Replication licensing and the DNA damage checkpoint

Accurate and timely duplication of chromosomal DNA requires that replication be coordinated with processes that ensure genome integrity. Significant advances in determining how the earliest steps in DNA replication are affected by DNA damage have highlighted some of the mechanisms to establish that coordination. Recent insights have expanded the relationship between the ATM and ATR-dependent checkpoint pathways and the proteins that bind and function at replication origins. These findings suggest that checkpoints and replication are more intimately associated than previously appreciated, even in the absence of exogenous DNA damage. This review summarizes some of these developments

G1 Dynamics at the Crossroads of Pluripotency and Cancer

G1 cell cycle phase dynamics are regulated by intricate networks involving cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors, which control G1 progression and ensure proper cell cycle transitions. Moreover, adequate origin licensing in G1 phase, the first committed step of DNA replication in the subsequent S phase, is essential to maintain genome integrity. In this review, we highlight the intriguing parallels and disparities in G1 dynamics between stem cells and cancer cells, focusing on their regulatory mechanisms and functional outcomes. Notably, SOX2, OCT4, KLF4, and the pluripotency reprogramming facilitator c-MYC, known for their role in establishing and maintaining stem cell pluripotency, are also aberrantly expressed in certain cancer cells. In this review, we discuss recent advances in understanding the regulatory role of these pluripotency factors in G1 dynamics in the context of stem cells and cancer cells, which may offer new insights into the interconnections between pluripotency and tumorigenesis

Nucleosomes in the neighborhood: New roles for chromatin modifications in replication origin control

The importance of local chromatin structure in regulating replication initiation has become increasingly apparent. Most recently, histone methylation and nucleosome positioning have been added to the list of modifications demonstrated to regulate origins. In particular, the methylation states of H3K4, H3K36 and H4K20 have been associated with establishing active, repressed or poised origins depending on the timing and extent of methylation. The stability and precise positioning of nucleosomes has also been demonstrated to affect replication efficiency. Although it is not yet clear how these modifications alter the behavior of specific replication factors, ample evidence establishes their role in maintaining coordinated replication. This review will summarize recent advances in understanding these aspects of chromatin structure in DNA replication origin control

Origin licensing and p53 status regulate Cdk2 activity during G1

Origins of DNA replication are licensed through the assembly of a chromatin-bound prereplication complex. Multiple regulatory mechanisms block new prereplication complex assembly after the G1/S transition to prevent rereplication. The strict inhibition of licensing after the G1/S transition means that all origins used in S phase must have been licensed in the preceding G1. Nevertheless mechanisms that coordinate S phase entry with the completion of origin licensing are still poorly understood. We demonstrate that depletion of either of two essential licensing factors, Cdc6 or Cdt1, in normal human fibroblasts induces a G1 arrest accompanied by inhibition of cyclin E/Cdk2 activity and hypophosphorylation of Rb. The Cdk2 inhibition is attributed to a reduction in the essential activating phosphorylation of T160 and an associated delay in Cdk2 nuclear accumulation. In contrast, licensing inhibition in the HeLa or U2OS cancer cell lines failed to regulate Cdk2 or Rb phosphorylation, and these cells died by apoptosis. Co-depletion of Cdc6 and p53 in normal cells restored Cdk2 activation and Rb phosphorylation, permitting them to enter S phase with a reduced rate of replication and also to accumulate markers of DNA damage. These results demonstrate dependence on origin licensing for multiple events required for G1 progression, and suggest a mechanism to prevent premature S phase entry that functions in normal cells but not in p53-deficient cells

Coordinated Activation of the Origin Licensing Factor CDC6 and CDK2 in Resting Human Fibroblasts Expressing SV40 Small T Antigen and Cyclin E

We have previously shown that SV40 small t antigen (st) cooperates with deregulated cyclin E to activate CDK2 and bypass quiescence in normal human fibroblasts (NHF). Here we show that st expression in serum-starved and density-arrested NHF specifically induces up-regulation and loading of CDC6 onto chromatin. Coexpression of cyclin E results in further accumulation of CDC6 onto chromatin concomitantly with phosphorylation of CDK2 on Thr-160 and CDC6 on Ser-54. Investigation of the mechanism leading to CDC6 accumulation and chromatin loading indicates that st is a potent inducer of cdc6 mRNA expression and increases CDC6 protein stability. We also show that CDC6 expression in quiescent NHF efficiently promotes cyclin E loading onto chromatin, but it is not sufficient to activate CDK2. Moreover, we show that CDC6 expression is linked to phosphorylation of the activating T loop of CDK2 in serum-starved NHF stimulated with mitogens or ectopically expressing cyclin E and st. Our data suggest a model where the combination of st and deregulated cyclin E result in cooperative and coordinated activation of both an essential origin licensing factor, CDC6, and an activity required for origin firing, CDK2, resulting in progression from quiescence to S phase

Replication licensing promotes cyclin D1 expression and G 1 progression in untransformed human cells

Defects in DNA replication are implicated as early and causal events in malignancy. However, the immediate effects of impaired DNA replication licensing on cell cycle progression of non-malignant human cells are unknown. Therefore, we have investigated the acute effects of Mcm7 ablation using synchronized cultures of untransformed Human Dermal Fibroblasts (HDF). Mcm7 ablation elicited a G1 delay associated with impaired activation of CDK4 and CDK2 and reduced Rb phosphorylation. The cell cycle delay of Mcm7-ablated cells was not associated with a DNA damage response. However, levels of cyclin D1 mRNA were specifically reduced and binding of RNA Polymerase II to the CYCD1 promoter was decreased in Mcm7-depleted cells. Similar to Mcm7-deficiency, Mcm2- or Cdc6-depletion led to impaired cyclin D expression. Ectopic overexpression of Cdc6 in quiescent cells promoted cyclin D1 expression, CDK4 activation and G1 progression. Therefore timely and efficient expression of cyclin D1 during G1 phase requires replication licensing. Reconstitution of cyclin D1 expression was insufficient to correct the G1 delay of Mcm7-depleted cells, indicating that additional cell cycle events during G1 are dependent on replication licensing. However, ectopic expression of the HPV-E7 oncoprotein, and the resulting bypass of the requirement for cyclin D1-Rb signaling enabled Mcm7-depleted cells to enter S-phase. HPV-E7-induced S-phase entry of Mcm7-depleted cells led to a DNA damage response, a hallmark of pre-malignancy. Taken together, our results suggest the existence of a ‘replication licensing restriction point’ that couples pre-RC assembly with G1 progression in normal cells to minimize replication stress, DNA damage and tumorigenesis

CDK4/6 inhibitors induce replication stress to cause long-term cell cycle withdrawal

CDK4/6 inhibitors arrest the cell cycle in G1‐phase. They are approved to treat breast cancer and are also undergoing clinical trials against a range of other tumour types. To facilitate these efforts, it is important to understand why a cytostatic arrest in G1 causes long‐lasting effects on tumour growth. Here, we demonstrate that a prolonged G1 arrest following CDK4/6 inhibition downregulates replisome components and impairs origin licencing. Upon release from that arrest, many cells fail to complete DNA replication and exit the cell cycle in a p53‐dependent manner. If cells fail to withdraw from the cell cycle following DNA replication problems, they enter mitosis and missegregate chromosomes causing excessive DNA damage, which further limits their proliferative potential. These effects are observed in a range of tumour types, including breast cancer, implying that genotoxic stress is a common outcome of CDK4/6 inhibition. This unanticipated ability of CDK4/6 inhibitors to induce DNA damage now provides a rationale to better predict responsive tumour types and effective combination therapies, as demonstrated by the fact that CDK4/6 inhibition induces sensitivity to chemotherapeutics that also cause replication stress

Cell Cycle-Regulated Protein Abundance Changes in Synchronously Proliferating HeLa Cells Include Regulation of Pre-mRNA Splicing Proteins

Cell proliferation involves dramatic changes in DNA metabolism and cell division, and control of DNA replication, mitosis, and cytokinesis have received the greatest attention in the cell cycle field. To catalogue a wider range of cell cycle-regulated processes, we employed quantitative proteomics of synchronized HeLa cells. We quantified changes in protein abundance as cells actively progress from G1 to S phase and from S to G2 phase. We also describe a cohort of proteins whose abundance changes in response to pharmacological inhibition of the proteasome. Our analysis reveals not only the expected changes in proteins required for DNA replication and mitosis but also cell cycle-associated changes in proteins required for biological processes not known to be cell-cycle regulated. For example, many pre-mRNA alternative splicing proteins are down-regulated in S phase. Comparison of this dataset to several other proteomic datasets sheds light on global mechanisms of cell cycle phase transitions and underscores the importance of both phosphorylation and ubiquitination in cell cycle changes

CDK1-dependent Inhibition of the E3 Ubiquitin Ligase CRL4 CDT2 Ensures Robust Transition from S Phase to Mitosis

Replication-coupled destruction of a cohort of cell cycle proteins ensures efficient and precise genome duplication. Three proteins destroyed during replication via the CRL4CDT2 ubiquitin E3 ligase, CDT1, p21, and SET8 (PR-SET7), are also essential or important during mitosis, making their reaccumulation after S phase a critical cell cycle event. During early and mid-S phase and during DNA repair, proliferating cell nuclear antigen (PCNA) loading onto DNA (PCNADNA) triggers the interaction between CRL4CDT2 and its substrates, resulting in their degradation. We have discovered that, beginning in late S phase, PCNADNA is no longer sufficient to trigger CRL4CDT2-mediated degradation. A CDK1-dependent mechanism that blocks CRL4CDT2 activity by interfering with CDT2 recruitment to chromatin actively protects CRL4CDT2 substrates. We postulate that deliberate override of replication-coupled destruction allows anticipatory accumulation in late S phase. We further show that (as for CDT1) de novo SET8 reaccumulation is important for normal mitotic progression. In this manner, CDK1-dependent CRL4CDT2 inactivation contributes to efficient transition from S phase to mitosis