Abstract
Replications forks are routinely hindered by different endogenous stresses. Because homologous recombination plays a pivotal role in the reactivation of arrested replication forks, defects in homologous recombination reveal the initial endogenous stress(es). Homologous recombination-defective cells consistently exhibit a spontaneously reduced replication speed, leading to mitotic extra centrosomes. Here, we identify oxidative stress as a major endogenous source of replication speed deceleration in homologous recombination-defective cells. The treatment of homologous recombination-defective cells with the antioxidant N-acetyl-cysteine or the maintenance of the cells at low O2 levels (3%) rescues both the replication fork speed, as monitored by single-molecule analysis (molecular combing), and the associated mitotic extra centrosome frequency. Reciprocally, the exposure of wild-type cells to H2O2 reduces the replication fork speed and generates mitotic extra centrosomes. Supplying deoxynucleotide precursors to H2O2-exposed cells rescued the replication speed. Remarkably, treatment with N-acetyl-cysteine strongly expanded the nucleotide pool, accounting for the replication speed rescue. Remarkably, homologous recombination-defective cells exhibit a high level of endogenous reactive oxygen species. Consistently, homologous recombination-defective cells accumulate spontaneous γH2AX or XRCC1 foci that are abolished by treatment with N-acetyl-cysteine or maintenance at 3% O2. Finally, oxidative stress stimulated homologous recombination, which is suppressed by supplying deoxynucleotide precursors. Therefore, the cellular redox status strongly impacts genome duplication and transmission. Oxidative stress should generate replication stress through different mechanisms, including DNA damage and nucleotide pool imbalance. These data highlight the intricacy of endogenous replication and oxidative stresses, which are both evoked during tumorigenesis and senescence initiation, and emphasize the importance of homologous recombination as a barrier against spontaneous genetic instability triggered by the endogenous oxidative/replication stress axis.
Highlights
The maintenance of genome stability relies on successful DNA replication and equal partitioning of the duplicated DNA during mitosis
Are both evoked during tumorigenesis and senescence initiation, and emphasize the importance of homologous recombination as a barrier against spontaneous genetic instability triggered by the endogenous oxidative/replication stress axis
Replication forks are routinely hindered by different endogenous stresses
Summary
The maintenance of genome stability relies on successful DNA replication and equal partitioning of the duplicated DNA during mitosis. Replication failure leads to mitotic anaphase bridges and breaks at common fragile sites [1,2,3] They do not contain DNA, active mitotic extra centrosomes (MECs) are a consequence of replication stress, leading to multipolar chromosomal segregation or chromosome lagging, amplifying chromosome instability from a local problem during replication to a genome-wide problem after mitosis [4]. Spontaneous activation of the DNA damage response (DDR) has been described as a consequence of endogenous DNA replication stress in pre-cancerous cells and during the early stages of malignancy or senescence [5,6,7,8]. Little is known regarding the nature of endogenous replication stress because this phenomenon is difficult to detect and analyze
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