Cyclin binding Cy motifs have multiple activities in the initiation of DNA replication

SummaryThe initiation of DNA replication involves the cell cycle-dependent assembly and disassembly of protein complexes, including the Origin Recognition Complex (ORC) and CDC6 AAA+ ATPases. We report that multiple short, linear protein motifs (SLiMs) within intrinsically disordered regions in ORC1 and CDC6, including Cyclin-binding (Cy) motifs, mediate Cyclin-CDK dependent and independent protein-protein interactions, conditional on cell cycle phase. The ORC1 Cy motif mediates an auto-regulatory self-interaction, and the same Cy motif prevents CDC6 binding to ORC1 in mitosis, but then facilitates the destruction of ORC1 in S phase. In contrast, in G1, the CDC6 Cy motif promotes ORC1-CDC6 interaction independent of Cyclin-CDK protein phosphorylation. CDC6 interaction with ORC also requires a basic region of ORC1 that in yeast mediates ORC-DNA interactions. We also demonstrate that protein phosphatase 1 binds directly to a SLiM in ORC1, causing de-phosphorylation upon mitotic exit. Thus, Cy-motifs have wider roles, functioning as a ligand and as a degron.

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A new class of disordered elements controls DNA replication through initiator self-assembly

eLife ◽

10.7554/elife.48562 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 22

Author(s):

Matthew W Parker ◽

Maren Bell ◽

Mustafa Mir ◽

Jonchee A Kao ◽

Xavier Darzacq ◽

...

Keyword(s):

Dna Replication ◽

Self Assembly ◽

Origin Recognition Complex ◽

Replication Initiation ◽

Linear Arrangement ◽

New Class ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Liquid Liquid Phase Separation

The initiation of DNA replication in metazoans occurs at thousands of chromosomal sites known as origins. At each origin, the Origin Recognition Complex (ORC), Cdc6, and Cdt1 co-assemble to load the Mcm2-7 replicative helicase onto chromatin. Current replication models envisage a linear arrangement of isolated origins functioning autonomously; the extent of inter-origin organization and communication is unknown. Here, we report that the replication initiation machinery of D. melanogaster unexpectedly undergoes liquid-liquid phase separation (LLPS) upon binding DNA in vitro. We find that ORC, Cdc6, and Cdt1 contain intrinsically disordered regions (IDRs) that drive LLPS and constitute a new class of phase separating elements. Initiator IDRs are shown to regulate multiple functions, including chromosome recruitment, initiator-specific co-assembly, and Mcm2-7 loading. These data help explain how CDK activity controls replication initiation and suggest that replication programs are subject to higher-order levels of inter-origin organization.

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Single-embryo phosphoproteomics reveals the importance of intrinsic disorder in cell cycle dynamics

10.1101/2021.08.29.458076 ◽

2021 ◽

Author(s):

Juan Manuel Valverde ◽

Geronimo Dubra ◽

Henk van den Toorn ◽

Guido van Mierlo ◽

Michiel Vermeulen ◽

...

Keyword(s):

Cell Cycle ◽

Intrinsically Disordered Proteins ◽

Protein Complexes ◽

Disordered Proteins ◽

Cell Cycles ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Egg Extracts ◽

Cell Cycle Dynamics

Switch-like cyclin-dependent kinase (CDK)-1 activation is thought to underlie the abruptness of mitotic onset, but how CDKs can simultaneously phosphorylate many diverse substrates is unknown, and direct evidence for such phosphorylation dynamics in vivo is lacking. Here, we analysed protein phosphorylation states in single Xenopus embryos throughout synchronous cell cycles. Over a thousand phosphosites were dynamic in vivo, and assignment of cell cycle phases using egg extracts revealed hundreds of S-phase phosphorylations. Targeted phosphoproteomics in single embryos showed switch-like mitotic phosphorylation of diverse protein complexes. The majority of cell cycle-regulated phosphosites occurred in CDK consensus motifs, and 72% located to intrinsically disordered regions. Dynamically phosphorylated proteins, and documented substrates of cell cycle kinases, are significantly more disordered than phosphoproteins in general. Furthermore, 30-50% are components of membraneless organelles. Our results suggest that phosphorylation of intrinsically disordered proteins by cell cycle kinases, particularly CDKs, allows switch-like mitotic cellular reorganisation.

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InterPep2: global peptide–protein docking using interaction surface templates

Bioinformatics ◽

10.1093/bioinformatics/btaa005 ◽

2020 ◽

Vol 36 (8) ◽

pp. 2458-2465 ◽

Cited By ~ 2

Author(s):

Isak Johansson-Åkhe ◽

Claudio Mirabello ◽

Björn Wallner

Keyword(s):

Protein Interactions ◽

Protein Complexes ◽

Structural Features ◽

Protein Docking ◽

Supplementary Information ◽

Peptide Ligand ◽

Protein Protein Interactions ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Improved Performance

Abstract Motivation Interactions between proteins and peptides or peptide-like intrinsically disordered regions are involved in many important biological processes, such as gene expression and cell life-cycle regulation. Experimentally determining the structure of such interactions is time-consuming and difficult because of the inherent flexibility of the peptide ligand. Although several prediction-methods exist, most are limited in performance or availability. Results InterPep2 is a freely available method for predicting the structure of peptide–protein interactions. Improved performance is obtained by using templates from both peptide–protein and regular protein–protein interactions, and by a random forest trained to predict the DockQ-score for a given template using sequence and structural features. When tested on 252 bound peptide–protein complexes from structures deposited after the complexes used in the construction of the training and templates sets of InterPep2, InterPep2-Refined correctly positioned 67 peptides within 4.0 Å LRMSD among top10, similar to another state-of-the-art template-based method which positioned 54 peptides correctly. However, InterPep2 displays a superior ability to evaluate the quality of its own predictions. On a previously established set of 27 non-redundant unbound-to-bound peptide–protein complexes, InterPep2 performs on-par with leading methods. The extended InterPep2-Refined protocol managed to correctly model 15 of these complexes within 4.0 Å LRMSD among top10, without using templates from homologs. In addition, combining the template-based predictions from InterPep2 with ab initio predictions from PIPER-FlexPepDock resulted in 22% more near-native predictions compared to the best single method (22 versus 18). Availability and implementation The program is available from: http://wallnerlab.org/InterPep2. Supplementary information Supplementary data are available at Bioinformatics online.

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A new class of disordered elements controls DNA replication through initiator self-assembly

10.1101/623058 ◽

2019 ◽

Cited By ~ 1

Author(s):

Matthew W. Parker ◽

Maren Bell ◽

Mustafa Mir ◽

Jonchee A. Kao ◽

Xavier Darzacq ◽

...

Keyword(s):

Dna Replication ◽

Self Assembly ◽

Origin Recognition Complex ◽

Replication Initiation ◽

Linear Arrangement ◽

New Class ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Liquid Liquid Phase Separation

SUMMARYThe initiation of DNA replication in metazoans occurs at thousands of chromosomal sites known as origins. At each origin, the Origin Recognition Complex (ORC), Cdc6, and Cdt1 co-assemble to load the Mcm2-7 replicative helicase onto chromatin. Current replication models envisage a linear arrangement of isolated origins functioning autonomously; the extent of inter-origin organization and communication is unknown. Here, we report that the replication initiation machinery of D. melanogaster unexpectedly undergoes liquid-liquid phase separation (LLPS) upon binding DNA in vitro. We find that ORC, Cdc6, and Cdt1 contain intrinsically disordered regions (IDRs) that drive LLPS and constitute a new class of phase separating elements. Initiator IDRs are shown to regulate multiple functions, including chromosome recruitment, initiator-specific co-assembly, and Mcm2-7 loading. These data help explain how CDK activity controls replication initiation and suggest that replication programs are subject to higher-order levels of inter-origin organization.

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Structure determination of protein-peptide complexes from NMR chemical shift data using MELD.

10.1101/2021.12.31.474671 ◽

2022 ◽

Author(s):

Arup Mondal ◽

G.V.T. Swapna ◽

Jingzhou Hao ◽

LiChung Ma ◽

Monica J. Roth ◽

...

Keyword(s):

Chemical Shift ◽

Protein Interactions ◽

Protein Complexes ◽

Bound State ◽

Nmr Chemical Shift ◽

Modeling Tools ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Chemical Shift Data ◽

Shift Data

Intrinsically disordered regions of proteins often mediate important protein-protein interactions. However, the folding upon binding nature of many polypeptide-protein interactions limits the ability of modeling tools to predict structures of such complexes. To address this problem, we have taken a tandem approach combining NMR chemical shift data and molecular simulations to determine structures of peptide-protein complexes. Here, we demonstrate this approach for polypeptide com-plexes formed with the extraterminal (ET) domain of bromo and extraterminal domain (BET) proteins, which exhibit a high degree of binding plasticity. This system is particularly challenging as the binding process includes allosteric changes across the ET receptor upon binding, and the polypeptide binding partners can form different conformations (e.g., helices and hair-pins) in the complex. In a blind study, the new approach successfully modeled bound-state conformations and binding pos-es, using only backbone chemical shift data, in excellent agreement with experimentally-determined structures. The approach also predicts relative binding affinities of different peptides. This hybrid MELD-NMR approach provides a powerful new tool for structural analysis of protein-polypeptide complexes in the low NMR information content regime, which can be used successfully for flexible systems where one polypeptide binding partner folds upon complex formation.

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Evolutionary Study of Disorder in Protein Sequences

Biomolecules ◽

10.3390/biom10101413 ◽

2020 ◽

Vol 10 (10) ◽

pp. 1413

Author(s):

Kristina Kastano ◽

Gábor Erdős ◽

Pablo Mier ◽

Gregorio Alanis-Lobato ◽

Vasilis J. Promponas ◽

...

Keyword(s):

Protein Interactions ◽

Intrinsically Disordered Proteins ◽

Protein Complexes ◽

Cell Types ◽

Disordered Proteins ◽

Protein Protein Interactions ◽

Globular Structure ◽

Large Variability ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions

Intrinsically disordered proteins (IDPs) contain regions lacking intrinsic globular structure (intrinsically disordered regions, IDRs). IDPs are present across the tree of life, with great variability of IDR type and frequency even between closely related taxa. To investigate the function of IDRs, we evaluated and compared the distribution of disorder content in 10,695 reference proteomes, confirming its high variability and finding certain correlation along the Euteleostomi (bony vertebrates) lineage to number of cell types. We used the comparison of orthologs to study the function of disorder related to increase in cell types, observing that multiple interacting subunits of protein complexes might gain IDRs in evolution, thus stressing the function of IDRs in modulating protein-protein interactions, particularly in the cell nucleus. Interestingly, the conservation of local compositional biases of IDPs follows residue-type specific patterns, with E- and K-rich regions being evolutionarily stable and Q- and A-rich regions being more dynamic. We provide a framework for targeted evolutionary studies of the emergence of IDRs. We believe that, given the large variability of IDR distributions in different species, studies using this evolutionary perspective are required.

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Geminin Inhibits a Late Step in the Formation of Human Pre-replicative Complexes

Journal of Biological Chemistry ◽

10.1074/jbc.m114.552935 ◽

2014 ◽

Vol 289 (44) ◽

pp. 30810-30821 ◽

Cited By ~ 8

Author(s):

Min Wu ◽

Wenyan Lu ◽

Ruth E. Santos ◽

Mark G. Frattini ◽

Thomas J. Kelly

Keyword(s):

Dna Replication ◽

Origin Recognition Complex ◽

Atp Hydrolysis ◽

Protein Complexes ◽

Initial Step ◽

High Salt ◽

Late Step ◽

Eukaryotic Dna ◽

Initiation Of Dna Replication

The initial step in initiation of eukaryotic DNA replication involves the assembly of pre-replicative complexes (pre-RCs) at origins of replication during the G1 phase of the cell cycle. In metazoans initiation is inhibited by the regulatory factor Geminin. We have purified the human pre-RC proteins, studied their interactions in vitro with each other and with origin DNA, and analyzed the effects of HsGeminin on formation of DNA-protein complexes. The formation of an initial complex containing the human origin recognition complex (HsORC), HsCdt1, HsCdc6, and origin DNA is cooperative, involving all possible binary interactions among the components. Maximal association of HsMCM2–7, a component of the replicative helicase, requires HsORC, HsCdc6, HsCdt1, and ATP, and is driven by interactions of HsCdt1 and HsCdc6 with multiple HsMCM2–7 subunits. Formation of stable complexes, resistant to high salt, requires ATP hydrolysis. In the absence of HsMCM proteins, HsGeminin inhibits the association of HsCdt1 with DNA or with HsORC-HsCdc6-DNA complexes. However, HsGeminin does not inhibit recruitment of HsMCM2–7 to DNA to form complexes containing all of the pre-RC proteins. In fact, HsGeminin itself is a component of such complexes, and interacts directly with the HsMcm3 and HsMcm5 subunits of HsMCM2–7, as well as with HsCdt1. Although HsGeminin does not prevent the initial formation of DNA-protein complexes containing the pre-RC proteins, it strongly inhibits the formation of stable pre-RCs that are resistant to high salt. We suggest that bound HsGeminin prevents transition of the pre-RC to a state that is competent for initiation of DNA replication.

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InterPep2: Global Peptide-Protein Docking with Structural Templates

10.1101/813238 ◽

2019 ◽

Cited By ~ 2

Author(s):

Isak Johansson-Åkhe ◽

Claudio Mirabello ◽

Björn Wallner

Keyword(s):

Protein Interactions ◽

Protein Complexes ◽

False Positive Rate ◽

Confidence Score ◽

Structural Features ◽

Protein Docking ◽

Second Best ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Positive Rate

AbstractMotivationInteractions between proteins and peptides or peptide-like intrinsically disordered regions are involved in many important biological processes, such as gene expression and cell life-cycle regulation. Experimentally determining the structure of such interactions is time-consuming, and because of the disordered nature of the ligand, the interactions are especially difficult to predict through software, requiring specialized solutions. Although several prediction-methods exist, most are limited in performance or availability.ResultsInterPep2 is a freely available method for predicting the structure of peptide-protein interactions. We have previously shown that structural templates can be used to accurately predict peptide-protein binding sites, and that using templates from regular protein-protein interactions will increase the number of sites found. Here, we show that the same principle can be extended to dock the peptide to the binding surface using InterPep2. A key component of InterPep2 is the ability to score plausible interaction templates using a RandomForest trained to predict the DockQ-score using sequence and structural features. InterPep2 is tested on a difficult dataset of 251 peptide-protein complexes, where it correctly positions 136 (54%) at the correct site compared to 114 (45%) for the second best method. Analyzing the confidence score InterPep2 recalls more true positives across all specificity levels compared to the second best method, for example at 10% False Positive Rate it correctly identifies 59% of the complexes compared to 44% for the second best method.AvailabilityThe program is available from: http://wallnerlab.org/InterPepContactBjörn Wallner [email protected]

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A murine replication protein accumulates temporarily in the heterochromatic regions of nuclei prior to initiation of DNA replication

Journal of Cell Science ◽

10.1242/jcs.108.3.927 ◽

1995 ◽

Vol 108 (3) ◽

pp. 927-934 ◽

Cited By ~ 2

Author(s):

M. Starborg ◽

E. Brundell ◽

K. Gell ◽

C. Larsson ◽

I. White ◽

...

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Mammalian Cells ◽

Mitotic Cell ◽

Yeast Cells ◽

Metaphase Chromosomes ◽

Licensing Factor ◽

Mammalian Homologue ◽

Initiation Of Dna Replication

We have analyzed the expression of the murine P1 gene, the mammalian homologue of the yeast MCM3 protein, during the mitotic cell cycle. The MCM3 protein has previously been shown to be of importance for initiation of DNA replication in Saccharomyces cerevisiae. We found that the murine P1 protein was present in the nuclei of mammalian cells throughout interphase of the cell cycle. This is in contrast to the MCM3 protein, which is located in the nuclei of yeast cells only between the M and the S phase of the cell cycle. Detailed analysis of the intranuclear localization of the P1 protein during the cell cycle revealed that it accumulates transiently in the heterochromatic regions towards the end of G1. The accumulation of the P1 protein in the heterochromatic regions prior to activation of DNA replication suggests that the mammalian P1 protein is also of importance for initiation of DNA replication. The MCM2-3.5 proteins have been suggested to represent yeast equivalents of a hypothetical replication licensing factor initially described in Xenopus. Our data support this model and indicate that the murine P1 protein could function as replication licensing factor. The chromosomal localization of the P1 gene was determined by fluorescence in situ hybridization to region 6p12 in human metaphase chromosomes.

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Cdc6p establishes and maintains a state of replication competence during G1 phase

Journal of Cell Science ◽

10.1242/jcs.110.6.753 ◽

1997 ◽

Vol 110 (6) ◽

pp. 753-763 ◽

Cited By ~ 1

Author(s):

C.S. Detweiler ◽

J.J. Li

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Transcriptional Repression ◽

Prolonged Exposure ◽

S Phase ◽

Restrictive Temperature ◽

Yeast Saccharomyces Cerevisiae ◽

Important Intermediate ◽

Initiation Of Dna Replication ◽

And Function

CDC6 is essential for the initiation of DNA replication in the budding yeast Saccharomyces cerevisiae. Here we examine the timing of Cdc6p expression and function during the cell cycle. Cdc6p is expressed primarily between mitosis and Start. This pattern of expression is due in part to posttranscriptional controls, since it is maintained when CDC6 is driven by a constitutively induced promoter. Transcriptional repression of CDC6 or exposure of cdc6-1(ts) cells to the restrictive temperature at mitosis blocks subsequent S phase, demonstrating that the activity of newly synthesized Cdc6p is required each cell cycle for DNA replication. In contrast, similar perturbations imposed on cells arrested in G(1) before Start have moderate or no effects on DNA replication. This suggests that, between mitosis and Start, Cdc6p functions in an early step of initiation, effectively making cells competent for replication. Prolonged exposure of cdc6-1(ts) cells to the restrictive temperature at the pre-Start arrest eventually does cripple S phase, indicating that Cdc6p also functions to maintain this initiation competence during G(1). The requirement for Cdc6p to establish and maintain initiation competence tightly correlates with the requirement for Cdc6p to establish and maintain the pre-replicative complex at a replication origin, strongly suggesting that the pre-replicative complex is an important intermediate for the initiation of DNA replication. Confining assembly of the complex to G(1) by restricting expression of Cdc6p to this period may be one way of ensuring precisely one round of replication per cell cycle.

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