Discrimination against the cytosine analog tC by Escherichia coli DNA polymerase IV DinB

The cytosine analog 1,3-diaza-2-oxophenothiazine (tC) is a fluorescent nucleotide that forms Watson-Crick base pairs with dG. The Klenow fragment of DNA polymerase I (an A-family polymerase) can efficiently bypass tC on the template strand and incorporate deoxyribose-triphosphate-tC into the growing primer terminus. Y-family DNA polymerases are known for their ability to accommodate bulky lesions and modified bases and to replicate beyond such nonstandard DNA structures in a process known as translesion synthesis. We probed the ability of the Escherichia coli Y-family DNA polymerase DinB (Pol IV) to copy DNA containing tC and to incorporate tC into a growing DNA strand. DinB selectively adds dGTP across from tC in template DNA but cannot extend beyond the newly formed G:tC base pair. However, we find that DinB incorporates the tC deoxyribonucleotide triphosphate opposite template G and extends from tC. Therefore, DinB displays asymmetry in terms of its ability to discriminate against the modification of the DNA template compared to the incoming nucleotide. In addition, our finding that DinB (a lesion-bypass DNA polymerase) specifically discriminates against tC in the template strand may suggest that DinB discriminates against template modifications in the major groove of DNA.     

Translesion synthesis of acetylaminofluorene-dG adducts by DNA polymerase zeta is stimulated by yeast Rev1 protein

Translesion synthesis is an important mechanism in response to unrepaired DNA lesions during replication. The DNA polymerase ζ (Polζ) mutagenesis pathway is a major error-prone translesion synthesis mechanism requiring Polζ and Rev1. In addition to its dCMP transferase, a non-catalytic function of Rev1 is suspected in cellular response to certain types of DNA lesions. However, it is not well understood about the non-catalytic function of Rev1 in translesion synthesis. We have analyzed the role of Rev1 in translesion synthesis of an acetylaminofluorene (AAF)-dG DNA adduct. Purified yeast Rev1 was essentially unresponsive to a template AAF-dG DNA adduct, in contrast to its efficient C insertion opposite a template 1,N⁶-ethenoadenine adduct. Purified yeast Polζ was very inefficient in the bypass of the AAF-dG adduct. Combining Rev1 and Polζ, however, led to a synergistic effect on translesion synthesis. Rev1 protein enhanced Polζ-catalyzed nucleotide insertion opposite the AAF-dG adduct and strongly stimulated Polζ-catalyzed extension from opposite the lesion. Rev1 also stimulated the deficient synthesis by Polζ at the very end of undamaged DNA templates. Deleting the C-terminal 205 aa of Rev1 did not affect its dCMP transferase activity, but abolished its stimulatory activity on Polζ-catalyzed extension from opposite the AAF-dG adduct. These results suggest that translesion synthesis of AAF-dG adducts by Polζ is stimulated by Rev1 protein in yeast. Consistent with the in vitro results, both Polζ and Rev1 were found to be equally important for error-prone translesion synthesis across from AAF-dG DNA adducts in yeast cells.     

The human REV1 gene codes for a DNA template-dependent dCMP transferase.

DNA is frequently damaged by various physical and chemical agents. DNA damage can lead to mutations during replication. In the yeast Saccharomyces cerevisiae, the damage-induced mutagenesis pathway requires the Rev1 protein. We have isolated a human cDNA homologous to the yeast REV1 gene. The human REV1 cDNA consists of 4255 bp and codes for a protein of 1251 amino acid residues with a calculated molecular weight of 138 248 Da. The human REV1 gene is localized between 2q11.1 and 2q11.2. We show that the human REV1 protein is a dCMP transferase that specifically inserts a dCMP residue opposite a DNA template G. In addition, the human REV1 transferase is able to efficiently and specifically insert a dCMP opposite a DNA template apurinic/apyrimidinic (AP) site or a uracil residue. These results suggest that the REV1 transferase may play a critical role during mutagenic translesion DNA synthesis bypassing a template AP site in human cells. Consistent with its role as a fundamental mutagenic protein, the REV1 gene is ubiquitously expressed in various human tissues.     

A non-catalytic function of Rev1 in translesion DNA synthesis and mutagenesis is mediated by its stable interaction with Rad5

DNA damage tolerance consisting of template switching and translesion synthesis is a major cellular mechanism in response to unrepaired DNA lesions during replication. The Rev1 pathway constitutes the major mechanism of translesion synthesis and base damage-induced mutagenesis in model cell systems. Rev1 is a dCMP transferase, but additionally plays non-catalytic functions in translesion synthesis. Using the yeast model system, we attempted to gain further insights into the non-catalytic functions of Rev1. Rev1 stably interacts with Rad5 (a central component of the template switching pathway) via the C-terminal region of Rev1 and the N-terminal region of Rad5. Supporting functional significance of this interaction, both the Rev1 pathway and Rad5 are required for translesion synthesis and mutagenesis of 1,N⁶-ethenoadenine. Furthermore, disrupting the Rev1–Rad5 interaction by mutating Rev1 did not affect its dCMP transferase, but led to inactivation of the Rev1 non-catalytic function in translesion synthesis of UV-induced DNA damage. Deletion analysis revealed that the C-terminal 21-amino acid sequence of Rev1 is uniquely required for its interaction with Rad5 and is essential for its non-catalytic function. Deletion analysis additionally implicated a C-terminal region of Rev1 in its negative regulation. These results show that a non-catalytic function of Rev1 in translesion synthesis and mutagenesis is mediated by its interaction with Rad5.     

Translesion DNA polymerases Pol ζ, Pol η, Pol ι, Pol κ and Rev1 are not essential for repeat-induced point mutation inNeurospora crassa

Pol ζ, Pol η, Pol ι, Pol κ and Rev1 are specialized DNA polymerases that are able to synthesize DNA across a damaged template. DNA synthesis by such translesion polymerases can be mutagenic due to the miscoding nature of most damaged nucleotides. In fact, many mutational and hypermutational processes in systems ranging from yeast to mammals have been traced to the activity of such polymerases. We show however, that the translesion polymerases are dispensable for repeat-induced point mutation (RIP) inNeurospora crassa. Additionally, we demonstrate that theupr-1 gene, which encodes the catalytic subunit of Pol ζ, is a highly polymorphic locus in Neurospora. Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession Nos DQ 231523, DQ 231524, DQ 235021, DQ 235525-DQ 235541, DQ 240287, DQ 240288, DQ 354228, DQ 354235-DQ 354237, DQ 386416-DQ 386422, DQ 387872, DQ494492-DQ494503 and DQ417211-DQ417220.     

Two translesion synthesis DNA polymerase genes, AtPOLH and AtREV1, are involved in development and UV light resistance in Arabidopsis

Plants are continually exposed to external and internal DNA-damaging agents. Although lesions can be removed by different repair processes, damages often remain in the DNA during replication. Synthesis of template damages requires the replacement of replicative enzymes by translesion synthesis polymerases, which are able to perform DNA synthesis opposite specific lesions. These proteins, in contrast to replicative polymerases, operate at low processivity and fidelity. DNA polymerase η and Rev 1 are two proteins found in eukaryotes that are involved in translesion DNA synthesis. In Arabidopsis, DNA polymerase η and Rev 1 are encoded by AtPOLH and AtREV1 genes, respectively. Transgenic plants over-expressing AtPOLH showed increased resistance to ultraviolet light. Only plants with moderate AtREV1 over-expression were obtained, indicating that this enzyme could be toxic at high levels. Transgenic plants that over-expressed or disrupted AtREV1 showed reduced germination percentage, but the former exhibited a higher stem growth rate than the wild type during development.     

Novel role for the C terminus of Saccharomyces cerevisiae Rev1 in mediating protein-protein interactions

The Saccharomyces cerevisiae REV3/7-encoded polymerase zeta and Rev1 are central to the replicative bypass of DNA lesions, a process called translesion synthesis (TLS). While yeast polymerase zeta extends from distorted DNA structures, Rev1 predominantly incorporates C residues from across a template G and a variety of DNA lesions. Intriguingly, Rev1 catalytic activity does not appear to be required for TLS. Instead, yeast Rev1 is thought to participate in TLS by facilitating protein-protein interactions via an N-terminal BRCT motif. In addition, higher eukaryotic homologs of Rev1 possess a C terminus that interacts with other TLS polymerases. Due to a lack of sequence similarity, the yeast Rev1 C-terminal region, located after the polymerase domain, had initially been thought not to play a role in TLS. Here, we report that elevated levels of the yeast Rev1 C terminus confer a strong dominant-negative effect on viability and induced mutagenesis after DNA damage, highlighting the crucial role that the C terminus plays in DNA damage tolerance. We show that this phenotype requires REV7 and, using immunoprecipitations from crude extracts, demonstrate that, in addition to the polymerase-associated domain, the extreme Rev1 C terminus and the BRCT region of Rev1 mediate interactions with Rev7.     

Complex formation of yeast Rev1 and Rev7 proteins: a novel role for the polymerase-associated domain

The Rev1 protein of Saccharomyces cerevisiae functions in translesion synthesis (TLS) together with DNA polymerase (Pol) zeta, which is comprised of the Rev3 catalytic and the Rev7 accessory subunits. Rev1, a member of the Y family of Pols, differs from other members in its high degree of specificity for incorporating a C opposite template G as well as opposite an abasic site. Although Rev1 is indispensable for Polzeta-dependent TLS, its DNA synthetic activity is not required for many of the Polzeta-dependent lesion bypass events. This observation has suggested a structural role for Rev1 in this process. Here we show that in yeast, Rev1 forms a stable complex with Rev7, and the two proteins copurify. Importantly, the polymerase-associated domain (PAD) of Rev1 mediates its binding to Rev7. These observations reveal a novel role for the PAD region of Rev1 in protein-protein interactions, and they raise the possibility of a similar involvement of the PAD of other Y family Pols in protein-protein interactions. We discuss the possible roles of Rev1 versus the Rev1-Rev7 complex in TLS.     

Human DNA polymerase N (POLN) is a low fidelity enzyme capable of error-free bypass of 5S-thymine glycol

Human DNA polymerase N (POLN or pol nu) is the most recently discovered nuclear DNA polymerase in the human genome. It is an A-family DNA polymerase related to Escherichia coli pol I, human POLQ, and Drosophila Mus308. We report the first purification of the recombinant enzyme and examination of its biochemical properties, as a step toward understanding the functions of POLN. Unusual for an A-family DNA polymerase, POLN is a low fidelity enzyme incorporating T opposite template G with a frequency of 0.45 and G opposite template T with a frequency of 0.021. The frequency of misincorporation of T opposite template G is higher than any other known DNA polymerase. POLN has a processivity of DNA synthesis (1-100 nucleotides) similar to the exonuclease-deficient Klenow fragment of E. coli pol I, is inhibited by dideoxynucleotides, and resistant to aphidicolin. The strand displacement activity of POLN was higher than exonuclease-deficient Klenow fragment. Furthermore, POLN can perform translesion synthesis past thymine glycol, a common endogenous and radiation-induced product of reactive oxygen species damage to DNA. Thymine glycol blocks DNA synthesis by most DNA polymerases, but POLN was particularly adept at efficient and accurate translesion synthesis past a 5S-thymine glycol.     

The catalytic function of the Rev1 dCMP transferase is required in a lesion-specific manner for translesion synthesis and base damage-induced mutagenesis

The Rev1-Polζ pathway is believed to be the major mechanism of translesion DNA synthesis and base damage-induced mutagenesis in eukaryotes. While it is widely believed that Rev1 plays a non-catalytic function in translesion synthesis, the role of its dCMP transferase activity remains uncertain. To determine the relevance of its catalytic function in translesion synthesis, we separated the Rev1 dCMP transferase activity from its non-catalytic function in yeast. This was achieved by mutating two conserved amino acid residues in the catalytic domain of Rev1, i.e. D467A/E468A, where its catalytic function was abolished but its non-catalytic function remained intact. In this mutant strain, whereas translesion synthesis and mutagenesis of UV radiation were fully functional, those of a site-specific 1,N⁶-ethenoadenine were severely deficient. Specifically, the predominant A→G mutations resulting from C insertion opposite the lesion were abolished. Therefore, translesion synthesis and mutagenesis of 1,N⁶-ethenoadenine require the catalytic function of the Rev1 dCMP transferase, in contrast to those of UV lesions, which only require the non-catalytic function of Rev1. These results show that the catalytic function of the Rev1 dCMP transferase is required in a lesion-specific manner for translesion synthesis and base damage-induced mutagenesis.     

Overexpression of DNA polymerase zeta reduces the mitochondrial mutability caused by pathological mutations in DNA polymerase gamma in yeast

In yeast, DNA polymerase zeta (Rev3 and Rev7) and Rev1, involved in the error-prone translesion synthesis during replication of nuclear DNA, localize also in mitochondria. We show that overexpression of Rev3 reduced the mtDNA extended mutability caused by a subclass of pathological mutations in Mip1, the yeast mitochondrial DNA polymerase orthologous to human Pol gamma. This beneficial effect was synergistic with the effect achieved by increasing the dNTPs pools. Since overexpression of Rev3 is detrimental for nuclear DNA mutability, we constructed a mutant Rev3 isoform unable to migrate into the nucleus: its overexpression reduced mtDNA mutability without increasing the nuclear one.     

Cryo-EM reveals conformational flexibility in apo DNA polymerase ζ

The translesion synthesis (TLS) DNA polymerases Rev1 and Polζ function together in DNA lesion bypass during DNA replication, acting as nucleotide inserter and extender polymerases, respectively. While the structural characterization of the Saccharomyces cerevisiae Polζ in its DNA-bound state has illuminated how this enzyme synthesizes DNA, a mechanistic understanding of TLS also requires probing conformational changes associated with DNA- and Rev1 binding. Here, we used single-particle cryo-electron microscopy to determine the structure of the apo Polζ holoenzyme. We show that compared with its DNA-bound state, apo Polζ displays enhanced flexibility that correlates with concerted motions associated with expansion of the Polζ DNA-binding channel upon DNA binding. We also identified a lysine residue that obstructs the DNA-binding channel in apo Polζ, suggesting a gating mechanism. The Polζ subunit Rev7 is a hub protein that directly binds Rev1 and is a component of several other protein complexes such as the shieldin DNA double-strand break repair complex. We analyzed the molecular interactions of budding yeast Rev7 in the context of Polζ and those of human Rev7 in the context of shieldin using a crystal structure of Rev7 bound to a fragment of the shieldin-3 protein. Overall, our study provides new insights into Polζ mechanism of action and the manner in which Rev7 recognizes partner proteins.     

Pre-steady-state kinetic studies of protein-template-directed nucleotide incorporation by the yeast Rev1 protein

The yeast Rev1 protein (Rev1p) is a member of the Y family of DNA polymerases that specifically catalyzes the incorporation of C opposite template G and several types of DNA damage. The X-ray crystal structure of the Rev1p-DNA-dCTP ternary complex showed that Rev1p utilizes an unusual mechanism of nucleotide incorporation whereby the template residue is displaced from the DNA double helix and the side chain of Arg-324 forms hydrogen bonds with the incoming dCTP. To better understand the impact of this protein-template-directed mechanism on the thermodynamics and kinetics of nucleotide incorporation, we have carried out pre-steady-state kinetic studies with Rev1p. Interestingly, we found that Rev1p's specificity for incorporating C is achieved solely at the initial nucleotide-binding step, not at the subsequent nucleotide-incorporation step. In this respect, Rev1p differs from all previously investigated DNA polymerases. We also found that the base occupying the template position in the DNA impacts nucleotide incorporation more at the nucleotide-binding step than at the nucleotide-incorporation step. These studies provide the first detailed, quantitative information regarding the mechanistic impact of protein-template-directed nucleotide incorporation by Rev1p. Moreover, on the basis of these findings and on structures of the unrelated Escherichia coli MutM DNA glycosylase, we suggest the possible structures for the ternary complexes of Rev1p with the other incoming dNTPs.     

Complex formation of yeast Rev1 with DNA polymerase eta

In Saccharomyces cerevisiae, Rev1 functions in translesion DNA synthesis (TLS) together with polymerase ζ (Polζ), comprised of the Rev3 catalytic and Rev7 accessory subunits. Rev1 plays an indispensable structural role in promoting Polζ function, and deletion of the Rev1-C terminal region that is involved in physical interactions with Rev3 inactivates Polζ function in TLS. In humans, however, Rev1 has been shown to physically interact with the Y-family polymerases Polη, Polι, and Polκ, and the Rev1 C terminus mediates these interactions. Since all the available genetic and biochemical evidence in yeast support the requirement of Rev1 as a structural element for Polζ and not for Polη, these observations have raised the possibility that in its structural role, Rev1 has diverged between yeast and humans. Here we show that although in yeast a stable Rev1-Polη complex can be formed, this complex formation involves the polymerase-associated domain of Rev1 and not the Rev1 C terminus as in humans. We also found that the DNA synthesis activity of Rev1 is enhanced in this complex. We discuss the implications of these and other observations for the possible divergence of Rev1's structural role between yeast and humans.     

Rev1 is essential for DNA damage tolerance and non-templated immunoglobulin gene mutation in a vertebrate cell line

The majority of DNA damage-induced mutagenesis in the yeast Saccharomyces cerevisiae arises as a result of translesion replication. This process is critically dependent on the deoxycytidyl transferase Rev1p, which forms a complex with the subunits of DNA polymerase zeta, Rev3p and Rev7p. To examine the role of Rev1 in vertebrate mutagenesis and the DNA damage response, we disrupted the gene in DT40 cells. Rev1-deficient DT40 grow slowly and are sensitive to a wide range of DNA-damaging agents. Homologous recombination repair is likely to be intact as basal and damage induced sister chromatid exchange and immunoglobulin gene conversion are unaffected. How ever, the mutant cells show a markedly reduced level of non-templated immunoglobulin gene mutation, indicating a defect in translesion bypass. Furthermore, ultraviolet exposure results in marked chromosome breakage, suggesting that replication gaps created in the absence of Rev1 cannot be efficiently repaired by recombination. Thus, Rev1-dependent translesion bypass and mutagenesis is likely to be a trade-off for the ability to complete replication of a damaged template and thereby maintain genome integrity.     

Protein-template-directed synthesis across an acrolein-derived DNA adduct by yeast Rev1 DNA polymerase

Acrolein is generated as the end product of lipid peroxidation and is also a ubiquitous environmental pollutant. Its reaction with the N2 of guanine leads to a cyclic gamma-HOPdG adduct that presents a block to normal replication. We show here that yeast Rev1 incorporates the correct nucleotide C opposite a permanently ring-closed form of gamma-HOPdG (PdG) with nearly the same efficiency as opposite an undamaged G. The structural basis of this action lies in the eviction of the PdG adduct from the Rev1 active site, and the pairing of incoming dCTP with a "surrogate" arginine residue. We also show that yeast Polzeta can carry out the subsequent extension reaction. Together, our studies reveal how the exocyclic PdG adduct is accommodated in a DNA polymerase active site, and they show that the combined action of Rev1 and Polzeta provides for accurate and efficient synthesis through this potentially carcinogenic DNA lesion.     

Translesion synthesis by the UmuC family of DNA polymerases.

Translesion synthesis is an important cellular mechanism to overcome replication blockage by DNA damage. To copy damaged DNA templates during replication, specialized DNA polymerases are required. Translesion synthesis can be error-free or error-prone. From E. coli to humans, error-prone translesion synthesis constitutes a major mechanism of DNA damage-induced mutagenesis. As a response to DNA damage during replication, translesion synthesis contributes to cell survival and induced mutagenesis. During 1999-2000, the UmuC superfamily had emerged, which consists of the following prototypic members: the E. coli UmuC, the E. coli DinB, the yeast Rad30, the human RAD30B, and the yeast Rev1. The corresponding biochemical activities are DNA polymerases V, IV, eta, iota, and dCMP transferase, respectively. Recent studies of the UmuC superfamily are summarized and evidence is presented suggesting that this family of DNA polymerases is involved in translesion DNA synthesis.     

DNA repair: DNA polymerase zeta and Rev1 break in

DNA polymerase zeta and Rev1 play key roles in replication past DNA lesions. New work shows that the yeast checkpoint kinase Mec1 recruits a complex consisting of polymerase zeta and Rev1 to DNA double-strand breaks. This study highlights the role of polymerases that mediate translesion synthesis in the response to DNA double-strand breaks.     

Proliferating cell nuclear antigen promotes translesion synthesis by DNA polymerase zeta

DNA polymerase zeta (Pol zeta), 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 zeta, 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 zeta on undamaged DNA. PCNA also stimulated translesion synthesis of a model abasic site by Pol zeta. 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 delta or eta (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 zeta or by DNA polymerase delta.     

Role of DNA polymerase eta in the bypass of abasic sites in yeast cells

Abasic (AP) sites are major DNA lesions and are highly mutagenic. AP site-induced mutagenesis largely depends on translesion synthesis. We have examined the role of DNA polymerase η (Polη) in translesion synthesis of AP sites by replicating a plasmid containing a site-specific AP site in yeast cells. In wild-type cells, AP site bypass resulted in preferred C insertion (62%) over A insertion (21%), as well as −1 deletion (3%), and complex event (14%) containing multiple mutations. In cells lacking Polη (rad30), Rev1, Polζ (rev3), and both Polη and Polζ, translesion synthesis was reduced to 30%, 30%, 15% and 3% of the wild-type level, respectively. C insertion opposite the AP site was reduced in rad30 mutant cells and was abolished in cells lacking Rev1 or Polζ, but significant A insertion was still detected in these mutant cells. While purified yeast Polα effectively inserted an A opposite the AP site in vitro, purified yeast Polδ was much less effective in A insertion opposite the lesion due to its 3′→5′ proofreading exonuclease activity. Purified yeast Polη performed extension synthesis from the primer 3′ A opposite the lesion. These results show that Polη is involved in translesion synthesis of AP sites in yeast cells, and suggest that an important role of Polη is to catalyze extension following A insertion opposite the lesion. Consistent with these conclusions, rad30 mutant cells were sensitive to methyl methanesulfonate (MMS), and rev1 rad30 or rev3 rad30 double mutant cells were synergistically more sensitive to MMS than the respective single mutant strains.