Eiji Ohashi

Interaction of hREV1 with three human Y-family DNA polymerases

Polκ is one of many DNA polymerases involved in translesion DNA synthesis (TLS). It belongs to the Y‐family of polymerases along with Polη, Polι and hREV1. Unlike Polη encoded by the xeroderma pigmentosum variant (XPV) gene, Polκ is unable to bypass UV‐induced DNA damage in vitro, but it is able to bypass benzo[a]pyrene (B[a]P)‐adducted guanines accurately and efficiently. In an attempt to identify factor(s) targeting Polκ to its cognate DNA lesion(s), we searched for Polκ‐interacting proteins by using the yeast two‐hybrid assay. We found that Polκ interacts with a C‐terminal region of hREV1. Polη and Polι were also found to interact with the same region of hREV1. The interaction between Polκ and hREV1 was confirmed by pull‐down and co‐immunoprecipitation assays. The C‐terminal region of hREV1 is known to interact with hREV7, a non‐catalytic subunit of Polζ that is another structurally unrelated TLS enzyme, and we show that Polκ and hREV7 bind to the same C‐terminal region of hREV1. Thus, our results suggest that hREV1 plays a pivotal role in the multi‐enzyme, multi‐step process of translesion DNA synthesis.

Fidelity and processivity of DNA synthesis by DNA polymerase κ, the product of the human DINB1 gene

Mammalian DNA polymerase κ (pol κ), a member of the UmuC/DinB nucleotidyl transferase superfamily, has been implicated in spontaneous mutagenesis. Here we show that human pol κ copies undamaged DNA with average single-base substitution and deletion error rates of 7 × 10−3 and 2 × 10−3, respectively. These error rates are high when compared to those of most other DNA polymerases. pol κ also has unusual error specificity, producing a high proportion of T·CMP mispairs and deleting and adding non-reiterated nucleotides at extraordinary rates. Unlike other members of the UmuC/DinB family, pol κ can processively synthesize chains of 25 or more nucleotides. This moderate processivity may reflect a contribution of C-terminal residues, which include two zinc clusters. The very low fidelity and moderate processivity of pol κ is novel in comparison to any previously studied DNA polymerase, and is consistent with a role in spontaneous mutagenesis.

Structural Basis for Novel Interactions between Human Translesion Synthesis Polymerases and Proliferating Cell Nuclear Antigen

Translesion synthesis (TLS) is a DNA damage tolerance mechanism that allows continued DNA synthesis, even in the presence of damaged DNA templates. Mammals have multiple DNA polymerases specialized for TLS, including Polη, Polι, and Polκ. These enzymes show preferential bypass for different lesions. Proliferating cell nuclear antigen (PCNA), which functions as a sliding clamp for the replicative polymerase Polδ, also interacts with the three TLS polymerases. Although many PCNA-binding proteins have a highly conserved sequence termed the PCNA-interacting protein box (PIP-box), Polη, Polι, and Polκ have a noncanonical PIP-box sequence. In response to DNA damage, Lys-164 of PCNA undergoes ubiquitination by the RAD6-RAD18 complex, and the ubiquitination is considered to facilitate TLS. Consistent with this, these three TLS polymerases have one or two ubiquitin binding domains and are recruited to replication forks via interactions with ubiquitinated PCNA involving the noncanonical PIP-box and ubiquitin binding domain. However, it is unclear how these TLS polymerases interact with PCNA. To address the structural basis for interactions between different TLS polymerases and PCNA, we determined crystal structures of PCNA bound to peptides containing the noncanonical PIP-box of these polymerases. We show that the three PIP-box peptides interact with PCNA in different ways, both from one another and from canonical PIP-box peptides. Especially, the PIP-box of Polι adopts a novel structure. Furthermore, these structures enable us to speculate how these TLS polymerases interact with Lys-164-monoubiquitinated PCNA. Our results will provide clues to understanding the mechanism of preferential recruitment of TLS polymerases to the stalled forks.

Identification of a novel REV1-interacting motif necessary for DNA polymerase κ function

When a replicative DNA polymerase (Pol) is stalled by damaged DNA, a “polymerase switch” recruits specialized translesion synthesis (TLS) DNA polymerase(s) to sites of damage. Mammalian cells have several TLS DNA polymerases, including the four Y‐family enzymes (Polη, Polι, Polκ and REV1) that share multiple primary sequence motifs, but show preferential bypass of different DNA lesions. REV1 interacts with Polη, Polι, and Polκ and therefore appears to play a central role during TLS in vivo. Here we have investigated the molecular basis for interactions between REV1 and Polκ. We have identified novel REV1‐interacting regions (RIRs) present in Polκ, Polι and Polη. Within the RIRs, the presence of two consecutive phenylalanines (FF) is essential for REV1‐binding. The consensus sequence for REV1‐binding is denoted by x‐x‐x‐F‐F‐y‐y‐y‐y (x, no specific residue and y, no specific residue but not proline). Our results identify structural requirements that are necessary for FF‐flanking residues to confer interactions with REV1. A Polκ mutant lacking REV1‐binding activity did not complement the genotoxin‐sensitivity of Polk‐null mouse embryonic fibroblast cells, thereby demonstrating that the REV1‐interaction is essential for Polκ function in vivo.

Interaction with DNA polymerase η is required for nuclear accumulation of REV1 and suppression of spontaneous mutations in human cells

Defects in the gene encoding human Poleta result in xeroderma pigmentosum variant (XP-V), an inherited cancer-prone syndrome. Poleta catalyzes efficient and accurate translesion DNA synthesis (TLS) past UV-induced lesions. In addition to Poleta, human cells have multiple TLS polymerases such as Poliota, Polkappa, Polzeta and REV1. REV1 physically interacts with other TLS polymerases, but the physiological relevance of the interaction remains unclear. Here we developed an antibody that detects the endogenous REV1 protein and found that human cells contain about 60,000 of REV1 molecules per cell as well as Poleta. In un-irradiated cells, formation of nuclear foci by ectopically expressed REV1 was enhanced by the co-expression of Poleta. Importantly, the endogenous REV1 protein accumulated at the UV-irradiated areas of nuclei in Poleta-expressing cells but not in Poleta-deficient XP-V cells. UV-irradiation induced nuclear foci of REV1 and Poleta proteins in both S-phase and G1 cells, suggesting that these proteins may function both during and outside S phase. We reconstituted XP-V cells with wild-type Poleta or with Poleta mutants harboring substitutions in phenylalanine residues critical for interaction with REV1. The REV1-interaction-deficient Poleta mutant failed to promote REV1 accumulation at sites of UV-irradiation, yet (similar to wild-type Poleta) corrected the UV sensitivity of XP-V cells and suppressed UV-induced mutations. Interestingly however, spontaneous mutations of XP-V cells were only partially suppressed by the REV1-interaction deficient mutant of Poleta. Thus, Poleta-REV1 interactions prevent spontaneous mutations, probably by promoting accurate TLS past endogenous DNA lesions, while the interaction is dispensable for accurate Poleta-mediated TLS of UV-induced lesions.

Penicilliols A and B, novel inhibitors specific to mammalian Y-family DNA polymerases

Penicilliols A (1) and B (2) are novel 5-methoxy-3(2H)-furanones isolated from cultures of a fungus (Penicillium daleae K.M. Zalessky) derived from a sea moss, and their structures were determined by spectroscopic analyses. These compounds selectively inhibited activities of eukaryotic Y-family DNA polymerases (pols) (i.e., pols eta, iota and kappa), and compound 1 was a stronger inhibitor than compound 2. Among mammalian Y-family pols, mouse pol iota activity was most strongly inhibited by compounds 1 and 2, with IC(50) values of 19.8 and 32.5 microM, respectively. On the other hand, activities of many other pols, such as A-family (i.e., pol gamma), B-family (i.e., pols alpha, delta and epsilon) or X-family (i.e., pols beta, lambda and terminal deoxynucleotidyl transferase), and some DNA metabolic enzymes, such as calf primase of pol alpha, human immunodeficiency virus type-1 (HIV-1) reverse transcriptase, human telomerase, T7 RNA polymerase, mouse IMP dehydrogenase (type II), human topoisomerases I and II, T4 polynucleotide kinase or bovine deoxyribonuclease I, are not influenced by these compounds. In conclusion, this is the first report on potent inhibitors of mammalian Y-family pols.

The Vital Role of Polymerase ζ and REV1 in Mutagenic, but Not Correct, DNA Synthesis across Benzo[a]pyrene-dG and Recruitment of Polymerase ζ by REV1 to Replication-stalled Site

Background: dG lesion derived from potent carcinogen benzo[a]pyrene causes mutations through DNA replication. Results: Pol ζ and REV1 are essential to mutagenic, but not accurate, translesion DNA synthesis. Conclusion: DNA synthesis across identical DNA damage can be catalyzed by a different set of polymerases. Significance: The results have revealed an important role for DNA polymerases, pol ζ and REV1, in inducing mutations. The DNA synthesis across DNA lesions, termed translesion synthesis (TLS), is a complex process influenced by various factors. To investigate this process in mammalian cells, we examined TLS across a benzo[a]pyrene dihydrodiol epoxide-derived dG adduct (BPDE-dG) using a plasmid bearing a single BPDE-dG and genetically engineered mouse embryonic fibroblasts (MEFs). In wild-type MEFs, TLS was extremely miscoding (>90%) with G → T transversions being predominant. Knockout of the Rev1 gene decreased both the TLS efficiency and the miscoding frequency. Knockout of the Rev3L gene, coding for the catalytic subunit of pol ζ, caused even greater decreases in these two TLS parameters; almost all residual TLS were error-free. Thus, REV1 and pol ζ are critical to mutagenic, but not accurate, TLS across BPDE-dG. The introduction of human REV1 cDNA into Rev1−/− MEFs restored the mutagenic TLS, but a REV1 mutant lacking the C terminus did not. Yeast and mammalian three-hybrid assays revealed that the REV7 subunit of pol ζ mediated the interaction between REV3 and the REV1 C terminus. These results support the hypothesis that REV1 recruits pol ζ through the interaction with REV7. Our results also predict the existence of a minor REV1-independent pol ζ recruitment pathway. Finally, although mutagenic TLS across BPDE-dG largely depends on RAD18, experiments using Polk−/− Polh−/− Poli−/− triple-gene knockout MEFs unexpectedly revealed that another polymerase(s) could insert a nucleotide opposite BPDE-dG. This indicates that a non-Y family polymerase(s) can insert a nucleotide opposite BPDE-dG, but the subsequent extension from miscoding termini depends on REV1-polζ in a RAD18-dependent manner.

Casein kinase 2-dependent phosphorylation of human Rad9 mediates the interaction between human Rad9-Hus1-Rad1 complex and TopBP1.

The checkpoint clamp Rad9‐Hus1‐Rad1 (9‐1‐1) is loaded by the Rad17–RFC complex onto chromatin after DNA damage and plays a key role in the ATR‐dependent checkpoint activation. Here, we demonstrate that in vitro casein kinase 2 (CK2) specifically interacts with human 9‐1‐1 and phosphorylates serines 341 and 387 (Ser‐341 and Ser‐387) in the C‐terminal tail of Rad9. Interestingly, phosphorylated Ser‐387 has previously been reported to be required for interacting with a checkpoint mediator TopBP1. Indeed, 9‐1‐1 purified from Escherichia coli and phosphorylated in vitro by CK2 physically interacts with TopBP1. Further analyses showed that phosphorylation at both serine residues occurs in vivo and is required for the efficient interaction with TopBP1 in vitro. Furthermore, when over‐expressed in HeLa cells, a mutant Rad9 harboring phospho‐deficient substitutions at both Ser‐341 and Ser‐387 residues causes hypersensitivity to UV and methyl methane sulfonate (MMS). Our observations suggest that CK2 plays a crucial role in the ATR‐dependent checkpoint pathway through its ability to phosphorylate Ser‐341 and Ser‐387 of the Rad9 subunit of the 9‐1‐1 complex.

Stable Interaction between the Human Proliferating Cell Nuclear Antigen Loader Complex Ctf18-Replication Factor C (RFC) and DNA Polymerase ϵ Is Mediated by the Cohesion-specific Subunits, Ctf18, Dcc1, and Ctf8

One of the proliferating cell nuclear antigen loader complexes, Ctf18-replication factor C (RFC), is involved in sister chromatid cohesion. To examine its relationship with factors involved in DNA replication, we performed a proteomics analysis of Ctf18-interacting proteins. We found that Ctf18 interacts with a replicative DNA polymerase, DNA polymerase ϵ (pol ϵ). Co-immunoprecipitation with recombinant Ctf18-RFC and pol ϵ demonstrated that their binding is direct and mediated by two distinct interactions, one weak and one stable. Three subunits that are specifically required for cohesion in yeast, Ctf18, Dcc1, and Ctf8, formed a trimeric complex (18-1-8) and together enabled stable binding with pol ϵ. The C-terminal 23-amino acid stretch of Ctf18 was necessary for the trimeric association of 18-1-8 and was required for the stable interaction. The weak interaction was observed with alternative loader complexes including Ctf18-RFC(5), which lacks Dcc1 and Ctf8, suggesting that the common loader structures, including the RFC small subunits (RFC2–5), are responsible for the weak interaction. The two interaction modes, mediated through distinguishable structures of Ctf18-RFC, both occurred through the N-terminal half of pol ϵ, which includes the catalytic domain. The addition of Ctf18-RFC or Ctf18-RFC(5) to the DNA synthesis reaction caused partial inhibition and stimulation, respectively. Thus, Ctf18-RFC has multiple interactions with pol ϵ that promote polymorphic modulation of DNA synthesis. We propose that their interaction alters the DNA synthesis mode to enable the replication fork to cooperate with the establishment of cohesion.

Error‐prone and inefficient replication across 8‐hydroxyguanine (8‐oxoguanine) in human and mouse ras gene fragments by DNA polymerase κ

Using fragments of human c‐Ha‐ras and mouse Ha‐ras1 genes containing 8‐hydroxyguanine (8‐OH‐G) in hypermutagenic codon 12, we analyzed the kinetics of DNA synthesis catalyzed by human Polκ. This translesion DNA polymerase, belonging to the Y‐family, was found to be moderately inhibited by the presence of 8‐OH‐G on either mouse or human templates. From our previous results, inhibition of various polymerases by 8‐OH‐G increases in the following order: Polη < Polκ < Polβ < Polα, showing that major replicative and repair polymerases are more sensitive to this lesion than enzymes belonging to the Y‐family. In the direct mutagenesis experiments, Polκ was found to be more mutagenic than Polη studied previously: it inserted dAMP more efficiently than dCMP opposite 8‐OH‐G. Polκ was also able to cause indirect mispair (‘action‐at‐a‐distance’ mutagenesis), this effect being more distinct on mouse templates. Two adjacent 8‐OH‐G residues in codon 12 inhibited Polκ moderately and induced misincorporation of dAMP. However, this effect was not comparable to the strong relaxation of the enzyme specificity, observed previously in the case of Polη. Polκ catalyzed incorporation (and misincorporation of dAMP) much more efficiently on mouse templates, human DNA fragments being distinctly worse substrates. Interestingly, in direct mutagenesis systems, the preference for dAMP over dCMP was nearly the same on mouse and human templates.