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.
Hum Mol Genet. 2010 May 1;19(9):1690-701. doi: 10.1093/hmg/ddq046. Epub 2010 Feb 1.
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.
DNA 聚合酶 eta（poleta）能够在紫外线（UV）光产物的基础上进行跨损伤合成，并且在易患皮肤癌的色素性干皮病变异型（XP-V）综合征中存在缺陷。低浓度的咖啡因可显著增强 XP-V 细胞对 UV 的轻微敏感性。到目前为止，这种特征的生物学解释仍然难以捉摸。通过 DNA 梳理，我们发现跨损伤合成缺陷会导致人细胞中活跃复制叉的数量明显减少，并且停滞叉的比例很高，这与芽殖酵母形成鲜明对比。此外，在受辐照的 XP-V 细胞中复制过程中会形成大量的单链 DNA 区域，导致低剂量 UVC 照射后 ATR/Chk1 途径过度激活。尽管低浓度咖啡因在辐照后不能有效地恢复 S 期进程，但它可以显著降低 Chk1 的激活并阻断 XP-V 细胞的 DNA 合成。虽然 UCN-01 抑制 Chk1 活性可防止野生型细胞中 UVC 诱导的 S 期延迟，但它会通过增加叉停滞来加剧 XP-V 细胞中的复制缺陷。因此，UCN-01 像咖啡因一样使 XP-V 细胞对 UVC 敏感。我们的研究结果表明，poleta 在停滞的叉上发挥作用以恢复其进展，从而防止低剂量 UVC 后对有效的复制检查点的需求。在没有 poleta 的情况下，Chk1 激酶通过叉稳定化通过替代途径对于复制恢复变得至关重要。
