Parie Garg

DNA Polymerases that Propagate the Eukaryotic DNA Replication Fork

Abstract Three DNA polymerases are thought to function at the eukaryotic DNA replication fork. Currently, a coherent model has been derived for the composition and activities of the lagging strand machinery. RNA-DNA primers are initiated by DNA polymerase α -primase. Loading of the proliferating cell nuclear antigen, PCNA, dissociates DNA polymerase α and recruits DNA polymerase δ and the flap endonuclease FEN1 for elongation and in preparation for its requirement during maturation, respectively. Nick translation by the strand displacement action of DNA polymerase δ, coupled with the nuclease action of FEN1, results in processive RNA degradation until a proper DNA nick is reached for closure by DNA ligase I. In the event of excessive strand displacement synthesis, other factors, such as the Dna2 nuclease/helicase, are required to trim excess flaps. Paradoxically, the composition and activity of the much simpler leading strand machinery has not been clearly established. The burden of evidence suggests that DNA polymerase ε normally replicates this strand, but under conditions of dysfunction, DNA polymerase δ may substitute.

The Pol32 Subunit of DNA Polymerase δ Contains Separable Domains for Processive Replication and Proliferating Cell Nuclear Antigen (PCNA) Binding

We have carried out a domain analysis of POL32, the third subunit of Saccharomyces cerevisiae DNA polymerase δ (Pol δ). Interactions with POL31, the second subunit of Pol δ, are specified by the amino-terminal 92 amino acids, whereas interactions with the replication clamp proliferating cell nuclear antigen (PCNA, POL30) reside at the extreme carboxyl-terminal region. Pol32 binding, in vivo and in vitro, to the large subunit of DNA polymerase α, POL1, requires the carboxyl-proximal region of Pol32. The amino-terminal region of Pol32 is essential for damage-induced mutagenesis. However, the presence of its carboxyl-terminal PCNA-binding domain enhances the efficiency of mutagenesis, particularly at high loads of DNA damage. In vitro, in the absence of effector DNA, the PCNA-binding domain of Pol32 is essential for PCNA-Pol δ interactions. However, this domain has minimal importance for processive DNA synthesis by the ternary DNA-PCNA-Pol δ complex. Rather, processivity is determined by PCNA-binding domains located in the Pol3 and/or Pol31 subunits. Using diagnostic PCNA mutants, we show that during DNA synthesis the carboxyl-terminal domain of Pol32 interacts with the carboxyl-terminal region of PCNA, whereas interactions of the other subunit(s) of Pol δ localize largely to a hydrophobic pocket at the interdomain connector loop region of PCNA.

A Ubiquitin-binding Motif in the Translesion DNA Polymerase Rev1 Mediates Its Essential Functional Interaction with Ubiquitinated Proliferating Cell Nuclear Antigen in Response to DNA Damage

During normal DNA replication, the proliferating cell nuclear antigen (PCNA) enhances the processivity of DNA polymerases at the replication fork. When DNA damage is encountered, PCNA is monoubiquitinated on Lys-164 by the Rad6–Rad18 complex as the initiating step of translesion synthesis. DNA damage bypass by the translesion synthesis polymerase Rev1 is enhanced by the presence of ubiquitinated PCNA. Here we have carried out a mutational analysis of Rev1, and we have identified the functional domain in the C terminus of Rev1 that mediates interactions with PCNA. We show that a unique motif within this domain binds the ubiquitin moiety of ubiquitinated PCNA. Point mutations within this ubiquitin-binding motif of Rev1 (L821A,P822A,I825A) abolish its functional interaction with ubiquitinated PCNA in vitro and strongly attenuate damage-induced mutagenesis in vivo. Taken together, these studies suggest a specific mechanism by which the interaction between Rev1 and ubiquitinated PCNA is stabilized during the DNA damage response.

The fidelity of DNA synthesis by yeast DNA polymerase zeta alone and with accessory proteins

DNA polymerase zeta (pol ζ) participates in several DNA transactions in eukaryotic cells that increase spontaneous and damage-induced mutagenesis. To better understand this central role in mutagenesis in vivo, here we report the fidelity of DNA synthesis in vitro by yeast pol ζ alone and with RFC, PCNA and RPA. Overall, the accessory proteins have little effect on the fidelity of pol ζ. Pol ζ is relatively accurate for single base insertion/deletion errors. However, the average base substitution fidelity of pol ζ is substantially lower than that of homologous B family pols α, δ and ɛ. Pol ζ is particularly error prone for substitutions in specific sequence contexts and generates multiple single base errors clustered in short patches at a rate that is unprecedented in comparison with other polymerases. The unique error specificity of pol ζ in vitro is consistent with Pol ζ-dependent mutagenic specificity reported in vivo. This fact, combined with the high rate of single base substitution errors and complex mutations observed here, indicates that pol ζ contributes to mutagenesis in vivo not only by extending mismatches made by other polymerases, but also by directly generating its own mismatches and then extending them.

Proliferating Cell Nuclear Antigen Promotes Translesion Synthesis by DNA Polymerase ζ

DNA polymerase ζ (Pol ζ), a heterodimer of Rev3 and Rev7, is essential for DNA damage provoked mutagenesis in eukaryotes. DNA polymerases that function in a processive complex with the replication clamp proliferating cell nuclear antigen (PCNA) have been shown to possess a close match to the consensus PCNA-binding motif QxxLxxFF. This consensus motif is lacking in either subunit of Pol ζ, yet its activity is stimulated by PCNA. In particular, translesion synthesis of UV damage-containing DNA is dramatically stimulated by PCNA such that translesion synthesis rates are comparable with replication rates by Pol ζ on undamaged DNA. PCNA also stimulated translesion synthesis of a model abasic site by Pol ζ. Efficient PCNA stimulation required that PCNA was prevented from sliding off the damage-containing model oligonucleotide template-primer through the use of biotin-streptavidin bumpers or other blocks. Under those experimental conditions, facile bypass of the abasic site was also detected by DNA polymerase δ or η (Rad30). The yeast DNA damage checkpoint clamp, consisting of Rad17, Mec3, and Ddc1, and an ortholog of human 9-1-1, has been implicated in damage-induced mutagenesis. However, this checkpoint clamp did not stimulate translesion synthesis by Pol ζ or by DNA polymerase δ.

A novel function of DNA polymerase ζ regulated by PCNA

DNA polymerase ζ (Polζ) participates in translesion DNA synthesis and is involved in the generation of the majority of mutations induced by DNA damage. The mechanisms that license access of Polζ to the primer terminus and regulate the extent of its participation in genome replication are poorly understood. The Polζ‐dependent damage‐induced mutagenesis requires monoubiquitination of proliferating cell nuclear antigen (PCNA) that is triggered by exposure to mutagens. We show that Polζ contributes to DNA replication and causes mutagenesis not only in response to DNA damage but also in response to malfunction of normal replicative machinery due to mutations in replication genes. These replication defects lead to ubiquitination of PCNA even in the absence of DNA damage. Unlike damage‐induced mutagenesis, the Polζ‐dependent spontaneous mutagenesis in replication mutants is reduced in strains defective in both ubiquitination and sumoylation of Lys164 of PCNA. Additionally, studies of a PCNA mutant defective for functional interactions with Polζ, but not for monoubiquitination by the Rad6/Rad18 complex demonstrate a role for PCNA in regulating the mutagenic activity of Polζ separate from its modification at Lys164.

The efficiency and fidelity of 8-oxo-guanine bypass by DNA polymerases δ and η

A DNA lesion created by oxidative stress is 7,8-dihydro-8-oxo-guanine (8-oxoG). Because 8-oxoG can mispair with adenine during DNA synthesis, it is of interest to understand the efficiency and fidelity of 8-oxoG bypass by DNA polymerases. We quantify bypass parameters for two DNA polymerases implicated in 8-oxoG bypass, Pols δ and η. Yeast Pol δ and yeast Pol η both bypass 8-oxoG and misincorporate adenine during bypass. However, yeast Pol η is 10-fold more efficient than Pol δ, and following bypass Pol η switches to less processive synthesis, similar to that observed during bypass of a cis-syn thymine-thymine dimer. Moreover, yeast Pol η is at least 10-fold more accurate than yeast Pol δ during 8-oxoG bypass. These differences are maintained in the presence of the accessory proteins RFC, PCNA and RPA and are consistent with the established role of Pol η in suppressing ogg1-dependent mutagenesis in yeast. Surprisingly different results are obtained with human and mouse Pol η. Both mammalian enzymes bypass 8-oxoG efficiently, but they do so less processively, without a switch point and with much lower fidelity than yeast Pol η. The fact that yeast and mammalian Pol η have intrinsically different catalytic properties has potential biological implications.

The Multiple Biological Roles of the 3′→5′ Exonuclease of Saccharomyces cerevisiae DNA Polymerase δ Require Switching between the Polymerase and Exonuclease Domains

ABSTRACT Until recently, the only biological function attributed to the 3′→5′ exonuclease activity of DNA polymerases was proofreading of replication errors. Based on genetic and biochemical analysis of the 3′→5′ exonuclease of yeast DNA polymerase δ (Pol δ) we have discerned additional biological roles for this exonuclease in Okazaki fragment maturation and mismatch repair. We asked whether Pol δ exonuclease performs all these biological functions in association with the replicative complex or as an exonuclease separate from the replicating holoenzyme. We have identified yeast Pol δ mutants at Leu523 that are defective in processive DNA synthesis when the rate of misincorporation is high because of a deoxynucleoside triphosphate (dNTP) imbalance. Yet the mutants retain robust 3′→5′ exonuclease activity. Based on biochemical studies, the mutant enzymes appear to be impaired in switching of the nascent 3′ end between the polymerase and the exonuclease sites, resulting in severely impaired biological functions. Mutation rates and spectra and synergistic interactions of the pol3-L523X mutations with msh2, exo1, and rad27/fen1 defects were indistinguishable from those observed with previously studied exonuclease-defective mutants of the Pol δ. We conclude that the three biological functions of the 3′→5′ exonuclease addressed in this study are performed intramolecularly within the replicating holoenzyme.

How the Cell Deals with DNA Nicks

During lagging strand DNA replication, the Okazaki fragment maturation machinery is requiredto degrade the initiator RNA with high speed and efficiency, and to generate with great accuracya proper DNA nick for closure by DNA ligase. Several operational parameters are important ingenerating and maintaining a ligatable nick. These are the strand opening capacity of the laggingstrand DNA polymerase ? (Pol ?), and its ability to limit strand opening to that of a fewnucleotides. In the presence of the flap endonuclease FEN1, Pol ? rapidly hands off the strandopenedproduct for cutting by FEN1, while in its absence, the ability of DNA polymerase ? toswitch to its 3’-5’-exonuclease domain in order to degrade back to the nick position is importantin maintaining a ligatable nick. This regulatory system has a built-in redundancy so thatdysfunction of one of these activities can be tolerated in the cell. However, further dysfunctionleads to uncontrolled strand displacement synthesis with deleterious consequences, as is revealedby genetic studies of exonuclease-defective mutants of S. cerevisiae Pol ?. These sameparameters are also important for other DNA metabolic processes, such as base excision repair,that depend on Pol ? for synthesis.