ATR/Chk1 pathway is essential for resumption of DNA synthesis and cell survival in UV-irradiated XP variant cells.
DNA polymerase eta (poleta) performs translesion synthesis past ultraviolet (UV) photoproducts and is deficient in cancer-prone xeroderma pigmentosum variant (XP-V) syndrome. The slight sensitivity of XP-V cells to UV is dramatically enhanced by low concentrations of caffeine. So far, the biological explanation for this feature remains elusive. Using DNA combing, we showed that translesion synthesis defect leads to a strong reduction in the number of active replication forks and a high proportion of stalled forks in human cells, which contrasts with budding yeast. Moreover, extensive regions of single-strand DNA are formed during replication in irradiated XP-V cells, leading to an over-activation of ATR/Chk1 pathway after low UVC doses. Addition of a low concentration of caffeine post-irradiation, although inefficient to restore S-phase progression, significantly decreases Chk1 activation and abrogates DNA synthesis in XP-V cells. While inhibition of Chk1 activity by UCN-01 prevents UVC-induced S-phase delay in wild-type cells, it aggravates replication defect in XP-V cells by increasing fork stalling. Consequently, UCN-01 sensitizes XP-V cells to UVC as caffeine does. Our findings indicate that poleta acts at stalled forks to resume their progression, preventing the requirement for efficient replication checkpoint after low UVC doses. In the absence of poleta, Chk1 kinase becomes essential for replication resumption by alternative pathways, via fork stabilization.
Despras
Emmanuelle
E
Centre Nationale de Recherche Scientifique (CNRS) UMR8200, Laboratoire Stabilité Génétique et Oncogenèse, Université Paris-Sud, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94800 Villejuif, France.
Daboussi
Fayza
F
Hyrien
Olivier
O
Marheineke
Kathrin
K
Kannouche
Patricia L
PL
D064007:Ataxia Telangiectasia Mutated Proteins; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D000071877:Checkpoint Kinase 1; D004247:DNA / Q000096:biosynthesis*; D004249:DNA Damage / Q000235:genetics*; D004261:DNA Replication / Q000235:genetics / Q000502:physiology*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection; D014466:Ultraviolet Rays / Q000009:adverse effects; D014983:Xeroderma Pigmentosum / Q000378:metabolism*; D000099270:Y-Family DNA Polymerases
D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins; D000071877:Checkpoint Kinase 1; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D034741:RNA, Small Interfering; C576597:ATR protein, human; C000605867:CHEK1 protein, human; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases