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The Licensing Factor Cdt1 Links Cell Cycle Progression to the DNA

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The Licensing Factor Cdt1 Links Cell Cycle Progression to the DNA ANTICANCER RESEARCH 40 : 2449-2456 (2020) doi:10.21873/anticanres.14214 Review The Licensing Factor Cdt1 Links Cell Cycle Progression to the DNA Damage Response ALEXANDRA KANELLOU, NICKOLAOS NIKIFOROS GIAKOUMAKIS, ANDREAS PANAGOPOULOS, SPYRIDON CHAMPERIS TSANIRAS and ZOI LYGEROU School of Medicine, University of Patras, Patras, Greece Abstract. The maintenance of genome integrity is essential (1, 2). In addition, mistakes made during DNA replication or for cellular survival and propagation. It relies upon the damage to DNA brought about by endogenous or exogenous accurate and timely replication of the genetic material, as well factors must be quickly sensed and repaired. For this reason, as the rapid sensing and repairing of damage to DNA. hundreds of cellular proteins constantly scan the DNA to Uncontrolled DNA replication and unresolved DNA lesions recognize damaged sites and recruit additional protein contribute to genomic instability and can lead to cancer. complexes to orchestrate repair and signal to the cell cycle Chromatin licensing and DNA replication factor 1 (Cdt1) is machinery the presence of damage and, therefore, the need to essential for loading the minichromosome maintenance 2-7 halt further cell cycle progression. This DNA damage response helicase complex onto chromatin exclusively during the G 1 (DDR) must be closely coordinated with the cell cycle phase of the cell cycle, thus limiting DNA replication to once machinery to ensure the maintenance of genomic stability. per cell cycle. Upon DNA damage, Cdt1 rapidly accumulates Factors linking the DDR to the cell cycle have attracted to sites of damage and is subsequently poly-ubiquitinated by significant attention in recent years (3-6), while defects in this the cullin 4-RING E3 ubiquitin ligase complex, in conjunction coordination can lead to genomic instability and are closely with the substrate recognition factor Cdt2 (CRL4 Cdt2 ), and linked to disease, most prominently to cancer (7, 8). targeted for degradation. We here discuss the cellular functions In this review, we focus on the chromatin licensing and of Cdt1 and how it may interlink cell cycle regulation and DNA DNA replication factor 1 (Cdt1), a cell cycle regulator damage response pathways, contributing to genome stability. essential for timely DNA replication. We discuss the cellular function of Cdt1, as well as its link with the pathways that During every cell division, cells must ensure that they pass sense and repair DNA damage. Moreover, we describe how down a full and accurate copy of their genetic material to their this link could affect both cell cycle progression and cellular daughter cells. To achieve this, all eukaryotic cells carry out DNA damage response. Experimental data from our group a strict series of events, known as the cell cycle, which permits (Figures 1-3) are used to illustrate key points, consistent with genomic DNA to be copied once and only once during the S earlier findings. phase of the cell cycle, before one copy of each chromosome is passed down to the daughter cells during mitosis (1, 2). Cdt1 Is a Central Regulator Multiple protein complexes associate with chromatin of DNA Replication Licensing throughout the cell cycle to coordinate cell cycle events and ensure timely DNA replication and chromosome segregation Cdt1 was originally identified in fission yeast as a gene regulated by the cell cycle transcription factor Cdc10, and was named Cdc10-dependent transcript 1 (9). It was shown to be important both for entry into S phase and for arresting This article is freely accessible online. progression to mitosis in the absence of DNA replication (9). Functional characterization of Cdt1 showed it to be required Correspondence to: Zoi Lygerou, School of Medicine, University for the loading of minichromosome maintenance (MCM) of Patras, 26504, Rio, Patras, Greece. Tel.: +30 2610 997610, Fax: +30 2610 997422, e-mail: [email protected] proteins onto DNA during the G 1 phase of the cell cycle in fission yeast (10) and Xenopus egg extracts (11). Cdt1 was Key Words: DNA replication, DNA repair, chromatin, cell cycle, also shown to enhance aberrant over-replication brought genome stability, Cdt1, review. about by ectopic expression of the licensing factor Cdc18 in 2449 ANTICANCER RESEARCH 40 : 2449-2456 (2020) fission yeast (10-14). Subsequently, it was revealed that its binds to proliferating cell nuclear antigen (PCNA) loaded function was conserved in all eukaryotes (15) and Cdt1 was onto damaged chromatin. Independent recruitment of Cdt1 accordingly renamed to chromatin licensing and DNA and Cdt2 onto DNA-bound PCNA was recently shown to be replication factor 1. required for the recognition of Cdt1 by CRL4 Cdt2 , both after In the normal cell cycle, DNA is licensed for a new round DNA damage and in S phase (37-39), ensuring that Cdt1 of DNA replication after passage through mitosis, through poly-ubiquitination takes place only on damaged or the association of the six-subunit MCM complex (MCM2-7) replicating DNA. Multiple DNA repair pathways bring about onto DNA replication origins (16, 17). Cdt1 and Cdc6 PCNA loading and Cdt1 proteolysis, including the (Cdc18 in fission yeast) are essential for MCM loading, and xeroderma pigmentosum pathway and the mismatch repair therefore for DNA replication licensing. In mammalian cells, pathway following UV-irradiation (36, 40, 41), or the cellular replication origins begin to be licensed in telophase, while response to double-strand breaks. the majority is licensed in late G 1, prior to S phase onset (16, Cdt1 proteolysis following DNA damage has been 18, 19). The MCM complex is activated as a helicase when suggested as a checkpoint, which inhibits entry into S phase, cells enter S phase, and moves ahead of the replication fork, to ensure time for repair prior to the initiation of DNA unwinding DNA for replication. Origins that have already replication. Indeed, Cdt1 depletion leads to decreased fired are not able to fire again within the same cell cycle, as licensing, while mammalian cells have been shown to licensing in inhibited and the MCM complex cannot load possess a checkpoint, which inhibits entry into S phase with onto chromatin from S phase until completion of mitosis. incomplete licensing (42-46). This checkpoint is dependent Thus, restriction of MCM complex loading exclusively in the on p53 (44). However, in cancer cells as well as in cells G1 phase is pivotal for the maintenance of genome integrity, entering the cell cycle from G 0 (47, 48), this checkpoint is and is ensured by the tight regulation of the loading factors dysfunctional contributing to the appearance of replication Cdt1 and Cdc6. stress (8). Cdt1 proteolysis following DNA damage has also Cdt1 is tightly regulated so as to be present within the been suggested as a means to inhibit re-replication in nucleus only during the G 1 phase of the cell cycle. In Figure damaged cells, where the checkpoints elicited as a response 1, it is shown that in cultured human breast cancer cells Cdt1 to the presence of damage culminate in the inhibition of is detected only in cells that are in G 1, consistent with earlier cyclin-dependent kinases (CDKs), thus alleviating CDK- work (20-22). This strict regulation of Cdt1 levels is evident mediated blocks to re-replication (49). in different tissues (23) and different species (15). Cdt1 protein levels are controlled through cell cycle-specific Cdt1 Rapidly Accumulates at Sites of DNA Damage proteolysis. More specifically, following S phase entry, Cdt1 is poly-ubiquitinated and targeted for proteasome-dependent Cdt1 accumulates on damaged chromatin prior to its proteolysis by two major E3 ubiquitin ligases, a Skp1-Cullin- proteolysis. By using a pulsed laser to induce DNA damage at F-box protein complex containing Skp2 (SCF Skp2 ) and cullin a specific subnuclear volume in living cells, Cdt1 was shown ring ligase CRL4 Cdt2 (15, 21). Aberrations in this control to be rapidly recruited to the site of DNA lesion, within bring about untimely licensing and origin refiring within the minutes of the induction of damage (50). This is also evident same cell cycle (15). It has been demonstrated that Cdt1 is when cells are locally irradiated with UV, through the use of misregulated in tumors (24-26), and this misregulation micropore filters with defined pores (37, 51). As shown in already occurs in early, precancerous lesions (27), while Figure 3, following laser micro-irradiation of MCF7 breast aberrant regulation of Cdt1 leads to dysplasia and cancer cells, Cdt1 accumulates at sites of damage, marked by malignancy in mice (28-30). Cdt1 is therefore believed to be the phosphorylated histone variant H2AX ( γH2AX). an important contributor to genomic instability during tumor PCNA and the PCNA interacting protein (PIP) box at the initiation and progression (8, 31, 32). N-terminus of Cdt1 are required for this recruitment, consistent with Cdt1 accumulation taking place through Cdt1 Is Proteolysed Following DNA Damage by binding to chromatin loaded PCNA (50, 51). Using the Cullin 4-RING E3 Ubiquitin Ligase CRL4 Cdt2 fluorescence recovery after photobleaching (FRAP) (52-54), Cdt1 was shown to exhibit dynamic binding to sites of Cdt1 has been shown to link cell cycle regulation with the damage (37, 50, 55), in contrast to PCNA, which maintains DNA damage response, as it is rapidly proteolyzed when stable interactions with damaged chromatin. The cells experience damage in G 1 (33), due to irradiation (33, accumulation of Cdt1 to sites of damage is followed by its 34), or drugs (35). As shown in Figure 2, Cdt1 levels drop proteolysis, which is usually completed within 30 min to 2 when MCF-7 breast cancer cells are exposed to ultraviolet h, depending on the cell type (37, 50). Therefore, Cdt1 irradiation. This DNA damage-dependent proteolysis of Cdt1 remains associated on damaged DNA for an appreciable time is mediated by the CRL4 Cdt2 ubiquitin ligase (21, 36), which prior to its proteolysis.
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