To cope with ultraviolet C (UVC)-stalled replication forks and restart DNA

To cope with ultraviolet C (UVC)-stalled replication forks and restart DNA synthesis cells either undergo DNA translesion synthesis (TLS) by specialised DNA polymerases or tolerate the lesions using homologous recombination (HR)-based mechanisms. on the FANC core complex act upstream of Rabbit Polyclonal to HSL (phospho-Ser855/554). FANCD2 and are required for the proper relocalisation of monoubiquitinylated FANCD2 (Ub-FANCD2) to subnuclear foci. Second during S-phase Ub-FANCD2 and MDC1 relocalise to UVC-damaged nuclear areas or foci simultaneously but independently of each other. Third Ub-FANCD2 and MDC1 are independently required for optimal BRCA1 relocalisation. While RPA32 phosphorylation (p-RPA32) and RPA foci formation were reduced in parallel with increasing levels of H2AX phosphorylation and MDC1 foci in UVC-irradiated FANC pathway-depleted cells MDC1 depletion was associated with increased UVC-induced Ub-FANCD2 and FANCD2 foci as well as p-RPA32 levels and p-RPA32 foci. On the basis of the previous observations we propose that the FANC pathway participates in the rescue of UVC-stalled replication forks in association with TLS by maintaining the integrity of ssDNA regions and by preserving genome stability and preventing the formation of DSBs the resolution of which would require Abacavir the intervention of MDC1. Introduction DNA damage is a primary source of cellular stress and a leading cause of cancer [1]. To cope with DNA lesions cells have developed an integrated and tightly regulated molecular network called the DNA damage response (DDR) in which cell cycle checkpoints and DNA repair pathways collaborate to efficiently restore the integrity of the genetic material [2]. To avoid the induction and fixation of mutations and to avoid the transmission of genetic modifications DNA lesions must be eliminated before DNA replication. Nevertheless replication forks will inevitably encounter DNA lesions and stall. To restore DNA synthesis and permit cells to progress into mitosis cells exploit DNA damage tolerance (DDT) pathways that involve either translesion synthesis Abacavir (TLS) by specialised DNA polymerases or using homologous recombination (HR)-based mechanisms such as template switching (TS) and break-induced replication (BIR) [3] [4]. DNA damage induced by ultraviolet C radiation (UVC) is a well-characterised roadblock for ongoing replication forks. UVC induces two major DNA lesions cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidone photoproducts (6 4 These lesions are primarily removed through the error-free nucleotide excision repair (NER) pathway [2]. Germline recessive mutations that lead to NER defects are responsible for the classic form of the skin cancer predisposition syndrome xeroderma pigmentosum (XP). The products of the seven cloned genes (to have been identified as the molecular defect underlying the skin cancer predisposition syndrome XP-variant (XP-V) [8]. Compared to classically XP-affected individuals XP-V patients’ Abacavir photosensitivity is reduced and skin Abacavir cancers develop later. XP-V cells repair UVC-induced lesions at a normal rate and display modest increase in sensitivity to UVC exposure. However these cells are unable to replicate past UVC lesions. Therefore XP-V cells accumulate mutations and small deletions [9] [10] Abacavir contributing to the cancer predisposition associated with XP-V. UVC exposure also activates the FANC pathway which is involved in safeguarding DNA replication and cell division in both unstressed and DNA-damaged cells [11]-[13]. Bi-allelic germline mutations in any of at least 15 genes (to was efficiently analysed by immunofluorescence following local irradiation of cells at 100 J/m2. Nuclear local irradiated regions (LIR) were easily visualised through the use of Abacavir specific antibodies directed against CPDs or 6 4 (Figure 1A and 1C). By co-staining cells with a DNA replication tracker (BrdU or EdU) an anti-UVC-induced lesion and/or an anti-FANCD2 antibody we observed that FANCD2 was recruited to LIR only in replicative and post-replicative primary or transformed cells (Figures 1A and S1A). This contrasts with the well-known response of NER proteins which rapidly relocalise to damaged LIR independently of the cell cycle phase (Figures S1C and S1D). These observations indicate that FANCD2 redistribution to damaged nuclear areas a.