The TRESLIN-MTBP complex couples completion of DNA replication with S/G2 transition.

It has been proposed that ATR kinase senses the completion of DNA replication to initiate the S/G2 transition. In contrast to this model, we show here that the TRESLIN-MTBP complex prevents a premature entry into G2 from early S-phase independently of ATR/CHK1 kinases. TRESLIN-MTBP acts transiently at pre-replication complexes (preRCs) to initiate origin firing and is released after the subsequent recruitment of CDC45. This dynamic behavior of TRESLIN-MTBP implements a monitoring system that checks the activation of replication forks and senses the rate of origin firing to prevent the entry into G2. This system detects the decline in the number of origins of replication that naturally occurs in very late S, which is the signature that cells use to determine the completion of DNA replication and permit the S/G2 transition. Our work introduces TRESLIN-MTBP as a key player in cell-cycle control independent of canonical checkpoints.

Intra-S phase checkpoint kinase Chk1 dissociates replication proteins Treslin and TopBP1 through multiple mechanisms during replication stress.

Replication stress impedes DNA polymerase progression causing activation of the ataxia telangiectasia and Rad3-related signaling pathway, which promotes the intra-S phase checkpoint activity through phosphorylation of checkpoint kinase 1 (Chk1). Chk1 suppresses replication origin firing, in part, by disrupting the interaction between the preinitiation complex components Treslin and TopBP1, an interaction that is mediated by TopBP1 BRCT domain-binding to two cyclin-dependent kinase (CDK) phosphorylation sites, T968 and S1000, in Treslin. Two nonexclusive models for how Chk1 regulates the Treslin-TopBP1 interaction have been proposed in the literature: in one model, these proteins dissociate due to a Chk1-induced decrease in CDK activity that reduces phosphorylation of the Treslin sites that bind TopBP1 and in the second model, Chk1 directly phosphorylates Treslin, resulting in dissociation of TopBP1. However, these models have not been formally examined. We show here that Treslin T968 phosphorylation was decreased in a Chk1-dependent manner, while Treslin S1000 phosphorylation was unchanged, demonstrating that T968 and S1000 are differentially regulated. However, CDK2-mediated phosphorylation alone did not fully account for Chk1 regulation of the Treslin-TopBP1 interaction. We also identified additional Chk1 phosphorylation sites on Treslin that contributed to disruption of the Treslin-TopBP1 interaction, including S1114. Finally, we showed that both of the proposed mechanisms regulate origin firing in cancer cell line models undergoing replication stress, with the relative roles of each mechanism varying among cell lines. This study demonstrates that Chk1 regulates Treslin through multiple mechanisms to promote efficient dissociation of Treslin and TopBP1 and furthers our understanding of Treslin regulation during the intra-S phase checkpoint.

Mutant p53 perturbs DNA replication checkpoint control through TopBP1 and Treslin.

Accumulating evidence supports the gain-of-function of mutant forms of p53 (mutp53s). However, whether mutp53 directly perturbs the DNA replication checkpoint remains unclear. Previously, we have demonstrated that TopBP1 forms a complex with mutp53s and mediates their gain-of-function through NF-Y and p63/p73. Akt phosphorylates TopBP1 and induces its oligomerization, which inhibits its ATR-activating function. Here we show that various contact and conformational mutp53s bypass Akt to induce TopBP1 oligomerization and attenuate ATR checkpoint response during replication stress. The effect on ATR response caused by mutp53 can be exploited in a synthetic lethality strategy, as depletion of another ATR activator, DNA2, in mutp53-R273H-expressing cancer cells renders cells hypersensitive to cisplatin. Expression of mutp53-R273H also makes cancer cells more sensitive to DNA2 depletion or DNA2 inhibitors. In addition to ATR-activating function during replication stress, TopBP1 interacts with Treslin in a Cdk-dependent manner to initiate DNA replication during normal growth. We find that mutp53 also interferes with TopBP1 replication function. Several contact, but not conformational, mutp53s enhance the interaction between TopBP1 and Treslin and promote DNA replication despite the presence of a Cdk2 inhibitor. Together, these data uncover two distinct mechanisms by which mutp53 enhances DNA replication: (i) Both contact and conformational mutp53s can bind TopBP1 and attenuate the checkpoint response to replication stress, and (ii) during normal growth, contact (but not conformational) mutp53s can override the Cdk2 requirement to promote replication by facilitating the TopBP1/Treslin interaction.

Dimerization of Firing Factors for Replication Origin Activation in Eukaryotes: A Crucial Process for Simultaneous Assembly of Bidirectional Replication Forks?

Controlling the activity of the heterohexameric Mcm2-7 replicative helicase is crucial for regulation of replication origin activity in eukaryotes. Because bidirectional replication forks are generated from every replication origin, when origins are licensed for replication in the first step of DNA replication, two inactive Mcm2-7 heterohexiameric complexes are loaded around double stranded DNA as a head-to-head double hexamer. The helicases are subsequently activated via a 'firing' reaction, in which the Mcm2-7 double hexamer is converted into two active helicase units, the CMG complex, by firing factors. Dimerization of firing factors may contribute to this process by allowing simultaneous activation of two sets of helicases and thus efficient assembly of bidirectional replication forks. An example of this is dimerization of the firing factor Sld3/Treslin/Ticrr via its binding partner, Sld7/MTBP. In organisms in which no Sld7 ortholog has been identified, such as the fission yeast Schizosaccharomyces pombe, Sld3 itself has a dimerization domain, and it has been suggested that this self-interaction is crucial for the firing reaction in this organism. Dimerization induces a conformational change in Sdl3 that appears to be critical for the firing reaction. Moreover, Mcm10 also seems to be regulated by self-interaction in yeasts. Although it is not yet clear to what extent dimerization of firing factors contributes to the firing reaction in eukaryotes, we discuss the possible roles of firing factor dimerization in simultaneous helicase activation.

The role of DDK and Treslin-MTBP in coordinating replication licensing and pre-initiation complex formation.

Treslin/Ticrr is required for the initiation of DNA replication and binds to MTBP (Mdm2 Binding Protein). Here, we show that in Xenopus egg extract, MTBP forms an elongated tetramer with Treslin containing two molecules of each protein. Immunodepletion and add-back experiments show that Treslin-MTBP is rate limiting for replication initiation. It is recruited onto chromatin before S phase starts and recruitment continues during S phase. We show that DDK activity both increases and strengthens the interaction of Treslin-MTBP with licensed chromatin. We also show that DDK activity cooperates with CDK activity to drive the interaction of Treslin-MTBP with TopBP1 which is a regulated crucial step in pre-initiation complex formation. These results suggest how DDK works together with CDKs to regulate Treslin-MTBP and plays a crucial in selecting which origins will undergo initiation.

Binding of the Treslin-MTBP Complex to Specific Regions of the Human Genome Promotes the Initiation of DNA Replication.

The processes that control where higher eukaryotic cells initiate DNA replication throughout the genome are not understood clearly. In metazoans, the Treslin-MTBP complex mediates critical final steps in formation of the activated replicative helicase prior to initiation of replication. Here, we map the genome-wide distribution of the MTBP subunit of this complex in human cells. Our results indicate that MTBP binds to at least 30,000 sites in the genome. A majority of these sites reside in regions of open chromatin that contain transcriptional-regulatory elements (e.g., promoters, enhancers, and super-enhancers), which are known to be preferred areas for initiation of replication. Furthermore, many binding sites encompass two genomic features: a nucleosome-free DNA sequence (e.g., G-quadruplex DNA or AP-1 motif) and a nucleosome bearing histone marks characteristic of open chromatin, such as H3K4me2. Taken together, these findings indicate that Treslin-MTBP associates coordinately with multiple genomic signals to promote initiation of replication.

Cell Cycle-Dependent Switch of TopBP1 Functions by Cdk2 and Akt.

Cdk2-dependent TopBP1-treslin interaction is critical for DNA replication initiation. However, it remains unclear how this association is terminated after replication initiation is finished. Here, we demonstrate that phosphorylation of TopBP1 by Akt coincides with cyclin A activation during S and G2 phases and switches the TopBP1-interacting partner from treslin to E2F1, which results in the termination of replication initiation. Premature activation of Akt in G1 phase causes an early switch and inhibits DNA replication. TopBP1 is often overexpressed in cancer and can bypass control by Cdk2 to interact with treslin, leading to enhanced DNA replication. Consistent with this notion, reducing the levels of TopBP1 in cancer cells restores sensitivity to a Cdk2 inhibitor. Together, our study links Cdk2 and Akt pathways to the control of DNA replication through the regulation of TopBP1-treslin interaction. These data also suggest an important role for TopBP1 in driving abnormal DNA replication in cancer.

Increased Expression of TICRR Predicts Poor Clinical Outcomes: A Potential Therapeutic Target for Papillary Renal Cell Carcinoma.

Background: Papillary renal cell carcinoma (PRCC), although the second-most common type of renal cell carcinoma, still lacks specific biomarkers for diagnosis, treatment, and prognosis. TopBP1-interacting checkpoint and replication regulator (TICRR) is a DNA replication initiation regulator upregulated in various cancers. We aimed to evaluate the role of TICRR in PRCC tumorigenesis and prognosis. Methods: Based on the Kidney Renal Papillary cell carcinoma Project (KIRP) on The Cancer Genome Atlas (TCGA) database, we determined the expression of TICRR using the Wilcoxon rank sum test. The biological functions of TICRR were evaluated using the Metascape database and Gene Set Enrichment Analysis (GSEA). The association between TICRR and immune cell infiltration was investigated by single sample GSEA. Logistic analysis was applied to study the correlation between TICRR expression and clinicopathological characteristics. Finally, Cox regression analysis, Kaplan-Meier analysis, and nomograms were used to determine the predictive value of TICRR on clinical outcomes in PRCC patients. Results: TICRR expression was significantly elevated in PRCC tumors (P < 0.001). Functional annotation indicated enrichment with negative regulation of cell division, cell cycle, and corresponding pathways in the high TICRR expression phenotype. High TICRR expression in PRCC was associated with female sex, younger age, and worse clinical stages. Cox regression analysis revealed that TICRR was a risk factor for overall survival [hazard ratio (HR): 2.80, P = 0.002], progression-free interval (HR: 2.86, P < 0.001), and disease-specific survival (HR: 7.03, P < 0.001), especially in patients with male sex, age below 60 years, clinical stages II-IV and clinical T stage T1-T2. Conclusion: Increased TICRR expression in PRCC might play a role in tumorigenesis by regulating the cell cycle and has prognostic value for clinical outcomes.

CKS Proteins Promote Checkpoint Recovery by Stimulating Phosphorylation of Treslin.

CKS proteins are small (9-kDa) polypeptides that bind to a subset of the cyclin-dependent kinases. The two paralogs expressed in mammals, Cks1 and Cks2, share an overlapping function that is essential for early development. However, both proteins are frequently overexpressed in human malignancy. It has been shown that CKS protein overexpression overrides the replication stress checkpoint, promoting continued origin firing. This finding has led to the proposal that CKS protein-dependent checkpoint override allows premalignant cells to evade oncogene stress barriers, providing a causal link to oncogenesis. Here, we provide mechanistic insight into how overexpression of CKS proteins promotes override of the replication stress checkpoint. We show that CKS proteins greatly enhance the ability of Cdk2 to phosphorylate the key replication initiation protein treslin in vitro Furthermore, stimulation of treslin phosphorylation does not occur by the canonical adapter mechanism demonstrated for other substrates, as cyclin-dependent kinase (CDK) binding-defective mutants are capable of stimulating treslin phosphorylation. This effect is recapitulated in vivo, where silencing of Cks1 and Cks2 decreases treslin phosphorylation, and overexpression of wild-type or CDK binding-defective Cks2 prevents checkpoint-dependent dephosphorylation of treslin. Finally, we provide evidence that the role of CKS protein-dependent checkpoint override involves recovery from checkpoint-mediated arrest of DNA replication.

Sld3-MCM Interaction Facilitated by Dbf4-Dependent Kinase Defines an Essential Step in Eukaryotic DNA Replication Initiation.

Sld3/Treslin is an evolutionarily conserved protein essential for activation of DNA helicase Mcm2-7 and replication initiation in all eukaryotes. Nevertheless, it remains elusive how Sld3 is recruited to origins. Here, we have identified the direct physical association of Sld3 with Mcm2 and Mcm6 subunits in vitro, which is significantly enhanced by DDK in vivo. The Sld3-binding domain (SBD) is mapped to the N-termini of Mcm2 and Mcm6, both of them are essential for cell viability and enriched with the DDK phosphorylation sites. Glutamic acid substitution of four conserved positively charged residues of Sld3 (sld3-4E), near the Cdc45-binding region, interrupts its interaction with Mcm2/6 and causes cell death. By using a temperature-inducible degron (td), we show that deletion of Mcm6 SBD (mcm6ΔN122) abolishes not only Sld3 enrichment at early origins in G1 phase, but also subsequent recruitment of GINS and RPA during S phase. These findings elucidate the in vivo molecular details of the DDK-dependent Sld3-MCM association, which plays a crucial role in MCM helicase activation and origin unwinding.