Jean-Sébastien Hoffmann

The human specialized DNA polymerases and non-B DNA: vital relationships to preserve genome integrity.

In addition to the canonical right-handed double helix, DNA molecule can adopt several other non-B DNA structures. Readily formed in the genome at specific DNA repetitive sequences, these secondary conformations present a distinctive challenge for progression of DNA replication forks. Impeding normal DNA synthesis, cruciforms, hairpins, H DNA, Z DNA and G4 DNA considerably impact the genome stability and in some instances play a causal role in disease development. Along with previously discovered dedicated DNA helicases, the specialized DNA polymerases emerge as major actors performing DNA synthesis through these distorted impediments. In their new role, they are facilitating DNA synthesis on replication stalling sites formed by non-B DNA structures and thereby helping the completion of DNA replication, a process otherwise crucial for preserving genome integrity and concluding normal cell division. This review summarizes the evidence gathered describing the function of specialized DNA polymerases in replicating DNA through non-B DNA structures.

Aberrant expression of alternative DNA polymerases: a source of mutator phenotype as well as replicative stress in cancer.

The cell life span depends on a subtle equilibrium between the accurate duplication of the genomic DNA and less stringent DNA transactions which allow cells to tolerate mutations associated with DNA damage. The physiological role of the alternative, specialized or TLS (translesion synthesis) DNA polymerases could be to favor the necessary flexibility of the replication machinery, by allowing DNA replication to occur even in the presence of blocking DNA damage. As these alternative DNA polymerases are inaccurate when replicating undamaged DNA, the regulation of their expression needs to be carefully controlled. Evidence in the literature supports that dysregulation of these error-prone enzymes contributes to the acquisition of a mutator phenotype that, along with defective cell cycle control or other genome stability pathways, could be a motor for accelerated tumor progression.

Proteomic Profiling Reveals a Specific Role for Translesion DNA Polymerase η in the Alternative Lengthening of Telomeres

SUMMARY Cancer cells rely on the activation of telomerase or the alternative lengthening of telomeres (ALT) pathways for telomere maintenance and survival. ALT involves homologous recombination (HR)-dependent exchange and/or HR-associated synthesis of telomeric DNA. Utilizing proximity-dependent biotinylation (BioID), we sought to determine the proteome of telomeres in cancer cells that employ these distinct telomere elongation mechanisms. Our analysis reveals that multiple DNA repair networks converge at ALT telomeres. These include the specialized translesion DNA synthesis (TLS) proteins FANCJ-RAD18-PCNA and, most notably, DNA polymerase eta (Polη). We observe that the depletion of Polη leads to increased ALT activity and late DNA polymerase δ (Polδ)-dependent synthesis of telomeric DNA in mitosis. We propose that Polη fulfills an important role in managing replicative stress at ALT telomeres, maintaining telomere recombination at tolerable levels and stimulating DNA synthesis by Polδ.

DNA polymerase beta from Trypanosoma cruzi is involved in kinetoplast DNA replication and repair of oxidative lesions.

Specific DNA repair pathways from Trypanosoma cruzi are believed to protect genomic DNA and kinetoplast DNA (kDNA) from mutations. Particular pathways are supposed to operate in order to repair nucleotides oxidized by reactive oxygen species (ROS) during parasite infection, being 7,8-dihydro-8-oxoguanine (8oxoG) a frequent and highly mutagenic base alteration. If unrepaired, 8oxoG can lead to cytotoxic base transversions during DNA replication. In mammals, DNA polymerase beta (Polβ) is mainly involved in base excision repair (BER) of oxidative damage. However its biological role in T. cruzi is still unknown. We show, by immunofluorescence localization, that T. cruzi DNA polymerase beta (Tcpolβ) is restricted to the antipodal sites of kDNA in replicative epimastigote and amastigote developmental stages, being strictly localized to kDNA antipodal sites between G1/S and early G2 phase in replicative epimastigotes. Nevertheless, this polymerase was detected inside the mitochondrial matrix of trypomastigote forms, which are not able to replicate in culture. Parasites over expressing Tcpolβ showed reduced levels of 8oxoG in kDNA and an increased survival after treatment with hydrogen peroxide when compared to control cells. However, this resistance was lost after treating Tcpolβ overexpressors with methoxiamine, a potent BER inhibitor. Curiously, a presumed DNA repair focus containing Tcpolβ was identified in the vicinity of kDNA of cultured wild type epimastigotes after treatment with hydrogen peroxide. Taken together our data suggest participation of Tcpolβ during kDNA replication and repair of oxidative DNA damage induced by genotoxic stress in this organelle.

The R438W polymorphism of human DNA polymerase lambda triggers cellular sensitivity to camptothecin by compromising the homologous recombination repair pathway

The human DNA polymerase lambda (Polλ) is a DNA repair polymerase, which is believed not only to play a role in base excision repair but also to contribute to DNA double-strand break repair by non-homologous end joining. We described here that cellular expression of the recently described natural polymorphic variant of Polλ, Polλ(R438W), affects the homologous recombination (HR) pathway and sister chromatid exchange (SCE) events. We show that the HR defect provoked by this polymorphism enhances cellular sensitivity to the anticancer agent camptothecin (CPT), most of whose DNA damage is repaired by HR. All these effects were dependent on the DNA polymerase activity of Polλ(R438W) as the expression of a catalytically inactive Polλ(R438W) did not affect either the HR and SCE frequencies or the cellular sensitivity to CPT. These results suggest that sensitivity to CPT could result from cancer-related mutation in specialized DNA repair polymerases.

Role of specialized DNA polymerases in the limitation of replicative stress and DNA damage transmission

Replication stress is a strong and early driving force for genomic instability and tumor development. Beside replicative DNA polymerases, an emerging group of specialized DNA polymerases is involved in the technical assistance of the replication machinery in order to prevent replicative stress and its deleterious consequences. During S-phase, altered progression of the replication fork by endogenous or exogenous impediments induces replicative stress, causing cells to reach mitosis with genomic regions not fully duplicated. Recently, specific mechanisms to resolve replication intermediates during mitosis with the aim of limiting DNA damage transmission to daughter cells have been identified. In this review, we detail the two major actions of specialized DNA polymerases that limit DNA damage transmission: the prevention of replicative stress by non-B DNA replication and the recovery of stalled replication forks.

Excess Polθ functions in response to replicative stress in homologous recombination-proficient cancer cells.

ABSTRACT DNA polymerase theta (Polθ) is a specialized A-family DNA polymerase that functions in processes such as translesion synthesis (TLS), DNA double-strand break repair and DNA replication timing. Overexpression of POLQ, the gene encoding Polθ, is a prognostic marker for an adverse outcome in a wide range of human cancers. While increased Polθ dosage was recently suggested to promote survival of homologous recombination (HR)-deficient cancer cells, it remains unclear whether POLQ overexpression could be also beneficial to HR-proficient cancer cells. By performing a short interfering (si)RNA screen in which genes encoding druggable proteins were knocked down in Polθ-overexpressing cells as a means to uncover genetic vulnerabilities associated with POLQ overexpression, we could not identify genes that were essential for viability in Polθ-overexpressing cells in normal growth conditions. We also showed that, upon external DNA replication stress, Polθ expression promotes cell survival and limits genetic instability. Finally, we report that POLQ expression correlates with the expression of a set of HR genes in breast, lung and colorectal cancers. Collectively, our data suggest that Polθ upregulation, besides its importance for survival of HR-deficient cancer cells, may be crucial also for HR-proficient cells to better tolerate DNA replication stress, as part of a global gene deregulation response, including HR genes. Summary: Our work suggests that Polθ upregulation may be crucial for homologous recombination (HR)-proficient cells to better tolerate DNA replication stress, as part of a global gene deregulation response, including HR genes.

A DNA repair variant in POLQ (c.-1060A > G) is associated to hereditary breast cancer patients: a case–control study

BackgroundOne of the hallmarks of cancer is the occurrence of high levels of chromosomal rearrangements as a result of inaccurate repair of double-strand breaks (DSB). Germline mutations in BRCA and RAD51 genes, involved in DSB repair, are strongly associated with hereditary breast cancer. Pol θ, a translesional DNA polymerase specialized in the replication of damaged DNA, has been also shown to contribute to DNA synthesis associated to DSB repair. It is noteworthy that POLQ is highly expressed in breast tumors and this expression is able to predict patient outcome. The objective of this study was to analyze genetic variants related to POLQ as new population biomarkers of risk in hereditary (HBC) and sporadic (SBC) breast cancer.MethodsWe analyzed through case–control study nine SNPs of POLQ in hereditary (HBC) and sporadic (SBC) breast cancer patients using Taqman Real Time PCR assays. Polymorphisms were systematically identified through the NCBI database and are located within exons or promoter regions. We recruited 204 breast cancer patients (101 SBC and 103 HBC) and 212 unaffected controls residing in Southern Brazil.ResultsThe rs581553 SNP located in the promoter region was strongly associated with HBC (c.-1060Au2009>u2009G; HBC GGu2009=u200915, Control TTu2009=u20098; ORu2009=u20095.67, CI95%u2009=u20092.26-14.20; pu2009<u20090.0001). Interestingly, 11 of 15 homozygotes for this polymorphism fulfilled criteria for Hereditary Breast and Ovarian Cancer (HBOC) syndrome. Furthermore, 12 of them developed bilateral breast cancer and one had a familial history of bilateral breast cancer. This polymorphism was also associated with bilateral breast cancer in 67 patients (ORu2009=u20099.86, CI95%u2009=u20093.81-25.54). There was no statistically significant difference of age at breast cancer diagnosis between SNP carriers and non-carriers.ConclusionsConsidering that Pol θ is involved in DBS repair, our results suggest that this polymorphism may contribute to the etiology of HBC, particularly in patients with bilateral breast cancer.

Pol κ in replication checkpoint.

Cells have evolved several mechanisms to deal with the constant challenge of DNA replication fork arrest during the S phase of the cell cycle. One important part of the cellular response to replication arrest or stalling by DNA damage is the induction of the ATR replication checkpoint pathway, which senses stalled replication forks and allows the independent translocation of multicomponent protein complexes, leading to the phosphorylation of the main ATR effector, the protein kinase Chk1.1 In addition, activation of the DNA damage tolerance pathway that includes translesional (TLS) Y-family DNA polymerases (Pol η, Pol κ, Pol ι, Rev1)2 facilitates bypass of DNA lesions. TLS polymerases promote damage tolerance through their ability to insert nucleotides opposite DNA lesions that block the replicative DNA polymerases or to fill in postreplication gaps containing lesions left behind replication forks. n nAlthough both TLS and replication checkpoint choreograph the response to fork stalling, whether these pathways are coordinated has remained an open question. We addressed this issue by focusing on Pol κ, one of the most highly conserved TLS DNA polymerases.3 We reasoned that the ubiquity of Pol κ, and the fact that the mice defective for the POL κ gene manifest a spontaneous genetic instability phenotype in absence of external stress, argue that this protein may contribute to additional aspects of cell physiology in addition to its role in TLS. We described a previously unrecognized and unexpected role for Pol κ in response to replication stress using 2 different experimental systems, Xenopus cell-free extracts and mammalian cultured cells.4 We have found that Pol κ is required for checkpoint activation after replication fork stalling with DNA polymerases inhibitors such as hydroxyurea or aphidicolin, or in the presence of UV-blocking lesions. These effects appear to be specific to Pol κ, since removal of another member of the Y-family, Pol η did not affect the efficiency of Chk1 phosphorylation. This novel function appears to depend upon Pol κ catalytic activity, and we showed that Pol κ fulfils this checkpoint function by participating in DNA synthesis on ssDNA at stalled replication forks. Indeed, recent work has demonstrated that short DNA products accumulate on ssDNA templates in response to fork stalling by aphidicolin and strongly contribute to checkpoint activation.5 These DNA products are longer than the size normally synthesized by Pol α, and a subset of them are generated by Pol δ, most probably on the lagging strand. The leading-strand replicative polymerase Pol e, in contrast, does not appear to play a significant role in the synthesis of these DNA products.5 Pol κ was found to contribute with replicative Pol α and δ to the synthesis of these short DNA intermediates, which, in turn, may facilitate recruitment of the 9-1-1 complex at stalled forks and consequently contribute to efficient activation of the replication checkpoint4 (see also Fig.xa01). Further studies will be required to determine the molecular bases of Pol κ recruitment at stalled forks for its checkpoint function. It will be of interest to explore whether the Pol κ domains required for TLS are similar to the critical domains involved in the checkpoint function. Whether Pol κ is recruited to particular chromosomal regions is also an interesting question. Pol κ has been recently shown to perform accurate DNA synthesis at microsatellite,6 one type of interspersed tandem repeat ubiquitously present throughout the genome that constitutes natural fork barriers. Pol κ could potentially promote microsatellite stability and limit microsatellite allele length variation by its recruitment at stalled forks and its checkpoint associated DNA synthesis. n n n nFigurexa01. Speculative model of Pol κ function in replication checkpoint. Upon replication fork stalling with replicative DNA polymerases inhibitors (hydroxyurea, aphidicolin) or UV-blocking lesions, ssDNA is generated by the action of ... n n n nWe have also observed that Pol κ downregulation in mammalian results in accumulation of DNA damage, thus revealing a function for Pol κ during DNA replication in unperturbed cells and further extending the role of this DNA polymerase outside TLS.4 Interestingly, in the absence of Pol κ, ssDNA persists upon recovery from a hydroxyurea block, suggesting that Pol κ may be also required for replication fork restart. This latter observation may suggest that Pol κ could play a role in repriming replication forks. Since replication intermediates are already present on the lagging strand, it is likely Pol κ may function on the leading strand, alone or in combination with an as-yet-unknown DNA primase (Fig.xa01), such as the very recently identified Polprim.9,10 n nOur work illustrates how 2 major pathways that respond to stalled replication forks could be coordinated to ensure high cell viability and genomic stability. It reinforces also the emerging concept that TLS may not be the sole function assigned to the Y-family TLS DNA polymerases. Pol η, best known for its role in responding to lesions generated by UV irradiation, has been also found to hold another function outside TLS in the stability of common fragile site (CFS) during unperturbed S phase.7,8

DNA replication stress triggers Rapid DNA Replication Fork Breakage by Artemis and XPF

DNA replication stress (DRS) leads to the accumulation of stalled DNA replication forks leaving a fraction of genomic loci incompletely replicated, a source of chromosomal rearrangements during their partition in mitosis. MUS81 is known to limit the occurrence of chromosomal instability by processing these unresolved loci during mitosis. Here, we unveil that the endonucleases ARTEMIS and XPF-ERCC1 can also induce stalled DNA replication forks cleavage through non-epistatic pathways all along S and G2 phases of the cell cycle. We also showed that both nucleases are recruited to chromatin to promote replication fork restart. Finally, we found that rapid chromosomal breakage controlled by ARTEMIS and XPF is important to prevent mitotic segregation defects. Collectively, these results reveal that Rapid Replication Fork Breakage (RRFB) mediated by ARTEMIS and XPF in response to DRS contributes to DNA replication efficiency and limit chromosomal instability.