Small molecule scaffolds that disrupt the Rev1-CT/RIR protein-protein interaction

Translesion synthesis (TLS) is a DNA damage tolerance mechanism that allows replicative bypass of DNA lesions, including DNA adducts formed by cancer chemotherapeutics. Previous studies demonstrated that suppression of TLS can increase sensitivity of cancer cells to first-line chemotherapeutics and decrease mutagenesis linked to the onset of chemoresistance, marking the TLS pathway as an emerging therapeutic target. TLS is mediated by a heteroprotein complex consisting of specialized DNA polymerases, including the Y-family DNA polymerase Rev1. Previously, we developed a screening assay to identify the first small molecules that disrupt the protein–protein interaction between the C-terminal domain of Rev1 (Rev1-CT) and the Rev1-interacting region (RIR) present in multiple DNA polymerases involved in TLS. Herein we report additional hit scaffolds that inhibit this key TLS PPI. In addition, through a series of biochemical, computational, and cellular studies we have identified preliminary structure–activity relationships and determined initial pharmacokinetic parameters for our original hits.     

Translesion synthesis in Escherichia coli: lessons from the NarI mutation hot spot

Duplication of DNA containing damaged bases is a challenge to DNA polymerases that normally replicate with high speed, high accuracy and high processivity undamaged templates only. When a replicative DNA polymerase encounters a chemically altered base that it is unable to copy, a process called translesion synthesis (TLS) takes place during which the replicative polymerase is transiently replaced by a so-called specialized or lesion bypass polymerase. In addition to the central players that are the replicative and translesion DNA polymerases, TLS pathways involve accessory factors such as the general replication processivity factor (i.e. the beta-clamp in prokaryotes and PCNA in eukaryotes). In Escherichia coli, besides the beta-clamp, RecA plays a fundamental role as a co-factor of Pol V the major bypass polymerase in this organism. An integrated view of TLS pathways necessarily requires both genetic and biochemical studies. In this review we will attempt to summarize the insights into TLS gained over the last 25 years by studying a frameshift mutation hot spot, the NarI site. This site was initially discovered by serendipity when establishing a forward mutation spectrum induced by a chemical hepatocarcinogen, N-2-acetylaminofluorene (AAF). Indeed, this chemical carcinogen covalently binds to DNA forming adducts with guanine residues. When bound to G* in the NarI site, 5'-GGCG*CC-, AAF induces the loss of the G*pC dinucleotide at a frequency that is approximately 10(7)-fold higher than the spontaneous frequency. In vivo studies showed that the NarI mutation hot spot is neither restricted to the NarI sequence itself, nor to the carcinogen AAF. Instead, the hot spot requires a sequence containing at least two GpC repeats and any of a family of aromatic amides and nitro aromatic compounds that form a large class of human carcinogens. Genetic analysis initially revealed that the NarI frameshift pathway is SOS dependent but umuDC (i.e. Pol V) independent. More recently, DNA Pol II was identified as the enzyme responsible of this frameshift pathway. Concurrently the AAF adduct in the NarI site can be bypassed in an error-free way by Pol V. The NarI site thus offers a unique possibility to study the interplay between two specialized DNA polymerases, Pol II and Pol V, that can both extend replication intermediates formed when the replicative Pol III dissociates in the vicinity of the damage. Full reconstitution of the two pathways led us to highlight a key feature for TLS pathways, namely that it is critical the specialized DNA polymerase synthesizes, during the course of a single binding event, a patch of DNA synthesis (TLS patch) that is long enough as to "hide the lesion induced distortion" from the proofreading activity upon reloading of the replicative DNA polymerase (or any exonuclease that may get access to the primer when the specialized DNA polymerase detaches). The beta-clamp, to which all DNA polymerases bind, plays a critical role in allowing the specialized DNA polymerases to synthesize TLS patches that are long enough to resist such "external proofreading" activities.     

Translesion DNA polymerases in eukaryotes: what makes them tick?

Life as we know it, simply would not exist without DNA replication. All living organisms utilize a complex machinery to duplicate their genomes and the central role in this machinery belongs to replicative DNA polymerases, enzymes that are specifically designed to copy DNA. “Hassle-free” DNA duplication exists only in an ideal world, while in real life, it is constantly threatened by a myriad of diverse challenges. Among the most pressing obstacles that replicative polymerases often cannot overcome by themselves are lesions that distort the structure of DNA. Despite elaborate systems that cells utilize to cleanse their genomes of damaged DNA, repair is often incomplete. The persistence of DNA lesions obstructing the cellular replicases can have deleterious consequences. One of the mechanisms allowing cells to complete replication is “Translesion DNA Synthesis (TLS)”. TLS is intrinsically error-prone, but apparently, the potential downside of increased mutagenesis is a healthier outcome for the cell than incomplete replication. Although most of the currently identified eukaryotic DNA polymerases have been implicated in TLS, the best characterized are those belonging to the “Y-family” of DNA polymerases (pols η, ι, κ and Rev1), which are thought to play major roles in the TLS of persisting DNA lesions in coordination with the B-family polymerase, pol ζ. In this review, we summarize the unique features of these DNA polymerases by mainly focusing on their biochemical and structural characteristics, as well as potential protein–protein interactions with other critical factors affecting TLS regulation.     

Co-localization in replication foci and interaction of human Y-family members, DNA polymerase pol eta and REVl protein

The progress of replicative DNA polymerases along the replication fork may be impeded by the presence of lesions in the genome. One way to circumvent such hurdles involves the recruitment of specialized DNA polymerases that perform limited incorporation of nucleotides in the vicinity of the damaged site. This process entails DNA polymerase switch between replicative and specialized DNA polymerases. Five eukaryotic proteins can carry out translesion synthesis (TLS) of damaged DNA in vitro, DNA polymerases zeta, eta, iota, and kappa, and REV1. To identify novel proteins that interact with hpol eta, we performed a yeast two-hybrid screen. In this paper, we show that hREV1 interacts with hpol eta as well as with hpol kappa and poorly with hpol iota. Furthermore, cellular localization analysis demonstrates that hREV1 is present, with hpol eta in replication factories at stalled replication forks and is tightly associated with nuclear structures. This hREV1 nuclear localization occurs independently of the presence of hpol eta. Taken together, our data suggest a central role for hREV1 as a scaffold that recruits DNA polymerases involved in TLS.     

Trading places: how do DNA polymerases switch during translesion DNA synthesis?

The replicative bypass of base damage in DNA (translesion DNA synthesis [TLS]) is a ubiquitous mechanism for relieving arrested DNA replication. The process requires multiple polymerase switching events during which the high-fidelity DNA polymerase in the replication machinery arrested at the primer terminus is replaced by one or more polymerases that are specialized for TLS. When replicative bypass is fully completed, the primer terminus is once again occupied by high-fidelity polymerases in the replicative machinery. This review addresses recent advances in our understanding of DNA polymerase switching during TLS in bacteria such as E. coli and in lower and higher eukaryotes.     

PCNA trimer instability inhibits translesion synthesis by DNA polymerase η and by DNA polymerase δ

Translesion synthesis (TLS), the process by which DNA polymerases replicate through DNA lesions, is the source of most DNA damage-induced mutations. Sometimes TLS is carried out by replicative polymerases that have evolved to synthesize DNA on non-damaged templates. Most of the time, however, TLS is carried out by specialized translesion polymerases that have evolved to synthesize DNA on damaged templates. TLS requires the mono-ubiquitylation of the replication accessory factor proliferating cell nuclear antigen (PCNA). PCNA and ubiquitin-modified PCNA (UbPCNA) stimulate TLS by replicative and translesion polymerases. Two mutant forms of PCNA, one with an E113G substitution and one with a G178S substitution, support normal cell growth but inhibit TLS thereby reducing mutagenesis in yeast. A re-examination of the structures of both mutant PCNA proteins revealed substantial disruptions of the subunit interface that forms the PCNA trimer. Both mutant proteins have reduced trimer stability with the G178S substitution causing a more severe defect. The mutant forms of PCNA and UbPCNA do not stimulate TLS of an abasic site by either replicative Pol δ or translesion Pol η. Normal replication by Pol η was also impacted, but normal replication by Pol δ was much less affected. These findings support a model in which reduced trimer stability causes these mutant PCNA proteins to occasionally undergo conformational changes that compromise their ability to stimulate TLS by both replicative and translesion polymerases.     

DNA damage induced Pol η recruitment takes place independently of the cell cycle phase

When DNA is damaged in cells progressing through S phase, replication blockage can be avoided by TLS (Translesion DNA synthesis). This is an auxiliary replication mechanism that relies on the function of specialized polymerases that accomplish DNA damage bypass. Intriguingly, recent evidence has linked TLS polymerases to processes that can also take place outside S phase such as nucleotide excision repair (NER). Here we show that Pol η is recruited to UV-induced DNA lesions in cells outside S phase including cells permanently arrested in G1. This observation was confirmed by different strategies including global UV irradiation, local UV irradiation and local multi-photon laser irradiation of single nuclei in living cells. The potential connection between Pol η recruitment to DNA lesions outside S phase and NER was further evaluated. Interestingly, the recruitment of Pol η to damage sites outside S phase did not depend on active NER, as UV-induced focus formation occurred normally in XPA, XPG and XPF deficient fibroblasts. Our data reveals that the re-localization of the TLS polymerase Pol η to photo-lesions might be temporally and mechanistically uncoupled from replicative DNA synthesis and from DNA damage processing.     

Crosstalk between replicative and translesional DNA polymerases: PDIP38 interacts directly with Polη

Replicative DNA polymerases duplicate genomes in a very efficient and accurate mode. However their progression can be blocked by DNA lesions since they are unable to accommodate bulky damaged bases in their active site. In response to replication blockage, monoubiquitination of PCNA promotes the switch between replicative and specialized polymerases proficient to overcome the obstacle. In this study, we characterize novel connections between proteins involved in replication and TransLesion Synthesis (TLS). We demonstrate that PDIP38 (Polδ interacting protein of 38 kDa) directly interacts with the TLS polymerase Polη. Interestingly, the region of Polη interacting with PDIP38 is found to be located within the ubiquitin-binding zinc finger domain (UBZ) of Polη. We show that the depletion of PDIP38 increases the number of cells with Polη foci in the absence of DNA damage and diminishes cell survival after UV irradiation. In addition, PDIP38 is able to interact directly not only with Polη but also with the specialized polymerases Rev1 and Polζ (via Rev7). We thus suggest that PDIP38 serves as a mediator protein helping TLS Pols to transiently replace replicative polymerases at damaged sites.     

Xeroderma pigmentosum variant and error-prone DNA polymerases

Replicative DNA synthesis is a faithful event which requires undamaged DNA and high fidelity DNA polymerases. If unrepaired damage remains in the template DNA during replication, specialised low fidelity DNA polymerases synthesises DNA past lesions (translesion synthesis, TLS). Current evidence suggests that the polymerase switch from replicative to translesion polymerases might be mediated by post-translational modifications involving ubiquitination processes. One of these TLS polymerases, polymerase eta carries out TLS past UV photoproducts and is deficient in the variant form of xeroderma pigmentosum (XP-V). The dramatic proneness to skin cancer of XP-V individuals highlights the importance of this DNA polymerase in cancer avoidance. The UV hypermutability of XP-V cells suggests that, in the absence of a functional poleta, UV-induced lesions are bypassed by inaccurate DNA polymerase(s) which remain to be identified.     

Defining the position of the switches between replicative and bypass DNA polymerases

Cells contain specialized DNA polymerases that are able to copy past lesions with an associated risk of generating mutations, the major cause of cancer. Here, we reconstitute translesion synthesis (TLS) using the replicative (Pol III) and major bypass (Pol V) DNA polymerases from Escherichia coli in the presence of accessory factors. When the replicative polymerase disconnects from the template in the vicinity of a lesion, Pol V binds the blocked replication intermediate and forms a stable complex by means of a dual interaction with the tip of the RecA filament and the beta-clamp, the processivity factor donated by the blocked Pol III holoenzyme. Both interactions are required to confer to Pol V the processivity that will allow it synthesize, in a single binding event, a TLS patch long enough to support further extension by Pol III. In the absence of these accessory factors, the patch synthesized by Pol V is too short, being degraded by the Pol III-associated exonuclease activity that senses the distortion induced by the lesion, thus leading to an aborted bypass process.     

Translesion synthesis in mammalian cells

DNA damage blocks the progression of the replication fork. In order to circumvent the damaged bases, cells employ specialized low stringency DNA polymerases, which are able to carry out translesion synthesis (TLS) past different types of damage. The five polymerases used in TLS in human cells have different substrate specificities, enabling them to deal with many different types of damaged bases. PCNA plays a central role in recruiting the TLS polymerases and effecting the polymerase switch from replicative to TLS polymerase. When the fork is blocked PCNA gets ubiquitinated. This increases its affinity for the TLS polymerases, which all have novel ubiquitin-binding motifs, thereby facilitating their engagement at the stalled fork to effect TLS.     

Mismatch repair protein MSH2 regulates translesion DNA synthesis following exposure of cells to UV radiation

Translesion DNA synthesis (TLS) can use specialized DNA polymerases to insert and/or extend nucleotides across lesions, thereby limiting stalled replication fork collapse and the potential for cell death. Recent studies have shown that monoubiquitinated proliferating cell nuclear antigen (PCNA) plays an important role in recruitment of Y-family TLS polymerases to stalled replication forks after DNA damage treatment. To explore the possible roles of other factors that regulate the ultraviolet (UV)-induced assembly of specialized DNA polymerases at arrested replication forks, we performed immunoprecipitation experiments combined with mass spectrometry and established that DNA polymerase kappa (Polκ) can partner with MSH2, an important mismatch repair protein associated with hereditary non-polyposis colorectal cancer. We found that depletion of MSH2 impairs PCNA monoubiquitination and the formation of foci containing Polκ and other TLS polymerases after UV irradiation of cells. Interestingly, expression of MSH2 in Rad18-deficient cells increased UV-induced Polκ and REV1 focus formation without detectable changes in PCNA monoubiquitination, indicating that MSH2 can regulate post-UV focus formation by specialized DNA polymerases in both PCNA monoubiquitination-dependent and -independent fashions. Moreover, we observed that MSH2 can facilitate TLS across cyclobutane pyrimidine dimers photoproducts in living cells, presenting a novel role of MSH2 in post-UV cellular responses.     

Translesion synthesis: Y-family polymerases and the polymerase switch

Replicative DNA polymerases are blocked at DNA lesions. Synthesis past DNA damage requires the replacement of the replicative polymerase by one of a group of specialised translesion synthesis (TLS) polymerases, most of which belong to the Y-family. Each of these has different substrate specificities for different types of damage. In eukaryotes mono-ubiquitination of PCNA plays a crucial role in the switch from replicative to TLS polymerases at stalled forks. All the Y-family polymerases have ubiquitin binding sites that increase their binding affinity for ubiquitinated PCNA at the sites of stalled forks.     

Multiple two-polymerase mechanisms in mammalian translesion DNA synthesis

The encounter of replication forks with DNA lesions may lead to fork arrest and/or the formation of single-stranded gaps. A major strategy to cope with these replication irregularities is translesion DNA replication (TLS), in which specialized error-prone DNA polymerases bypass the blocking lesions. Recent studies suggest that TLS across a particular DNA lesion may involve as many as four different TLS polymerases, acting in two-polymerase reactions in which insertion by a particular polymerase is followed by extension by another polymerase. Insertion determines the accuracy and mutagenic specificity of the TLS reaction, and is carried out by one of several polymerases such as polη, polκ or polι. In contrast, extension is carried out primarily by polζ. In cells from XPV patients, which are deficient in TLS across cyclobutane pyrimidine dimers (CPD) due to a deficiency in polη, TLS is carried out by at least two backup reactions each involving two polymerases: One reaction involves polκ and polζ, and the other polι and polζ. These mechanisms may also assist polη in normal cells under an excessive amount of UV lesions.     

Postreplication repair mechanisms in the presence of DNA adducts in Escherichia coli

During bacterial replication, DNA polymerases may encounter DNA lesions that block processive DNA synthesis. Uncoupling the replicative helicase from the stalled DNA polymerase results in the formation of single-stranded DNA (ssDNA) gaps, which are repaired by postreplication repair (PRR), a process that involves at least three mechanisms that collectively remove, circumvent or bypass lesions. RecA mediated excision repair (RAMER) and homologous recombination (HR) are strand-exchange mechanisms that appear to be the predominant strategies for gap repair in the absence of prolonged SOS induction. During RAMER, RecA mediates pairing of damaged ssDNA with an undamaged homologous duplex and subsequent exchange of strands between the damaged and undamaged DNA. Repair of the lesion occurs in the context of the strand-exchange product and is initiated by UvrABC excinuclease; the resulting patch is filled by DNA synthesis using the complementary strand of the homologous duplex as a template. HR uses a complementary strand of an undamaged homologous duplex as a transient template for DNA synthesis. HR requires the formation and resolution of Holliday junctions, and is a mechanism to circumvent the lesion; lesions persisting in one of the daughter DNA duplexes will normally be repaired prior to subsequent rounds of replication/cell division. Translesion DNA Synthesis (TLS) does not involve strand-exchange mechanisms; it is carried out by specialized DNA polymerases that are able to catalyze nucleotide incorporation opposite lesions that cannot be bypassed by high-fidelity replicative polymerases. Maximum levels of TLS occur during prolonged SOS induction generally associated with increased mutagenesis. RAMER, HR and TLS are alternative mechanisms for processing a common intermediate-the ssDNA gap containing a RecA nucleofilament. The actual pathway that is utilized will be strongly influenced by multiple factors, including the blocking/coding capacity of the lesion, the nature of the gene products that can be assembled at the ssDNA gap, the availability of a homologous partner for RAMER and HR, and protein:protein interactions and post-translational modifications that modulate the mutagenic activity of Pol-IV and Pol-V.     

Differential Roles for DNA Polymerases Eta,Zeta, and REV1 in Lesion Bypass of Intrastrand versus Interstrand DNA Cross-Links

Translesion DNA synthesis (TLS) is a process whereby specialized DNA polymerases are recruited to bypass DNA lesions that would otherwise stall high-fidelity polymerases. We provide evidence that TLS across cisplatin intrastrand cross-links is performed by multiple translesion DNA polymerases. First, we determined that PCNA monoubiquitination by RAD18 is necessary for efficient bypass of cisplatin adducts by the TLS polymerases eta (Polη), REV1, and zeta (Polζ) based on the observations that depletion of these proteins individually leads to decreased cell survival, cell cycle arrest in S phase, and activation of the DNA damage response. Second, we showed that in addition to PCNA monoubiquitination by RAD18, the Fanconi anemia core complex is also important for recruitment of REV1 to stalled replication forks in cisplatin treated cells. Third, we present evidence that REV1 and Polζ are uniquely associated with protection against cisplatin and mitomycin C-induced chromosomal aberrations, and both are necessary for the timely resolution of DNA double-strand breaks associated with repair of DNA interstrand cross-links. Together, our findings indicate that REV1 and Polζ facilitate repair of interstrand cross-links independently of PCNA monoubiquitination and Polη, whereas RAD18 plus Polη, REV1, and Polζ are all necessary for replicative bypass of cisplatin intrastrand DNA cross-links.Maintenance of genomic integrity involves the activation of cell cycle checkpoints coupled with DNA repair. Despite these sophisticated mechanisms to remove DNA lesions prior to DNA replication, replication forks may inevitably encounter nonrepaired lesions that block high fidelity polymerases, potentially leading to replication fork instability, gaps in replicated DNA, and the generation of DNA double-strand breaks (DSBs). In order to preserve replication fork stability by allowing replication through polymerase blocking lesions, template DNA containing a damaged base or abasic site can be replicated through the actions of specialized translesion DNA synthesis (TLS) polymerases (61). A key event in the regulation of TLS is the monoubiquitination of PCNA, a homotrimeric protein that functions as an auxiliary factor for DNA polymerases (28, 31, 57, 60). The RAD6 (E2)-RAD18 (E3) complex specifically monoubiquitinates PCNA on Lys-164 in response to replication fork stalling. This event is thought to operate as a molecular switch from normal DNA replication to the TLS pathway based on the observations that association of Y-family TLS polymerases with monoubiquitinated PCNA is strengthened through the cooperative binding of one or more ubiquitin-binding domains (UBM or UBZ) plus a PCNA-interacting domain (6, 25).Extensive biochemical evidence suggests that replication through a large variety of lesions requires the sequential action of two TLS polymerases (44). The Y-family polymerase eta (Polη) plays a key role in the efficient and error-free bypass of cyclobutane pyrimidine (TT) dimers, one of the major lesions resulting from exposure to UV radiation (45). In contrast, Polη can only insert a nucleotide directly opposite other lesions and requires an additional TLS polymerase, such as Polζ, to extend beyond the insertion (45). Polζ is comprised of the REV3 catalytic subunit that shares homology with B-family polymerases plus the REV7 accessory subunit (34). Polζ is unusual compared to other TLS polymerases due to the fact that it is relatively efficient at extending beyond mispaired primer termini and nucleotides inserted opposite a variety of DNA lesions, although this may occur in a potentially mutagenic manner (45). Genetic evidence in yeast suggest that Polζ activity is regulated by the Y family REV1 polymerase (21). In addition to a UBM domain that directly interacts with monoubiquitinated PCNA, REV1 possesses an N-terminal BRCT motif that directly contacts PCNA and potentially other proteins (24, 25). In addition, REV1 possesses a unique protein interaction domain in its carboxy terminus that interacts with the Polζ accessory subunit, REV7, and other TLS polymerases, including Polη and the Polζ catalytic subunit, REV3 (1, 18, 23, 40, 58). The characterization of these protein-protein interaction domains has led to the proposal that REV1 facilitates polymerase switching from a polymerase that directly inserts a nucleotide opposite a damaged base and Polζ, which subsequently performs the extension step beyond the inserted nucleotide opposite the damaged base (21).In addition to facilitating direct lesion bypass and filling in postreplicative gaps in DNA, REV1 and Polζ may also play an important role in the repair of interstrand cross-links (46, 63). Deletion of REV1, REV3, or REV7 in chicken DT40 cells leads to remarkable hypersensitivity to a wide variety of genotoxic stresses, most notably cisplatin and other DNA cross-linking agents such as mitomycin C (MMC) (38, 41, 55, 56). The genetic epistasis observed between REV1, REV3, and the Fanconi anemia (FA) complementation group C (FANCC) gene for cisplatin sensitivity further implicates TLS in the interstrand cross-link repair pathway (38). Current models suggest that when two replication forks converge upon an interstrand cross-link, the MUS81-EME1 endonuclease recognizes and cleaves the resulting branched DNA structure by making an incision at one side of the interstrand cross-link creating a replication-associated DSB (26). The XPF-ERCC1 endonuclease uncouples the cross-linked cDNA strands by making an incision on the other side of the interstrand cross-link (37). Recent biochemical evidence suggests that Polζ performs DNA synthesis opposite the DNA strand containing the residual cross-link and this process may be necessary to prepare the daughter strand for subsequent homologous recombination repair of the replication-associated DSB (46).Agents that introduce intra- and interstrand cross-links are widely used in cancer chemotherapy, and thus understanding the means by which cells repair or cope with these lesions will be instrumental in identifying novel mechanisms leading to drug resistance and designing new agents refractory to DNA damage tolerance mechanisms. Polη, REV1, and Polζ have all been implicated in mediating TLS past cisplatin intrastrand cross-links since lowering their expression increases sensitivity and reduces cisplatin-induced mutagenesis in human cancer cells (2, 5, 12, 42, 62). Furthermore, biochemical and structural analyses of Polη identified this polymerase as being capable of efficiently inserting dCTP opposite the 3′dG of a 1,2-d(GpG) cisplatin intrastrand cross-link (3). Here, we demonstrate that RAD18, Polη, and REV1 all localized to sites of replication stress marked by PCNA and γ-H2AX foci after treatment of cells with cisplatin. However, REV1 focus formation is specifically dependent upon both RAD18 and a functional FA core complex, suggesting FA core proteins are also necessary for directing REV1 to cisplatin-induced stalled replication forks. In addition, depletion of RAD18, Polη, REV1, or Polζ proteins lead to the induction of cellular responses indicative of inefficient lesion bypass of cisplatin adducts. Unexpectedly, we found that REV1- or Polζ-depleted cells displayed a greater loss in cell viability and the accumulation of chromosome aberrations and failed to resolve DSBs after cisplatin treatment. These results lead us to hypothesize that REV1 and Polζ may be necessary for the repair of cisplatin interstrand cross-links in addition to performing lesion bypass of cisplatin intrastrand cross-links. In agreement with this concept, we found that REV1 and Polζ-depleted cells were uniquely hypersensitive to MMC, accumulated greater numbers of chromosome aberrations, and failed to resolve replication-associated DSBs induced by MMC treatment.Together our findings support a model where replicative bypass of cisplatin intrastrand cross-links requires cooperation of multiple TLS polymerases in mammalian cells and is triggered by PCNA monoubiquitination. Our results also provide evidence that REV1 and Polζ facilitate repair of interstrand cross-links in human cells, and this process is likely independent of PCNA monoubiquitination.     

Quantification of pyrophosphate as a universal approach to determine polymerase activity and assay polymerase inhibitors

The importance of DNA polymerases in biology and biotechnology, and their recognition as potential therapeutic targets, drives development of methods for deriving kinetic characteristics of polymerases and their propensity to perform polynucleotide synthesis over modified DNA templates. Among various polymerases, translesion synthesis (TLS) polymerases enable cells to avoid the cytotoxic stalling of replicative DNA polymerases at chemotherapy-induced DNA lesions, thereby leading to drug resistance. Identification of TLS inhibitors to overcome drug-resistance necessitates the development of appropriate high-throughput assays. Since polymerase-mediated DNA synthesis involves the release of inorganic pyrophosphate (PPi), we established a universal and fast method for monitoring the progress of DNA polymerases based on the quantification of PPi with a fluorescence-based assay that we coupled to in vitro primer extension reactions. The established assay has a nanomolar detection limit in PPi and enables the evaluation of single nucleotide incorporation and DNA synthesis progression kinetics. The results demonstrated that the developed assay is a reliable method for monitoring TLS and identifying nucleoside and nucleotide-based TLS inhibitors.     

Pivotal role of the beta-clamp in translesion DNA synthesis and mutagenesis in E. coli cells

The genetic information is continuously subjected to the attack by endogenous and exogenous chemical and physical carcinogens that damage the DNA template, thus compromising its biochemical functions. Despite the multiple and efficient DNA repair systems that have evolved to cope with the large variety of damages, some lesions may persist and, as a consequence, interfere with DNA replication. By essence, the damaged-DNA replication process (hereafter termed translesion synthesis or TLS) is a major source of point mutations and is therefore deeply involved in the onset of human diseases such as cancer. Recent identification of numerous DNA polymerases involved in TLS has shed new light onto the molecular mechanisms of mutagenesis. Here, we show that in vivo, both error-free and mutagenic bypass activities of the three DNA polymerases known to be involved in TLS in Escherichia coli (PolII, PolIV and PolV) strictly depend upon the integrity of small peptidic sequences identified as their beta-clamp binding motif. Thus, in addition to its crucial role as the processivity factor of the PolIII replicase, the beta-clamp plays a pivotal role during the TLS process.     

Def1 Promotes the Degradation of Pol3 for Polymerase Exchange to Occur During DNA-Damage–Induced Mutagenesis in Saccharomyces cerevisiae

DNA damages hinder the advance of replication forks because of the inability of the replicative polymerases to synthesize across most DNA lesions. Because stalled replication forks are prone to undergo DNA breakage and recombination that can lead to chromosomal rearrangements and cell death, cells possess different mechanisms to ensure the continuity of replication on damaged templates. Specialized, translesion synthesis (TLS) polymerases can take over synthesis at DNA damage sites. TLS polymerases synthesize DNA with a high error rate and are responsible for damage-induced mutagenesis, so their activity must be strictly regulated. However, the mechanism that allows their replacement of the replicative polymerase is unknown. Here, using protein complex purification and yeast genetic tools, we identify Def1 as a key factor for damage-induced mutagenesis in yeast. In in vivo experiments we demonstrate that upon DNA damage, Def1 promotes the ubiquitylation and subsequent proteasomal degradation of Pol3, the catalytic subunit of the replicative polymerase δ, whereas Pol31 and Pol32, the other two subunits of polymerase δ, are not affected. We also show that purified Pol31 and Pol32 can form a complex with the TLS polymerase Rev1. Our results imply that TLS polymerases carry out DNA lesion bypass only after the Def1-assisted removal of Pol3 from the stalled replication fork.     

NMR Structure and Dynamics of the C-Terminal Domain from Human Rev1 and Its Complex with Rev1 Interacting Region of DNA Polymerase η

Rev1 is a translesion synthesis (TLS) DNA polymerase essential for DNA damage tolerance in eukaryotes. In the process of TLS stalled high-fidelity replicative DNA polymerases are temporarily replaced by specialized TLS enzymes that can bypass sites of DNA damage (lesions), thus allowing replication to continue or postreplicational gaps to be filled. Despite its limited catalytic activity, human Rev1 plays a key role in TLS by serving as a scaffold that provides an access of Y-family TLS polymerases polη, ι, and κ to their cognate DNA lesions and facilitates their subsequent exchange to polζ that extends the distorted DNA primer-template. Rev1 interaction with the other major human TLS polymerases, polη, ι, κ, and the regulatory subunit Rev7 of polζ, is mediated by Rev1 C-terminal domain (Rev1-CT). We used NMR spectroscopy to determine the spatial structure of the Rev1-CT domain (residues 1157-1251) and its complex with Rev1 interacting region (RIR) from polη (residues 524-539). The domain forms a four-helix bundle with a well-structured N-terminal β-hairpin docking against helices 1 and 2, creating a binding pocket for the two conserved Phe residues of the RIR motif that upon binding folds into an α-helix. NMR spin-relaxation and NMR relaxation dispersion measurements suggest that free Rev1-CT and Rev1-CT/polη-RIR complex exhibit μs-ms conformational dynamics encompassing the RIR binding site, which might facilitate selection of the molecular configuration optimal for binding. These results offer new insights into the control of TLS in human cells by providing a structural basis for understanding the recognition of the Rev1-CT by Y-family DNA polymerases.