Dihydropyrimidinase protects from DNA replication stress caused by cytotoxic metabolites.

Imbalance in the level of the pyrimidine degradation products dihydrouracil and dihydrothymine is associated with cellular transformation and cancer progression. Dihydropyrimidines are degraded by dihydropyrimidinase (DHP), a zinc metalloenzyme that is upregulated in solid tumors but not in the corresponding normal tissues. How dihydropyrimidine metabolites affect cellular phenotypes remains elusive. Here we show that the accumulation of dihydropyrimidines induces the formation of DNA-protein crosslinks (DPCs) and causes DNA replication and transcriptional stress. We used Xenopus egg extracts to recapitulate DNA replication invitro. We found that dihydropyrimidines interfere directly with the replication of both plasmid and chromosomal DNA. Furthermore, we show that the plant flavonoid dihydromyricetin inhibits human DHP activity. Cellular exposure to dihydromyricetin triggered DPCs-dependent DNA replication stress in cancer cells. This study defines dihydropyrimidines as potentially cytotoxic metabolites that may offer an opportunity for therapeutic-targeting of DHP activity in solid tumors.

MCM8- and MCM9 Deficiencies Cause Lifelong Increased Hematopoietic DNA Damage Driving p53-Dependent Myeloid Tumors.

Hematopoiesis is particularly sensitive to DNA damage. Myeloid tumor incidence increases in patients with DNA repair defects and after chemotherapy. It is not known why hematopoietic cells are highly vulnerable to DNA damage. Addressing this question is complicated by the paucity of mouse models of hematopoietic malignancies due to defective DNA repair. We show that DNA repair-deficient Mcm8- and Mcm9-knockout mice develop myeloid tumors, phenocopying prevalent myelodysplastic syndromes. We demonstrate that these tumors are preceded by a lifelong DNA damage burden in bone marrow and that they acquire proliferative capacity by suppressing signaling of the tumor suppressor and cell cycle controller RB, as often seen in patients. Finally, we found that absence of MCM9 and the tumor suppressor Tp53 switches tumorigenesis to lymphoid tumors without precedent myeloid malignancy. Our results demonstrate that MCM8/9 deficiency drives myeloid tumor development and establishes a DNA damage burdened mouse model for hematopoietic malignancies.

Ddx19 links mRNA nuclear export with progression of transcription and replication and suppresses genomic instability upon DNA damage in proliferating cells.

The DEAD-box Helicase 19 (Ddx19) gene codes for an RNA helicase involved in both mRNA (mRNA) export from the nucleus into the cytoplasm and in mRNA translation. In unperturbed cells, Ddx19 localizes in the cytoplasm and at the cytoplasmic face of the nuclear pore. Here we review recent findings related to an additional Ddx19 function in the nucleus in resolving RNA:DNA hybrids (R-loops) generated during collision between transcription and replication, and upon DNA damage. Activation of a DNA damage response pathway dependent upon the ATR kinase, a major regulator of replication fork progression, stimulates translocation of the Ddx19 protein from the cytoplasm into the nucleus. Only nuclear Ddx19 is competent to resolve R-loops, and down regulation of Ddx19 expression induces DNA double strand breaks only in proliferating cells. Overall these observations put forward Ddx19 as an important novel mediator of the crosstalk between transcription and replication.

An ATR-dependent function for the Ddx19 RNA helicase in nuclear R-loop metabolism.

Coordination between transcription and replication is crucial in the maintenance of genome integrity. Disturbance of these processes leads to accumulation of aberrant DNA:RNA hybrids (R-loops) that, if unresolved, generate DNA damage and genomic instability. Here we report a novel, unexpected role for the nucleopore-associated mRNA export factor Ddx19 in removing nuclear R-loops formed upon replication stress or DNA damage. We show, in live cells, that Ddx19 transiently relocalizes from the nucleopore to the nucleus upon DNA damage, in an ATR/Chk1-dependent manner, and that Ddx19 nuclear relocalization is required to clear R-loops. Ddx19 depletion induces R-loop accumulation, proliferation-dependent DNA damage and defects in replication fork progression. Further, we show that Ddx19 resolves R-loops in vitro via its helicase activity. Furthermore, mutation of a residue phosphorylated by Chk1 in Ddx19 disrupts its interaction with Nup214 and allows its nuclear relocalization. Finally, we show that Ddx19 operates in resolving R-loops independently of the RNA helicase senataxin. Altogether these observations put forward a novel, ATR-dependent function for Ddx19 in R-loop metabolism to preserve genome integrity in mammalian cells.

PIP degron proteins, substrates of CRL4Cdt2, and not PIP boxes, interfere with DNA polymerase η and κ focus formation on UV damage.

Proliferating cell nuclear antigen (PCNA) is a well-known scaffold for many DNA replication and repair proteins, but how the switch between partners is regulated is currently unclear. Interaction with PCNA occurs via a domain known as a PCNA-Interacting Protein motif (PIP box). More recently, an additional specialized PIP box has been described, the « PIP degron ¼, that targets PCNA-interacting proteins for proteasomal degradation via the E3 ubiquitin ligase CRL4(Cdt2). Here we provide evidence that CRL4(Cdt2)-dependent degradation of PIP degron proteins plays a role in the switch of PCNA partners during the DNA damage response by facilitating accumulation of translesion synthesis DNA polymerases into nuclear foci. We show that expression of a nondegradable PIP degron (Cdt1) impairs both Pol η and Pol κ focus formation on ultraviolet irradiation and reduces cell viability, while canonical PIP box-containing proteins have no effect. Furthermore, we identify PIP degron-containing peptides from several substrates of CRL4(Cdt2) as efficient inhibitors of Pol η foci formation. By site-directed mutagenesis we show that inhibition depends on a conserved threonine residue that confers high affinity for PCNA-binding. Altogether these findings reveal an important regulative role for the CRL4(Cdt2) pathway in the switch of PCNA partners on DNA damage.

Combination of arsenic and interferon-α inhibits expression of KSHV latent transcripts and synergistically improves survival of mice with primary effusion lymphomas.

BACKGROUND: Kaposi sarcoma-associated herpesvirus (KSHV) is the etiologic agent of primary effusion lymphomas (PEL). PEL cell lines infected with KSHV, but negative for Epstein-Barr virus have a tumorigenic potential in non-obese diabetic/severe combined immunodeficient mice and result in efficient engraftment and formation of malignant ascites with notable abdominal distension, consistent with the clinical manifestations of PEL in humans. METHODOLOGY/PRINCIPAL FINDINGS: Using this preclinical mouse model, we demonstrate that the combination of arsenic trioxide and interferon-alpha (IFN) inhibits proliferation, induces apoptosis and downregulates the latent viral transcripts LANA-1, v-FLIP and v-Cyc in PEL cells derived from malignant ascites. Furthermore, this combination decreases the peritoneal volume and synergistically increases survival of PEL mice. CONCLUSION/SIGNIFICANCE: These results provide a promising rationale for the therapeutic use of arsenic/IFN in PEL patients.