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Faithful genome duplication requires appropriately controlled replication origin firing. The metazoan origin firing regulation hub Treslin/TICRR and its yeast orthologue Sld3 share the Sld3-Treslin domain and the adjacent TopBP1/Dpb11 interaction domain. We report a revised domain architecture model of Treslin/TICRR. Protein sequence analyses uncovered a conserved Ku70-homologous β-barrel fold in the Treslin/TICRR middle domain (M domain) and in Sld3. Thus, the Sld3-homologous Treslin/TICRR core comprises its three central domains, M domain, Sld3-Treslin domain, and TopBP1/Dpb11 interaction domain, flanked by non-conserved terminal domains, the CIT (conserved in Treslins) and the C terminus. The CIT includes a von Willebrand factor type A domain. Unexpectedly, MTBP, Treslin/TICRR, and Ku70/80 share the same N-terminal domain architecture, von Willebrand factor type A and Ku70-like β-barrels, suggesting a common ancestry. Binding experiments using mutants and the Sld3-Sld7 dimer structure suggest that the Treslin/Sld3 and MTBP/Sld7 β-barrels engage in homotypic interactions, reminiscent of Ku70-Ku80 dimerization. Cells expressing Treslin/TICRR domain mutants indicate that all Sld3-core domains and the non-conserved terminal domains fulfil important functions during origin firing in human cells. Thus, metazoa-specific and widely conserved molecular processes cooperate during metazoan origin firing., (© 2022 Ferreira et al.)

In mammalian females, germ cells remain arrested as primordial follicles. Resumption of meiosis is heralded by germinal vesicle breakdown, condensation of chromosomes, and their eventual alignment on metaphase plates. At the first meiotic division, anaphase-promoting complex/cyclosome associated with Cdc20 (APC/C Cdc20 ) activates separase and thereby destroys cohesion along chromosome arms. Because cohesion around centromeres is protected by shugoshin-2, sister chromatids remain attached through centromeric/pericentromeric cohesin. We show here that, by promoting proteolysis of cyclins and Cdc25B at the germinal vesicle (GV) stage, APC/C associated with the Cdh1 protein (APC/C Cdh1 ) delays the increase in Cdk1 activity, leading to germinal vesicle breakdown (GVBD). More surprisingly, by moderating the rate at which Cdk1 is activated following GVBD, APC/C Cdh1 creates conditions necessary for the removal of shugoshin-2 from chromosome arms by the Aurora B/C kinase, an event crucial for the efficient resolution of chiasmata., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: TPX2 (Targeting Protein for Xklp2) is essential for spindle assembly, activation of the mitotic kinase Aurora A and for triggering microtubule nucleation. Homologs of TPX2 in Chordata and plants were previously identified. Currently, proteins of the TPX2 family have little structural information and only small parts are covered by defined protein domains., Methods: We have used computational sequence analyses and structural predictions of proteins of the TPX2 family, supported with Circular Dichroism (CD) measurements., Results: Here, we report our finding that the C-terminal domain of TPX2, which is responsible of its microtubule nucleation capacity and is conserved in all members of the family, is actually formed by tandem repeats, covering well above 2/3 of the protein. We propose that this region forms a flexible solenoid involved in protein-protein interactions. Structural prediction and molecular modeling, combined with Circular Dichroism (CD) measurements reveal a predominant alpha-helical content. Furthermore, we identify full length homologs in fungi and shorter homologs with a different domain organization in diptera (including a paralogous expansion in Drosophila)., Conclusions: Our results, represent the first computational and biophysical analysis of the TPX2 proteins family and help understand the structure and evolution of this conserved protein family to direct future structural studies.

DNA replication precisely duplicates the genome to ensure stable inheritance of genetic information. Impaired licensing of origins of replication during the G1 phase of the cell cycle has been implicated in Meier-Gorlin syndrome (MGS), a disorder defined by the triad of short stature, microtia, and a/hypoplastic patellae. Biallelic partial loss-of-function mutations in multiple components of the pre-replication complex (preRC; ORC1, ORC4, ORC6, CDT1, or CDC6) as well as de novo stabilizing mutations in the licensing inhibitor, GMNN, cause MGS. Here we report the identification of mutations in CDC45 in 15 affected individuals from 12 families with MGS and/or craniosynostosis. CDC45 encodes a component of both the pre-initiation (preIC) and CMG helicase complexes, required for initiation of DNA replication origin firing and ongoing DNA synthesis during S-phase itself, respectively, and hence is functionally distinct from previously identified MGS-associated genes. The phenotypes of affected individuals range from syndromic coronal craniosynostosis to severe growth restriction, fulfilling diagnostic criteria for Meier-Gorlin syndrome. All mutations identified were biallelic and included synonymous mutations altering splicing of physiological CDC45 transcripts, as well as amino acid substitutions expected to result in partial loss of function. Functionally, mutations reduce levels of full-length transcripts and protein in subject cells, consistent with partial loss of CDC45 function and a predicted limited rate of DNA replication and cell proliferation. Our findings therefore implicate the preIC as an additional protein complex involved in the etiology of MGS and connect the core cellular machinery of genome replication with growth, chondrogenesis, and cranial suture homeostasis., (Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

The Caenorhabditis elegans gene rec-1 was the first genetic locus identified in metazoa to affect the distribution of meiotic crossovers along the chromosome. We report that rec-1 encodes a distant paralog of HIM-5, which was discovered by whole-genome sequencing and confirmed by multiple genome-edited alleles. REC-1 is phosphorylated by cyclin-dependent kinase (CDK) in vitro, and mutation of the CDK consensus sites in REC-1 compromises meiotic crossover distribution in vivo. Unexpectedly, rec-1; him-5 double mutants are synthetic-lethal due to a defect in meiotic double-strand break formation. Thus, we uncovered an unexpected robustness to meiotic DSB formation and crossover positioning that is executed by HIM-5 and REC-1 and regulated by phosphorylation., (© 2015 Chung et al.; Published by Cold Spring Harbor Laboratory Press.)

Precise positioning of the mitotic spindle determines the correct cell division axis and is crucial for organism development. Spindle positioning is mediated through a cortical machinery by capturing astral microtubules, thereby generating pushing/pulling forces at the cell cortex. However, the molecular link between these two structures remains elusive. Here we describe a previously uncharacterized protein, MISP (C19orf21), as a substrate of Plk1 that is required for correct mitotic spindle positioning. MISP is an actin-associated protein throughout the cell cycle. MISP depletion led to an impaired metaphase-to-anaphase transition, which depended on phosphorylation by Plk1. Loss of MISP induced mitotic defects including spindle misorientation accompanied by shortened astral microtubules. Furthermore, we find that MISP formed a complex with and regulated the cortical distribution of the +TIP binding protein p150(glued), a subunit of the dynein-dynactin complex. We propose that Plk1 phosphorylates MISP, thus stabilizing cortical and astral microtubule attachments required for proper mitotic spindle positioning.

Repo-Man targets protein phosphatase 1 γ (PP1γ) to chromatin at anaphase onset and regulates chromosome structure during mitotic exit. Here, we show that a Repo-Man:PP1 complex forms in anaphase following dephosphorylation of Repo-Man. Upon activation, the complex localizes to chromosomes and causes the dephosphorylation of histone H3 (Thr3, Ser10, and Ser28). In anaphase, Repo-Man has both catalytic and structural functions that are mediated by two separate domains. A C-terminal domain localizes Repo-Man to bulk chromatin in early anaphase. There, it targets PP1 for the dephosphorylation of histone H3 and possibly other chromosomal substrates. An N-terminal domain localizes Repo-Man to the chromosome periphery later in anaphase. There, it is responsible for the recruitment of nuclear components such as Importin β and Nup153 in a PP1-independent manner. These observations identify Repo-Man as a key factor that coordinates chromatin remodeling and early events of nuclear envelope reformation during mitotic exit., (Copyright © 2011 Elsevier Inc. All rights reserved.)

DNA replication is one of the most ancient of cellular processes and functional similarities among its molecular machinery are apparent across all cellular life. Cdc45 is one of the essential components of the eukaryotic replication fork and is required for the initiation and elongation of DNA replication, but its molecular function is currently unknown. In order to trace its evolutionary history and to identify functional domains, we embarked on a computational sequence analysis of the Cdc45 protein family. Our findings reveal eukaryotic Cdc45 and prokaryotic RecJ to possess a common ancestry and Cdc45 to contain a catalytic site within a predicted exonuclease domain. The likely orthology between Cdc45 and RecJ reveals new lines of enquiry into DNA replication mechanisms in eukaryotes.

Cyclin-dependent kinases (CDKs) play crucial roles in promoting DNA replication and preventing rereplication in eukaryotic cells [1-4]. In budding yeast, CDKs promote DNA replication by phosphorylating two proteins, Sld2 and Sld3, which generates binding sites for pairs of BRCT repeats (breast cancer gene 1 [BRCA1] C terminal repeats) in the Dpb11 protein [5, 6]. The Sld3-Dpb11-Sld2 complex generated by CDK phosphorylation is required for the assembly and activation of the Cdc45-Mcm2-7-GINS (CMG) replicative helicase. In response to DNA replication stress, the interaction between Sld3 and Dpb11 is blocked by the checkpoint kinase Rad53 [7], which prevents late origin firing [7, 8]. Here we show that the two key CDK sites in Sld3 are conserved in the human Sld3-related protein Treslin/ticrr and are essential for DNA replication. Moreover, phosphorylation of these two sites mediates interaction with the orthologous pair of BRCT repeats in the human Dpb11 ortholog, TopBP1. Finally, we show that DNA replication stress prevents the interaction between Treslin/ticrr and TopBP1 via the Chk1 checkpoint kinase. Our results indicate that Treslin/ticrr is a genuine ortholog of Sld3 and that the Sld3-Dpb11 interaction has remained a critical nexus of S phase regulation through eukaryotic evolution., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The growth arrest-specific gene 1 (Gas1) protein has been proposed to function during development as an inhibitor of growth and a mediator of cell death and is also re-expressed in adult neurons during excitotoxic insult. Here we have demonstrated that the Gas1 protein shows high structural similarity to the glial cell-derived neurotrophic factor (GDNF) family receptors alpha, which mediate GDNF responses through the receptor tyrosine kinase Ret. We found that Gas1 binds Ret in a ligand-independent manner and sequesters Ret in lipid rafts. Signaling downstream of Ret is thus modified through a mechanism that involves the adaptor protein Shc as well as ERK, eventually blocking Akt activation. Consequently, when Gas1 is induced, Ret-mediated GDNF-dependent survival effects are compromised.