Stéphane Coulon

Increase in dNTP pool size during the DNA damage response plays a key role in spontaneous and induced-mutagenesis in Escherichia coli.

Exposure of Escherichia coli to UV light increases expression of NrdAB, the major ribonucleotide reductase leading to a moderate increase in dNTP levels. The role of elevated dNTP levels during translesion synthesis (TLS) across specific replication-blocking lesions was investigated. Here we show that although the specialized DNA polymerase PolV is necessary for replication across UV-lesions, such as cyclobutane pyrimidine dimers or pyrimidine(6-4)pyrimidone photoproduct, Pol V per se is not sufficient. Indeed, efficient TLS additionally requires elevated dNTP levels. Similarly, for the bypass of an N-2-acetylaminofluorene-guanine adduct that requires Pol II instead of PolV, efficient TLS is only observed under conditions of high dNTP levels. We suggest that increased dNTP levels transiently modify the activity balance of Pol III (i.e., increasing the polymerase and reducing the proofreading functions). Indeed, we show that the stimulation of TLS by elevated dNTP levels can be mimicked by genetic inactivation of the proofreading function (mutD5 allele). We also show that spontaneous mutagenesis increases proportionally to dNTP pool levels, thus defining a unique spontaneous mutator phenotype. The so-called “dNTP mutator” phenotype does not depend upon any of the specialized DNA polymerases, and is thus likely to reflect an increase in Pol IIIs own replication errors because of the modified activity balance of Pol III. As up-regulation of the dNTP pool size represents a common physiological response to DNA damage, the present model is likely to represent a general and unique paradigm for TLS pathways in many organisms.

Regulation of Mus81–Eme1 Holliday junction resolvase in response to DNA damage

Structure-specific DNA endonucleases have critical roles during DNA replication, repair and recombination, yet they also have the potential for causing genome instability. Controlling these enzymes may be essential to ensure efficient processing of ad hoc substrates and to prevent random, unscheduled processing of other DNA structures, but it is unknown whether structure-specific endonucleases are regulated in response to DNA damage. Here, we uncover DNA damage–induced activation of Mus81–Eme1 Holliday junction resolvase in fission yeast. This new regulation requires both Cdc2CDK1- and Rad3ATR-dependent phosphorylation of Eme1. Mus81–Eme1 activation prevents gross chromosomal rearrangements in cells lacking the BLM-related DNA helicase Rqh1. We propose that linking Mus81–Eme1 DNA damage–induced activation to cell-cycle progression ensures efficient resolution of Holliday junctions that escape dissolution by Rqh1–TopIII while preventing unnecessary DNA cleavages.

RPA prevents G‐rich structure formation at lagging‐strand telomeres to allow maintenance of chromosome ends

Replication protein A (RPA) is a highly conserved heterotrimeric single‐stranded DNA‐binding protein involved in DNA replication, recombination, and repair. In fission yeast, the Rpa1‐D223Y mutation provokes telomere shortening. Here, we show that this mutation impairs lagging‐strand telomere replication and leads to the accumulation of secondary structures and recruitment of the homologous recombination factor Rad52. The presence of these secondary DNA structures correlates with reduced association of shelterin subunits Pot1 and Ccq1 at telomeres. Strikingly, heterologous expression of the budding yeast Pif1 known to efficiently unwind G‐quadruplex rescues all the telomeric defects of the D223Y cells. Furthermore, in vitro data show that the identical D to Y mutation in human RPA specifically affects its ability to bind G‐quadruplex. We propose that RPA prevents the formation of G‐quadruplex structures at lagging‐strand telomeres to promote shelterin association and facilitate telomerase action at telomeres.

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 (Poldelta interacting protein of 38kDa) directly interacts with the TLS polymerase Poleta. Interestingly, the region of Poleta interacting with PDIP38 is found to be located within the ubiquitin-binding zinc finger domain (UBZ) of Poleta. We show that the depletion of PDIP38 increases the number of cells with Poleta 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 Poleta but also with the specialized polymerases Rev1 and Polzeta (via Rev7). We thus suggest that PDIP38 serves as a mediator protein helping TLS Pols to transiently replace replicative polymerases at damaged sites.

RPA facilitates telomerase activity at chromosome ends in budding and fission yeasts

In Saccharomyces cerevisiae, the telomerase complex binds to chromosome ends and is activated in late S‐phase through a process coupled to the progression of the replication fork. Here, we show that the single‐stranded DNA‐binding protein RPA (replication protein A) binds to the two daughter telomeres during telomere replication but only its binding to the leading‐strand telomere depends on the Mre11/Rad50/Xrs2 (MRX) complex. We further demonstrate that RPA specifically co‐precipitates with yKu, Cdc13 and telomerase. The interaction of RPA with telomerase appears to be mediated by both yKu and the telomerase subunit Est1. Moreover, a mutation in Rfa1 that affects both the interaction with yKu and telomerase reduces the dramatic increase in telomere length of a rif1Δ, rif2Δ double mutant. Finally, we show that the RPA/telomerase association and function are conserved in Schizosaccharomyces pombe. Our results indicate that in both yeasts, RPA directly facilitates telomerase activity at chromosome ends.

Rad8Rad5/Mms2–Ubc13 ubiquitin ligase complex controls translesion synthesis in fission yeast

Many DNA lesions cause pausing of replication forks at lesion sites; thus, generating gaps in the daughter strands that are filled‐in by post‐replication repair (PRR) pathways. In Saccharomyces cerevisiae, PRR involves translesion synthesis (TLS) mediated by Polη or Polζ, or Rad5‐dependent gap filling through a poorly characterized error‐free mechanism. We have developed an assay to monitor error‐free and mutagenic TLS across single DNA lesions in Schizosaccharomyces pombe. For both main UV photolesions, we have delineated a major error‐free pathway mediated by a distinct combination of TLS polymerases. Surprisingly, these TLS pathways require enzymes needed for poly‐ubiquitination of proliferating cell nuclear antigen (PCNA) as well as those required for mono‐ubiquitination. For pathways that require several TLS polymerases the poly‐ubiquitin chains of PCNA may facilitate their recruitment through specific interactions with their multiple ubiquitin‐binding motifs. These error‐free TLS pathways may at least partially account for the previously described poly‐ubiquitination‐dependent error‐free branch of PRR. This work highlights major differences in the control of lesion tolerance pathways between S. pombe and S. cerevisiae despite the homologous sets of PRR genes these organisms share.

Solving the Telomere Replication Problem

Telomeres are complex nucleoprotein structures that protect the extremities of linear chromosomes. Telomere replication is a major challenge because many obstacles to the progression of the replication fork are concentrated at the ends of the chromosomes. This is known as the telomere replication problem. In this article, different and new aspects of telomere replication, that can threaten the integrity of telomeres, will be reviewed. In particular, we will focus on the functions of shelterin and the replisome for the preservation of telomere integrity.

Ubiquitin-PCNA fusion as a mimic for mono-ubiquitinated PCNA in Schizosaccharomyces pombe

Translesion synthesis is a major mechanism with which eukaryotic cells deal with DNA damage during replication. Mono-ubiquitinated PCNA is a key regulator of this process. We have investigated whether a ubiquitin-PCNA fusion can mimic ubiquitinated PCNA, by transforming plasmids expressing this fusion protein into different mutants of Schizosaccharomyces pombe. We show that the fusion protein is able to form PCNA trimers and that it can reduce the UV sensitivity and increase translesion synthesis in mutants in which PCNA cannot be ubiquitinated (pcn1-K164R and rhp18), but not of the rad8 mutant in which PCNA can be mono-ubiquitinated but not poly-ubiquitinated. We conclude that the fusion protein is a mimic of mono-ubiquitinated PCNA but it cannot be poly-ubiquitinated. Expression of the fusion protein at levels similar to that of endogenous unmodified protein has little effect on the spontaneous mutation rate of S. pombe. Replacement of the pcn1 locus with PCNA N-terminally tagged with different epitopes resulted in lethality, probably because the tagged proteins were expressed at substantially reduced levels.

Eroded telomeres are rearranged in quiescent fission yeast cells through duplications of subtelomeric sequences

While the mechanisms of telomere maintenance has been investigated in dividing cells, little is known about the stability of telomeres in quiescent cells and how dysfunctional telomeres are processed in non-proliferating cells. Here we examine the stability of telomeres in quiescent cells using fission yeast. While wild type telomeres are stable in quiescence, we observe that eroded telomeres were highly rearranged during quiescence in telomerase minus cells. These rearrangements depend on homologous recombination (HR) and correspond to duplications of subtelomeric regions. HR is initiated at newly identified subtelomeric homologous repeated sequences (HRS). We further show that TERRA (Telomeric Repeat-containing RNA) is increased in post-mitotic cells with short telomeres and correlates with telomere rearrangements. Finally, we demonstrate that rearranged telomeres prevent cells to exit properly from quiescence. Taken together, we describe in fission yeast a mode of telomere repair mechanism specific to post-mitotic cells that is likely promoted by transcription.How both telomere stability is regulated and dysfunctional telomeres processed in quiescent cells is poorly understood. Here, the authors provide evidence that eroded telomeres in quiescent fission yeast are rearranged by homologous recombination through duplications of subtelomeric sequences.

Improving the Specificity of an Anti-Estradiol Antibody by Random Mutagenesis and Phage Display

Estradiol is a steroid hormone secreted by endocrine glands (ovary, testis) [3]. The variation of estradiol levels in blood or urine may be associated with many pathologies such as hormone-dependent cancers and diseases of sexual behaviour [5]. Measurements of the concentration of estradiol in blood or urine is of great importance both for clinical diagnosis and during therapy. Competitive immunoassays using an antisteroid antibody and a steroid tracer have become the main routine method to measure steroid concentrations. Obviously, the antibody must be very specific to be able to discriminate between estradiol and other steroid analogues. At present, none of the available antibodies are able to provide unambiguous measurements of very low concentrations of estradiol directly in blood or urine [2]. First, the molecular structures of steroids are very similar, and consequently, the isolation of a specific steroid antibody devoid of cross-reactions with analogues is difficult. Second, the steroids are small molecules unable to cause an immunogenic reaction. They need to be coupled to an immunogenic protein like bovine serum albumin. Consequently, the antibody has often a weaker affinity for the free steroid than for the immunogen [8] and exhibits a lower specificity for the site of coupling.