SUMMARY Eukaryotic chromosomal DNA is faithfully replicated in a complex series of cell-cycle-regulated events that are incompletely understood. Here we report the reconstitution of DNA replication free in solution with purified proteins from the budding yeast Saccharomyces cerevisiae. The system recapitulates regulated bidirectional origin activation; synthesis of leading and lagging strands by the three replicative DNA polymerases Pol α, Pol δ, and Pol ε; and canonical maturation of Okazaki fragments into continuous daughter strands. We uncover a dual regulatory role for chromatin during DNA replication: promoting origin dependence and determining Okazaki fragment length by restricting Pol δ progression. This system thus provides a functional platform for the detailed mechanistic analysis of eukaryotic chromosome replication.

PTEN (phosphatase and tensin homolog), a tumor suppressor frequently mutated in human cancer, has various cytoplasmic and nuclear functions. PTEN translocates to the nucleus from the cytoplasm in response to oxidative stress. However, the mechanism and function of the translocation are not completely understood. In this study, topotecan (TPT), a topoisomerase I inhibitor, and cisplatin (CDDP) were employed to induce DNA damage. The results indicate that TPT or CDDP activates ATM (ATM serine/threonine kinase), which phosphorylates PTEN at serine 113 and further regulates PTEN nuclear translocation in A549 and HeLa cells. After nuclear translocation, PTEN induces autophagy, in association with the activation of the p-JUN-SESN2/AMPK pathway, in response to TPT. These results identify PTEN phosphorylation by ATM as essential for PTEN nuclear translocation and the subsequent induction of autophagy in response to DNA damage.

Tumor‐associated enzyme‐activated prodrugs can potentially improve the selectivity of chemotherapeutics. However, the paucity of tumor‐associated enzymes which are essential for prodrug activation usually limits the antitumor potency. A cooperative strategy that utilizes combretastatin A4 nanodrug (CA4‐NPs) and matrix metalloproteinase 9 (MMP9)‐activated doxorubicin prodrug (MMP9‐DOX‐NPs) is developed. CA4 is a typical vascular disrupting agent that can selectively disrupt immature tumor blood vessels and exacerbate the tumor hypoxia state. After treatment with CA4‐NPs, MMP9 expression can be significantly enhanced by 5.6‐fold in treated tumors, which further boosts tumor‐selective active drug release of MMP9‐DOX‐NPs by 3.7‐fold in an orthotopic 4T1 mammary adenocarcinoma mouse model. The sequential delivery of CA4‐NPs and MMP9‐DOX‐NPs exhibits enhanced antitumor efficacy with reduced systemic toxicity compared with the noncooperative controls.