Deregulated Cdc6 inhibits DNA replication and suppresses Cdc7-mediated phosphorylation of Mcm2–7 complex

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 Cdc7, but before Cdk2-dependent pathways and deregulating Cdc6 after this step does not impair activation of origin firing or elongation. Detailed analyses revealed that Cdc6 deregulation leads to strong suppression of Cdc7-mediated hyperphosphorylation of Mcm4 and subsequent chromatin loading of Cdc45, Sld5 and DNA polymerase α. Mcm2 phosphorylation is also repressed although to a lesser extent. Remarkably, Cdc6 itself does not directly inhibit Cdc7 kinase activity towards Mcm2–4–6–7 in purified systems, rather modulates Mcm2–7 phosphorylation on chromatin context. Taken together, we propose that Cdc6 on chromatin acts as a modulator of Cdc7-mediated phosphorylation of Mcm2–7, and thus destabilization of Cdc6 from chromatin after licensing is a key event ensuring proper transition to the initiation of DNA replication.     

Dpb11 Protein Helps Control Assembly of the Cdc45·Mcm2-7·GINS Replication Fork Helicase

Dpb11 is required for the initiation of DNA replication in budding yeast. Dpb11 binds to S-phase cyclin-dependent kinase-phosphorylated Sld2 and Sld3 to form a ternary complex during S phase. The replication fork helicase in eukaryotes is composed of Cdc45, Mcm2-7, and GINS. We show here, using purified proteins from budding yeast, that Dpb11 alone binds to Mcm2-7 and that Dpb11 also competes with GINS for binding to Mcm2-7. Furthermore, Dpb11 binds directly to single-stranded DNA (ssDNA), and ssDNA inhibits the Dpb11 interaction with Mcm2-7. We also found that Dpb11 can recruit Cdc45 to Mcm2-7. We identified a mutant of the BRCT4 motif of Dpb11 that remains bound to Mcm2-7 in the presence of ssDNA (dpb11-m1,m2,m3,m5), and this mutant exhibits a DNA replication defect when expressed in budding yeast cells. Expression of this mutant results in increased interaction between Dpb11 and Mcm2-7 during S phase, impaired GINS interaction with Mcm2-7 during S phase, and decreased replication protein A (RPA) interaction with origin DNA during S phase. We propose a model in which Dpb11 first recruits Cdc45 to Mcm2-7. Dpb11, although bound to Cdc45·Mcm2-7, can block the interaction between GINS and Mcm2-7. Upon extrusion of ssDNA from the central channel of Mcm2-7, Dpb11 dissociates from Mcm2-7, and Dpb11 binds to ssDNA, thereby allowing GINS to bind to Cdc45·Mcm2-7. Finally, we propose that Dpb11 functions with Sld2 and Sld3 to help control the assembly of the replication fork helicase.     

DAMpred: Recognizing Disease-Associated nsSNPs through Bayes-Guided Neural-Network Model Built on Low-Resolution Structure Prediction of Proteins and Protein–Protein Interactions

Nearly one-third of non-synonymous single-nucleotide polymorphism (nsSNPs) are deleterious to human health, but recognition of the disease-associated mutations remains a significant unsolved problem. We proposed a new algorithm, DAMpred, to identify disease-causing nsSNPs through the coupling of evolutionary profiles with structure predictions of proteins and protein–protein interactions. The pipeline was trained by a novel Bayes-guided artificial neural network algorithm that incorporates posterior probabilities of distinct feature classifiers with the network training process. DAMpred was tested on a large-scale data set involving 10,635 nsSNPs from 2154 ORFs in the human genome and recognized disease-associated nsSNPs with an accuracy 0.80 and a Matthews correlation coefficient of 0.601, which is 9.1% higher than the best of other state-of-the-art methods. In the blind test on the TP53 gene, DAMpred correctly recognized the mutations causative of Li–Fraumeni-like syndrome with a Matthews correlation coefficient that is 27% higher than the control methods. The study demonstrates an efficient avenue to quantitatively model the association of nsSNPs with human diseases from low-resolution protein structure prediction, which should find important usefulness in diagnosis and treatment of genetic diseases.     

DNA Replication Checkpoint Signaling Depends on a Rad53–Dbf4 N-Terminal Interaction in Saccharomyces cerevisiae

Dbf4-dependent kinase (DDK) and cyclin-dependent kinase (CDK) are essential to initiate DNA replication at individual origins. During replication stress, the S-phase checkpoint inhibits the DDK- and CDK-dependent activation of late replication origins. Rad53 kinase is a central effector of the replication checkpoint and both binds to and phosphorylates Dbf4 to prevent late-origin firing. The molecular basis for the Rad53–Dbf4 physical interaction is not clear but occurs through the Dbf4 N terminus. Here we found that both Rad53 FHA1 and FHA2 domains, which specifically recognize phospho-threonine (pT), interacted with Dbf4 through an N-terminal sequence and an adjacent BRCT domain. Purified Rad53 FHA1 domain (but not FHA2) bound to a pT Dbf4 peptide in vitro, suggesting a possible phospho-threonine-dependent interaction between FHA1 and Dbf4. The Dbf4–Rad53 interaction is governed by multiple contacts that are separable from the Cdc5- and Msa1-binding sites in the Dbf4 N terminus. Importantly, abrogation of the Rad53–Dbf4 physical interaction blocked Dbf4 phosphorylation and allowed late-origin firing during replication checkpoint activation. This indicated that Rad53 must stably bind to Dbf4 to regulate its activity.     

Keep Your Fingers Off My DNA: Protein–Protein Interactions Mediated by C2H2 Zinc Finger Domains

Cys2-His2 (C2H2) zinc finger domains (ZFs) were originally identified as DNA-binding domains, and uncharacterized domains are typically assumed to function in DNA binding. However, a growing body of evidence suggests an important and widespread role for these domains in protein binding. There are even examples of zinc fingers that support both DNA and protein interactions, which can be found in well-known DNA-binding proteins such as Sp1, Zif268, and Ying Yang 1 (YY1). C2H2 protein–protein interactions (PPIs) are proving to be more abundant than previously appreciated, more plastic than their DNA-binding counterparts, and more variable and complex in their interactions surfaces. Here we review the current knowledge of over 100 C2H2 zinc finger-mediated PPIs, focusing on what is known about the binding surface, contributions of individual fingers to the interaction, and function. An accurate understanding of zinc finger biology will likely require greater insights into the potential protein interaction capabilities of C2H2 ZFs.     

Breast Cancer Proteins PALB2 and BRCA2 Stimulate Polymerase η in Recombination-Associated DNA Synthesis at Blocked Replication Forks

1. Download : Download full-size image

     

The Cdc45·Mcm2-7·GINS protein complex in trypanosomes regulates DNA replication and interacts with two Orc1-like proteins in the origin recognition complex

Accurate DNA replication requires a complex interplay of many regulatory proteins at replication origins. The CMG (Cdc45·Mcm2-7·GINS) complex, which is composed of Cdc45, Mcm2-7, and the GINS (Go-Ichi-Ni-San) complex consisting of Sld5 and Psf1 to Psf3, is recruited by Cdc6 and Cdt1 onto origins bound by the heterohexameric origin recognition complex (ORC) and functions as a replicative helicase. Trypanosoma brucei, an early branched microbial eukaryote, appears to express an archaea-like ORC consisting of a single Orc1/Cdc6-like protein. However, unlike archaea, trypanosomes possess components of the eukaryote-like CMG complex, but whether they form an active helicase complex, associate with the ORC, and regulate DNA replication remains unknown. Here, we demonstrated that the CMG complex is formed in vivo in trypanosomes and that Mcm2-7 helicase activity is activated by the association with Cdc45 and the GINS complex in vitro. Mcm2-7 and GINS proteins are confined to the nucleus throughout the cell cycle, whereas Cdc45 is exported out of the nucleus after DNA replication, indicating that nuclear exclusion of Cdc45 constitutes one mechanism for preventing DNA re-replication in trypanosomes. With the exception of Mcm4, Mcm6, and Psf1, knockdown of individual CMG genes inhibits DNA replication and cell proliferation. Finally, we identified a novel Orc1-like protein, Orc1b, as an additional component of the ORC and showed that both Orc1b and Orc1/Cdc6 associate with Mcm2-7 via interactions with Mcm3. All together, we identified the Cdc45·Mcm2-7·GINS complex as the replicative helicase that interacts with two Orc1-like proteins in the unusual origin recognition complex in trypanosomes.     

Widdrol activates DNA damage checkpoint through the signaling Chk2–p53–Cdc25A–p21–MCM4 pathway in HT29 cells

Widdrol is an odorant compound isolated from Juniperus chinensis. We previously reported that widdrol induces Gap 1 (G1) phase cell cycle arrest and leads to apoptosis in human colon adenocarcinoma HT29 cells. It was also reported that this cell cycle arrest is associated with the induction of checkpoint kinase 2 (Chk2), p53 phosphorylation and cyclin dependent kinase (Cdk) inhibitor p21 expression. In this paper, we investigated the molecular mechanisms of widdrol on the activation of G1 DNA damage checkpoint at early phase when DNA damages occurred in HT29 cells. First of all, we examined that widdrol breaks DNA directly or not. As the results of DNA electrophoresis and formation of phosphorylated histone H2AX (γH2AX) foci in HT29 cells, widdrol generates DNA double-strand breaks directly within 0.5?h both in vitro and in vivo. Based on this result, the change of proteins related in checkpoint pathway was examined over a time course of 0.5-24?h. Treatment of HT29 cells with widdrol elicits the following: (1) phosphorylation of Chk2 and p53, (2) reduction of cell division cycle 25A (Cdc25A) expression, (3) increase of Cdk inhibitor p21 expression, and (4) decrease of the levels of Cdk2 and cyclin E expression in a time-dependent manner. Moreover, only the expression level of mini-chromosome maintenance 4 (MCM4) protein, a subunit of the eukaryotic DNA replicative helicase, is rapidly down-regulated in HT29 cells treated with widdrol over the same time course, but those of the other MCM proteins are unchanged. Overall, our results indicated that widdrol breaks DNA directly in HT29 cells, and this DNA damage results in checkpoint activation via Chk2-p53-Cdc25A-p21-MCM4 pathway and finally cells go to G1-phase cell cycle arrest and apoptosis.     

SARS-COV-2 Coronavirus Papain-like Protease PLpro as an Antiviral Target for Inhibitors of Active Site and Protein–Protein Interactions

Biophysics - The papain-like protease PLpro of the SARS-CoV-2 coronavirus is a multifunctional enzyme that catalyzes the proteolytic processing of two viral polyproteins, pp1a and pp1ab. PLpro also...     

α7 and β2 Nicotinic Acetylcholine Receptor Subunits Form Heteromeric Receptor Complexes that Are Expressed in the Human Cortex and Display Distinct Pharmacological Properties

The existence of α7β2 nicotinic acetylcholine receptors (nAChRs) has recently been demonstrated in both the rodent and human brain. Since α7-containing nAChRs are promising drug targets for schizophrenia and Alzheimer’s disease, it is critical to determine whether α7β2 nAChRs are present in the human brain, in which brain areas, and whether they differ functionally from α7 nAChR homomers. We used α-bungarotoxin to affinity purify α7-containing nAChRs from surgically excised human temporal cortex, and found that α7 subunits co-purify with β2 subunits, indicating the presence of α7β2 nAChRs in the human brain. We validated these results by demonstrating co-purification of β2 from wild-type, but not α7 or β2 knock-out mice. The pharmacology and kinetics of human α7β2 nAChRs differed significantly from that of α7 homomers in response to nAChR agonists when expressed in Xenopus oocytes and HEK293 cells. Notably, α7β2 heteromers expressed in HEK293 cells display markedly slower rise and decay phases. These results demonstrate that α7 subunits in the human brain form heteromeric complexes with β2 subunits, and that human α7β2 nAChR heteromers respond to nAChR agonists with a unique pharmacology and kinetic profile. α7β2 nAChRs thus represent an alternative mechanism for the reported clinical efficacy of α7 nAChR ligands.     

Noncanonical Gβ Gib2 Is a Scaffolding Protein Promoting cAMP Signaling through Functions of Ras1 and Cac1 Proteins in Cryptococcus neoformans

Gβ-like/RACK1 functions as a key mediator of various pathways and contributes to numerous cellular functions in eukaryotic organisms. In the pathogenic fungus Cryptococcus neoformans, noncanonical Gβ Gib2 promotes cAMP signaling in cells lacking normal Gpa1 function while displaying versatility in interactions with Gα Gpa1, protein kinase Pkc1, and endocytic intersectin Cin1. To elucidate the Gib2 functional mechanism(s), we demonstrate that Gib2 is required for normal growth and virulence. We show that Gib2 directly binds to Gpa1 and Gγ Gpg1/Gpg2 and that it interacts with phosphodiesterase Pde2 and monomeric GTPase Ras1. Pde2 remains functionally dispensable, but Ras1 is found to associate with adenylyl cyclase Cac1 through the conserved Ras association domain. In addition, the ras1 mutant exhibits normal capsule formation, whereas the ras1 gpa1 mutant displays enhanced capsule formation, and the ras1 gpa1 cac1 mutant is acapsular. Collectively, these findings suggest that Gib2 promotes cAMP levels by relieving an inhibitory function of Ras1 on Cac1 in the absence of Gpa1. In addition, using GST affinity purification combined with mass spectrometry, we identified 47 additional proteins that interact with Gib2. These proteins have putative functions ranging from signal transduction, energy generation, metabolism, and stress response to ribosomal function. After establishing and validating a protein-protein interactive network, we believe Gib2 to be a key adaptor/scaffolding protein that drives the formation of various protein complexes required for growth and virulence. Our study reveals Gib2 as an essential component in deciphering the complexity of regulatory networks that control growth and virulence in C. neoformans.     

Sulforaphane inhibits pancreatic cancer through disrupting Hsp90–p50Cdc37 complex and direct interactions with amino acids residues of Hsp90

Sulforaphane [1-isothiocyanato-4-(methyl-sulfinyl) butane)], an isothiocyanate derived from cruciferous vegetables, has been shown to possess potent chemopreventive activity. We analyzed the effect of sulforaphane on the proliferation of pancreatic cancer cells. Sulforaphane inhibited pancreatic cancer cell growth in vitro with IC₅₀s of around 10–15 μM and induced apoptosis. In pancreatic cancer xenograft mouse model, administration of sulforaphane showed remarkable inhibition of tumor growth without apparent toxicity noticed. We found that sulforaphane induced the degradation of heat shock protein 90 (Hsp90) client proteins and blocked the interaction of Hsp90 with its cochaperone p50^Cdc37 in pancreatic cancer cells. Using nuclear magnetic resonance spectroscopy (NMR) with an isoleucine-specific labeling strategy, we overcame the protein size limit of conventional NMR and studied the interaction of sulforaphane with full-length Hsp90 dimer (170 kDa) in solution. NMR revealed multiple chemical shifts in sheet 2 and the adjacent loop in Hsp90 N-terminal domain after incubation of Hsp90 with sulforaphane. Liquid chromatography coupled to mass spectrometry further mapped a short peptide in this region that was tagged with sulforaphane. These data suggest a new mechanism of sulforaphane that disrupts protein–protein interaction in Hsp90 complex for its chemopreventive activity.     

Potyviral VPg and HC-Pro Proteins and the Cellular Translation Initiation Factor eIF(iso)4E Interact with Exoribonuclease Rrp6 and a Small α-Heat Shock Protein

The helper-component proteinase (HC-Pro) of potyvirus is a multifunctional protein involved in many mechanisms of viral life cycle. In addition, HC-Pro protein was the first identified suppressor of RNA silencing in plants. However, the identities and functions of direct targets toward the pathways of RNA-silencing suppression mediated by HC-Pro are still to be determined. Here, a yeast two-hybrid search for potyviral HC-Pro interacting tobacco proteins was done to identify host partners and potential silencing suppressors. Two interacting cDNA clones were isolated. One of them encodes an Rrp6-like protein, a subunit of the exosome complex that belongs to the RNase D family of the DEDD superfamily of 3′–5′ hydrolytic exoribonucleases. The other clone codes for a small α-heat shock protein (α-Hsp). The interactions were validated by cross interaction assays with other potyviral HC-Pro proteins. Moreover, both identified clones also interacted with pathogenic viral protein-linked genomes (VPgs) and with translation eukaryotic initiation factors (iso) 4E (eIF(iso)4E) which are host determinants of resistance or susceptibility to potyvirus infections. All together, these findings emphasize the role of the potyviral HC-Pro and VPg proteins and the translation initiation factor eIF(iso)4E, as key players of the plant–virus interplay, where the exoribonuclease Rrp6 and a small α-heat shock protein appear as novel sharing targets.     

Phosphorylation Sites Within α4 Subunits of α4β2 Neuronal Nicotinic Receptors: A Comparison of Substrate Specificities for cAMP-Dependent Protein Kinase (PKA) and Protein Kinase C (PKC)

The present study determined whether putative phosphorylation sites within the M3/M4 cytoplasmic domain of the human 4 subunit of 42 neuronal nicotinic receptors are substrates for cAMP-dependent protein kinase (PKA) or protein kinase C (PKC). Five peptides corresponding to predicted phosphorylation sequences were synthesized, and phosphorylation was compared with standard peptide substrates for each kinase, that is, Kemptide for PKA and glycogen synthase (GS) 1-8 for PKC. VRCRSRSI had the highest affinity for PKA, with a Km of 44.5 M; Kemptide had a Km of 7.7 M. LMKRPSVVK and KARSLSVQH were also phosphorylated by PKA, but had lower affinities of 593 M and 2896 M, respectively. LMKRPSVVK had the highest affinity for PKC with a Km of 182 M; GS 1–8 had a Km of 2.1 M. VRCRSRSI had a comparative affinity for PKC with a Km of 327 M. PCKCTCKK was not phosphorylated by PKA, but was a substrate for PKC with a Km of 1392 M, whereas PGPSCKSP was not phosphorylated by either kinase. Based on these findings, results suggest that Ser-362 and Ser-486 on the human 4 subunit may be phosphorylated by either PKA or PKC, Ser-467 is a putative PKA site, and Thr-532 represents a likely PKC substrate; Ser-421 does not appear to be phosphorylated by either kinase.