Mitotic DNA synthesis is a recently discovered mechanism that resolves late replication intermediates, thereby supporting cell proliferation under replication stress. This unusual form of DNA synthesis occurs in the absence of RAD51 or BRCA2, which led to the identification of RAD52 as a key player in this process. Notably, mitotic DNA synthesis is predominantly observed at chromosome loci that colocalize with FANCD2 foci. However, the role of this protein in mitotic DNA synthesis remains largely unknown. In this study, we investigated the role of FANCD2 and its interplay with RAD52 in mitotic DNA synthesis using aphidicolin as a universal inducer of this process. After examining eight human cell lines, we provide evidence for FANCD2 rather than RAD52 as a fundamental supporter of mitotic DNA synthesis. In cancer cell lines, FANCD2 exerts this role independently of RAD52. Surprisingly , RAD52 is dispensable for mitotic DNA synthesis in noncancerous cell lines, but these cells strongly depend on FANCD2 for this process. Therefore, RAD52 functions selectively in cancer cells as a secondary regulator in addition to FANCD2 to facilitate mitotic DNA synthesis. As an alternative to aphidicolin, we found partial inhibition of origin licensing as an effective way to induce mitotic DNA synthesis preferentially in cancer cells. Importantly, cancer cells still perform mitotic DNA synthesis by dual regulation of FANCD2 and RAD52 under such conditions. Implications: These key differences in mitotic DNA synthesis between cancer and noncancerous cells advance our understanding of this mechanism and can be exploited for cancer therapies.

DNA replication is a prerequisite for cell proliferation, yet it can be increasingly challenging for a eukaryotic cell to faithfully duplicate its genome as its size and complexity expands. Dormant origins now emerge as a key component for cells to successfully accomplish such a demanding but essential task. In this perspective, we will first provide an overview of the fundamental processes eukaryotic cells have developed to regulate origin licensing and firing. With a special focus on mammalian systems, we will then highlight the role of dormant origins in preventing replication-associated genome instability and their functional interplay with proteins involved in the DNA damage repair response for tumor suppression. Lastly, deficiencies in the origin licensing machinery will be discussed in relation to their influence on stem cell maintenance and human diseases.

<p>S1. Depletion of RAD52 or FANCD2 in WT HCT116 cells phenocopies R52KO or D2KO cells, respectively. S2. Depletion of MCM4 is relatively inefficient in inducing EdU spot formation in HCT116 cells. S3. The formation of Aph- or siORC1-induced EdU spots depends both on RAD52 and FANCD2 in U2OS cells. S4. The formation of EdU spots depends both on RAD52 and FANCD2 in HeLa cells. S5. Depletion of FANCD2 or RAD52 confers similar cell cycle profiles. S6. EdU spots induced by BRCA2 depletion display the characteristics of mitotic DNA synthesis.</p>

<p>S1. Depletion of RAD52 or FANCD2 in WT HCT116 cells phenocopies R52KO or D2KO cells, respectively. S2. Depletion of MCM4 is relatively inefficient in inducing EdU spot formation in HCT116 cells. S3. The formation of Aph- or siORC1-induced EdU spots depends both on RAD52 and FANCD2 in U2OS cells. S4. The formation of EdU spots depends both on RAD52 and FANCD2 in HeLa cells. S5. Depletion of FANCD2 or RAD52 confers similar cell cycle profiles. S6. EdU spots induced by BRCA2 depletion display the characteristics of mitotic DNA synthesis.</p>

<div>Abstract<p>Mitotic DNA synthesis is a recently discovered mechanism that resolves late replication intermediates, thereby supporting cell proliferation under replication stress. This unusual form of DNA synthesis occurs in the absence of RAD51 or BRCA2, which led to the identification of RAD52 as a key player in this process. Notably, mitotic DNA synthesis is predominantly observed at chromosome loci that colocalize with FANCD2 foci. However, the role of this protein in mitotic DNA synthesis remains largely unknown. In this study, we investigated the role of FANCD2 and its interplay with RAD52 in mitotic DNA synthesis using aphidicolin as a universal inducer of this process. After examining eight human cell lines, we provide evidence for FANCD2 rather than RAD52 as a fundamental supporter of mitotic DNA synthesis. In cancer cell lines, FANCD2 exerts this role independently of RAD52. Surprisingly, RAD52 is dispensable for mitotic DNA synthesis in noncancerous cell lines, but these cells strongly depend on FANCD2 for this process. Therefore, RAD52 functions selectively in cancer cells as a secondary regulator in addition to FANCD2 to facilitate mitotic DNA synthesis. As an alternative to aphidicolin, we found partial inhibition of origin licensing as an effective way to induce mitotic DNA synthesis preferentially in cancer cells. Importantly, cancer cells still perform mitotic DNA synthesis by dual regulation of FANCD2 and RAD52 under such conditions.</p>Implications:<p>These key differences in mitotic DNA synthesis between cancer and noncancerous cells advance our understanding of this mechanism and can be exploited for cancer therapies.</p></div>