4) dipyrimidine photoproducts, intrastrand and the Tg lesion is encountered (Fig. 1B). Although the rep- interstrand DNAcross-links, abasic sites, and oxidized licative polymerase Pol␦ is able to incorporate a nucleo- purines and pyrimidines (Huang et al. 1994; Sancar 1996; tide opposite the Tg lesion, it is unable to carry out DNA Reardon et al. 1997; Prakash and Prakash 2000; Torres- synthesis beyond it (Fig. 1B, lane 2). In comparison to Ramos et al. 2000). If the oxidized base is not repaired by undamaged DNA, wherein Pol␦ synthesized past ∼40% base excision repair (BER) or NER, it can be a block to of the unmodified T residue (Fig. 1B, lane 1), there was no replicative DNApolymerases and can be potentially le- observed bypass of the Tg (Fig. 1B, lane 2). The absence of thal, unless the progression of the replication fork is res- any significant stall site just before the lesion indicates cued by lesion bypass processes. Alternatively, the lesion that Pol␦ can efficiently insert a nucleotide opposite the would be mutagenic if it is bypassed by the replicative Tg lesion and that the inserted nucleotide is not subject DNApolymerases by mispairing. to its 3Ј→ 5Ј exonuclease activity. Of the oxidized pyrimidine bases that have been stud- Although Pol␨ is required for the mutagenic bypass of ied to date, only Tg presents a strong block to DNA DNAlesions, we previously showed that Pol ␨ is highly polymerases in vitro. For example, synthesis by E. coli inefficient at inserting nucleotides opposite a variety of DNApolymerase I Klenow fragment and T4 DNApoly- DNAlesions; rather, it promotes the bypass of lesions by merase is arrested at the site of the lesion; both polymer- extending from the nucleotides inserted opposite the le- ases incorporate the correct nucleotide Aopposite Tg, sion site by another polymerase. On the Tg-containing but no extension from the inserted nucleotide occurs substrate, however, Pol␨ is able to carry out unassisted (Clark and Beardsley 1987).
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