Localization of Y‐Family Polymerases and the DNA Polymerase Switch in Mammalian Cells

During translesion synthesis past sites of damaged DNA, specialized Y-family polymerases are employed by the cell to replace the high stringency replicative polymerases and synthesize DNA past the damaged site. These polymerases are localized in replication factories during the S phase of the cell cycle. When progress of the replication fork is blocked, the polymerase accessory protein, proliferating cell nuclear antigen (PCNA), becomes ubiquitinated and the monoubiquitinated PCNA has an increased affinity for Y-family DNA polymerase ¿ (pol¿). This chapter describes methods for visualizing the polymerases in replication factories, for analyzing the ubiquitination status of PCNA, and for measuring its interaction with pol¿ in chromatin extracts

Caractérisation du rôle de la polymérase translésionnelle REV1 dans les cellules humaines

LE KREMLIN-B.- PARIS 11-BU Méd (940432101) / SudocSudocFranceF

Régulation de l'hélicase FBH1 et conséquences sur le maintien de la stabilité génétique chez l'homme

Bien que la recombinaison homologue (RH) soit requise, notamment, pour la réparation fidèle des cassures double brins et la prise en charge des fourches de réplication bloquées, une mauvaise régulation de ce mécanisme peut provoquer des réarrangements chromosomiques importants et des pertes d hétérozygoties. Chez la levure S. cerevisiae, la forme SUMOylée du facteur de processivité des polymérases réplicatives, PCNA, recruterait l hélicase Srs2 au niveau des fourches de réplication bloquées afin de prévenir les évènements de RH inappropriés via la dissociation du nucléofilament de Rad51. Préalablement à ce projet, notre équipe a montré que la SUMOylation de PCNA sur la lysine 164 existe chez l homme et qu elle est présente en particulier dans les cellules déficientes en polymérase translésionnelle (Pol ). Au cours de cette thèse, nous avons d abord examiné la localisation de cette forme SUMOylée et montrons qu elle s accumule au niveau des dommages induits par une irradiation aux ultra-violets (UV), ce qui suggère son implication dans la réponse à ce type de lésions.Dans le but de préciser la fonction de cette forme modifiée, nous nous sommes demandé si celle-ci était impliquée dans le recrutement d une hélicase anti-recombinogène.L hélicase humaine FBH1 a été proposée récemment comme homologue fonctionnel potentiel de Srs2 : elle complémente partiellement les levures déficientes en Srs2, possède une activité anti-recombinogène et s accumule aux sites de cassures double brins ou de stress réplicatif. Afin de caractériser plus précisément la fonction et le mode de régulation de l hélicase FBH1 dans les cellules humaines, nous avons examiné sa localisation subcellulaire en l absence de dommage et après traitement aux UV et à un agent méthylant l ADN. Nous montrons que FBH1 est recrutée au niveau des foyers de réplication où elle est colocalisée avec PCNA. Après traitement génotoxique, FBH1 s accumule aux sites de dommages de l ADN de façon précoce et transitoire. Nous montrons que PCNA contrôle l accumulation de FBH1 pendant la réplication et en réponse à des dommages via une interaction directe par le biais de deux motifs distincts d interaction à PCNA : PIP et APIM. FBH1 n interagit cependant pas de façon préférentielle avec PCNA-SUMO.De plus, nous montrons que le recrutement de FBH1 est suivi de sa polyubiquitination et de sa dégradation par la voie du protéasome. Cette dégradation dépend de l action conjuguée de PCNA et du complexe ubiquitine-ligase CRL4Cdt2. Elle est nécessaire au recrutement optimal de Pol .Notre hypothèse est donc que FBH1 serait recrutée sur l ADN via une interaction avec PCNA au moment de la réplication ou en réponse à un stress génotoxique, afin de limiter les événements de recombinaison non programmés dépendant de RAD51. Par la suite, PCNA et CRL4Cdt2 provoqueraient la dégradation de FBH1 afin de limiter le temps de résidence de l hélicase qui pourrait interférer avec la prise en charge des dommages par le mécanisme de synthèse translésionnelle.Although Homologous Recombination (HR) is required for error-free repair of double-strand breaks and stalled or collapsed replication forks, it has to be highly regulated to prevent unscheduled genome rearrangements and loss of heterozygosity. In yeast S. cerevisiae, the SUMOylated form of Proliferating Cell Nuclear Antigen (PCNA) recruits the DNA helicase Srs2 at stalled replication forks to prevent unscheduled HR events by disrupting Rad51 nucleoprotein. In our laboratory, previous results showed that PCNA is also SUMOylated in human on lysine 164, especially in translesion polymerase (Pol ) deficient cells.During my phD, I first studied the localization of SUMO-PCNA and showed that it accumulates at UV-induced DNA damage. It suggests that PCNA is involved in the DNA damage response to this kind of lesions. To characterize the function of this modified form of PCNA, we wondered whether it could recruit an anti-recombinogenic helicase.The human FBH1 helicase was recently thought to act as a functional homolog of Srs2, since it can partially complement Srs2-deficient S. cerevisiae strains. Besides, hFBH1 has an anti-recombinogenic activity and accumulates at sites of DNA breaks or replication stress.To further characterize the function and regulation of hFBH1 in human cells, we examined its subcellular localization in response to several DNA damaging agents. Our results showed that, without external treatment, FBH1 accumulates into replication foci where it colocalizes with PCNA. After genotoxic treatment, FBH1 accumulates early ant transiently to DNA damage. We show that PCNA coordinates the accumulation of FBH1 during replication and after DNA damage through direct interaction via two distinct PCNA interaction motifs: PIP and APIM. However, FBH1 does not interact preferentially with SUMO-PCNA.We also show that FBH1 recruitment is followed by its polyubiquitination and degradation by the proteasome. This degradation depends on PCNA and the ubiquitin-ligase CRL4Cdt2 and is required for Pol proper recruitment to UV-induced DNA damage. These findings suggest that PCNA recruits FBH1 at stalled replication forks or in response to DNA damage to limit unscheduled RAD51-dependent recombination. Then, PCNA and CRL4Cdt2 would promote FBH1 degradation to enable translesion synthesis.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Localisation of human Y-family DNA polymerase κ: relationship to PCNA foci

DNA polymerases of the Y-family are involved in translesion DNA synthesis past different types of DNA damage. Previous work has shown that DNA polymerases {eta} and {iota} are localised in replication factories during S phase, where they colocalise one-to-one with PCNA. Cells with factories containing these polymerases accumulate after treatment with DNA damaging agents because replication forks are stalled at sites of damage. We now show that DNA polymerase {kappa} (pol{kappa}) has a different localisation pattern. Although, like the other Y-family polymerases, it is exclusively localised in the nucleus, pol{kappa} is found in replication foci in only a small proportion of S-phase cells. It does not colocalise in those foci with proliferating cell nuclear antigen (PCNA) in the majority of cells. This reduced number of cells with pol{kappa} foci, when compared with those containing pol{eta} foci, is observed both in untreated cells and in cells treated with hydroxyurea, UV irradiation or benzo[a]pyrene. The C-terminal 97 amino acids of pol{kappa} are sufficient for this limited localisation into nuclear foci, and include a C2HC zinc finger, bipartite nuclear localisation signal and putative PCNA binding site

Why is immunotherapy effective (or not) in patients with MSI/MMRD tumors?

International audienc

Localisation of human Y-family DNA polymerase ?: relationship to PCNA foci

DNA polymerases of the Y-family are involved in translesion DNA synthesis past different types of DNA damage. Previous work has shown that DNA polymerases {eta} and {iota} are localised in replication factories during S phase, where they colocalise one-to-one with PCNA. Cells with factories containing these polymerases accumulate after treatment with DNA damaging agents because replication forks are stalled at sites of damage. We now show that DNA polymerase {kappa} (pol{kappa}) has a different localisation pattern. Although, like the other Y-family polymerases, it is exclusively localised in the nucleus, pol{kappa} is found in replication foci in only a small proportion of S-phase cells. It does not colocalise in those foci with proliferating cell nuclear antigen (PCNA) in the majority of cells. This reduced number of cells with pol{kappa} foci, when compared with those containing pol{eta} foci, is observed both in untreated cells and in cells treated with hydroxyurea, UV irradiation or benzo[a]pyrene. The C-terminal 97 amino acids of pol{kappa} are sufficient for this limited localisation into nuclear foci, and include a C2HC zinc finger, bipartite nuclear localisation signal and putative PCNA binding site

Role of DNA Polymerase ? in the UV Mutation Spectrum in Human Cells

In humans, inactivation of the DNA polymerase eta gene (pol eta) results in sunlight sensitivity and causes the cancer-prone xeroderma pigmentosum variant syndrome (XP-V). Cells from XP-V individuals have a reduced capacity to replicate UV-damaged DNA and show hypermutability after UV exposure. Biochemical assays have demonstrated the ability of pol eta to bypass cis-syn-cyclobutane thymine dimers, the most common lesion generated in DNA by UV. In most cases, this bypass is error-free. To determine the actual requirement of pol eta in vivo, XP-V cells (XP30RO) were complemented by the wild type pol eta gene. We have used two pol eta-corrected clones to study the in vivo characteristics of mutations produced by DNA polymerases during DNA synthesis of UV-irradiated shuttle vectors transfected into human host cells, which had or had not been exposed previously to UV radiation. The functional complementation of XP-V cells by pol eta reduced the mutation frequencies both at CG and TA base pairs and restored UV mutagenesis to a normal level. UV irradiation of host cells prior to transfection strongly increased the mutation frequency in undamaged vectors and, in addition, especially in the pol eta-deficient XP30RO cells at 5'-TT sites in UV-irradiated plasmids. These results clearly show the protective role of pol eta against UV-induced lesions and the activation by UV of pol eta-independent mutagenic processes

Ubiquitin-binding motif of human DNA polymerase η is required for correct localization

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