Abstract:Mcm2–7 is recruited to eukaryotic origins of DNA replication by origin recognition complex, Cdc6 and Cdt1 thereby licensing the origins. Cdc6 is essential for origin licensing during DNA replication and is readily destabilized from chromatin after Mcm2–7 loading. Here, we show that after origin licensing, deregulation of Cdc6 suppresses DNA replication in Xenopus egg extracts without the involvement of ATM/ATR-dependent checkpoint pathways. DNA replication is arrested specifically after chromatin binding of Cd… Show more
“…We found that FACT associates with chromatin in at least two distinct phases: an initial binding phase unrelated to DNA replication events and a second phase of chromatin binding that initiated after origin licensing. Initially loaded FACT is susceptible to removal from licensed chromatin but not from unlicensed chromatin, and the later association of FACT is inhibited if DNA replication is blocked before origin !ring by the addition of Cdc6 [24]. Our results also suggest that FACT binding after origin licensing is necessary for the efficient replication of eukaryotic chromatin.…”
Section: Introductionsupporting
confidence: 52%
“…Interphase extracts were prepared as described previously [3,24,25]. Xenopus egg extracts were supplemented with 250 "g/ml cycloheximide, 25 mM phosphocreatine, and 15 "g/ml creatine phosphokinase before use.…”
The facilitates chromatin transcription (FACT) complex affects nuclear DNA transactions in a chromatin context. Though the involvement of FACT in eukaryotic DNA replication has been revealed, a clear understanding of its biochemical behavior during DNA replication still remains elusive. Here, we analyzed the chromatin-binding dynamics of FACT using Xenopus egg extract cell-free system. We found that FACT has at least two distinct chromatin-binding phases: (1) a rapid chromatin-binding phase at the onset of DNA replication that did not involve origin licensing and (2) a second phase of chromatin binding that initiated after origin licensing. Intriguingly, early-binding FACT dissociated from chromatin when DNA replication was blocked by the addition of Cdc6 in the licensed state before origin firing. Cdc6-induced removal of FACT was blocked by the inhibition of origin licensing with geminin, but not by suppressing the activity of DNA polymerases, CDK, or Cdc7. Furthermore, chromatin transfer experiments revealed that impairing the later binding of FACT severely compromises DNA replication activity. Taken together, we propose that even though FACT has rapid chromatin-binding activity, the binding pattern of FACT on chromatin changes after origin licensing, which may contribute to the establishment of its functional link to the DNA replication machinery.
“…We found that FACT associates with chromatin in at least two distinct phases: an initial binding phase unrelated to DNA replication events and a second phase of chromatin binding that initiated after origin licensing. Initially loaded FACT is susceptible to removal from licensed chromatin but not from unlicensed chromatin, and the later association of FACT is inhibited if DNA replication is blocked before origin !ring by the addition of Cdc6 [24]. Our results also suggest that FACT binding after origin licensing is necessary for the efficient replication of eukaryotic chromatin.…”
Section: Introductionsupporting
confidence: 52%
“…Interphase extracts were prepared as described previously [3,24,25]. Xenopus egg extracts were supplemented with 250 "g/ml cycloheximide, 25 mM phosphocreatine, and 15 "g/ml creatine phosphokinase before use.…”
The facilitates chromatin transcription (FACT) complex affects nuclear DNA transactions in a chromatin context. Though the involvement of FACT in eukaryotic DNA replication has been revealed, a clear understanding of its biochemical behavior during DNA replication still remains elusive. Here, we analyzed the chromatin-binding dynamics of FACT using Xenopus egg extract cell-free system. We found that FACT has at least two distinct chromatin-binding phases: (1) a rapid chromatin-binding phase at the onset of DNA replication that did not involve origin licensing and (2) a second phase of chromatin binding that initiated after origin licensing. Intriguingly, early-binding FACT dissociated from chromatin when DNA replication was blocked by the addition of Cdc6 in the licensed state before origin firing. Cdc6-induced removal of FACT was blocked by the inhibition of origin licensing with geminin, but not by suppressing the activity of DNA polymerases, CDK, or Cdc7. Furthermore, chromatin transfer experiments revealed that impairing the later binding of FACT severely compromises DNA replication activity. Taken together, we propose that even though FACT has rapid chromatin-binding activity, the binding pattern of FACT on chromatin changes after origin licensing, which may contribute to the establishment of its functional link to the DNA replication machinery.
“…S8). p53 (TP53) contributes to CDC6 inhibition 24 , which results in a decreased binding affinity to origin-recognition complexes (ORC), and the formation of the pre-replication complexes (pre-RC), followed by the recruitment of the DNA replicative helicase MCM complex 25 . In addition, the phosphorylation of POLA and MCM protein binding at pre-RC is decreased as a result of the decreased transcript or protein expression of the CDC7/DBF4 complex 26 , and these interaction flows reflect the impairment of normal DNA replication processes.Figure 5Regulatory network model of DNA replication coupled with nicotinamide.…”
Triple negative breast cancer (TNBC) is characterized by an aggressive biological behavior in the absence of a specific target agent. Nicotinamide has recently been proven to be a novel therapeutic agent for skin tumors in an ONTRAC trial. We performed combinatory transcriptomic and in-depth proteomic analyses to characterize the network of molecular interactions in TNBC cells treated with nicotinamide. The multi-omic profiles revealed that nicotinamide drives significant functional alterations related to major cellular pathways, including the cell cycle, DNA replication, apoptosis and DNA damage repair. We further elaborated the global interaction networks of molecular events via nicotinamide-inducible expression changes at the mRNA and functional protein levels. This approach indicated that nicotinamide treatment rewires interaction networks toward dysfunction in DNA damage repair and away from a pro-growth state in TNBC. To our knowledge, the high-resolution network interactions identified in the present study provide the first evidence to comprehensively support the hypothesis of nicotinamide as a novel therapeutic agent in TNBC.
“…Cdc6 inhibits helicase activation by blocking Cdc7-mediated phosphorylation of the MCM helicase (26). We hypothesized that the Cdc6-mediated increase in MCM chromatin loading was associated with an inhibition of helicase activation.…”
Section: Resultsmentioning
confidence: 99%
“…ORC subsequently binds Cdc6 (22) and Cdt1 (23), leading to recruitment of the minichromosome maintenance (MCM) helicase (24), which is a hexamer consisting of MCM2 to 7, that functions to unwind DNA during replication (25). However, Cdc6 and Cdt1 inhibit activation of the MCM helicase until the start of S phase (26), when Cdc6 is phosphorylated by S phase CDKs, leading to its nuclear export and degradation (27, 28). Inactivation of Cdc6 and Cdt1 allows Cdc7 to phosphorylate the MCM helicase at the start of S phase (29), leading to its activation.…”
Cell cycle arrest in response to hypoxia is a fundamental physiological mechanism to maintain a balance between O2 supply and demand. Many of the cellular responses to reduced O2 availability are mediated through the transcriptional activity of hypoxia-inducible factor 1 (HIF-1). We report a role for the isolated HIF-1α subunit as an inhibitor of DNA replication, and this role was independent of HIF-1β and transcriptional regulation. In response to hypoxia, HIF-1α bound to Cdc6, a protein that is essential for loading of the minichromosome maintenance (MCM) complex (which has DNA helicase activity) onto DNA, and promoted the interaction between Cdc6 and the MCM complex. Although the interaction between Cdc6 and the MCM complex increased the association of the MCM proteins with chromatin, the binding of HIF-1α to the complex decreased phosphorylation and activation of the MCM complex by the kinase Cdc7. As a result, HIF-1α inhibited firing of replication origins, decreased DNA replication, and induced cell cycle arrest in various cell types. These findings establish a transcription-independent mechanism by which the stabilization of HIF-1α leads to cell cycle arrest in response to hypoxia.
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