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

Open Biology ◽  
2021 ◽  
Vol 11 (10) ◽  
Author(s):  
Ilaria Volpi ◽  
Peter J. Gillespie ◽  
Gaganmeet Singh Chadha ◽  
J. Julian Blow
Keyword(s):  
Dna Replication ◽  
Complex Formation ◽  
S Phase ◽  
Replication Initiation ◽  
Initiation Complex ◽  
Crucial Step ◽  
Egg Extract ◽  
Initiation Of Dna Replication ◽  
Rate Limiting

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.

scholarly journals The role of DDK and Treslin-MTBP in coordinating replication licensing and pre-Initiation Complex formation

2021 ◽  
Author(s):  
Ilaria Volpi ◽  
Peter J. Gillespie ◽  
Gaganmeet Singh Chadha ◽  
J. Julian Blow
Keyword(s):  
Dna Replication ◽  
Complex Formation ◽  
S Phase ◽  
Replication Initiation ◽  
Initiation Complex ◽  
Crucial Step ◽  
Egg Extract ◽  
Initiation Of Dna Replication ◽  
Rate Limiting

AbstractTreslin/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.

scholarly journals HA95 and LAP2β mediate a novel chromatin–nuclear envelope interaction implicated in initiation of DNA replication

2003 ◽  
Vol 160 (2) ◽  
pp. 177-188 ◽  
Author(s):  
Sandra Martins ◽  
Sissel Eikvar ◽  
Kazuhiro Furukawa ◽  
Philippe Collas
Keyword(s):  
Dna Replication ◽  
Nuclear Envelope ◽  
Proteasome Inhibitors ◽  
S Phase ◽  
Binding Domain ◽  
Replication Initiation ◽  
Lamin B ◽  
Initiation Of Dna Replication ◽  
Inner Nuclear Membrane Protein

HA95 is a chromatin-associated protein that interfaces the nuclear envelope (NE) and chromatin. We report an interaction between HA95 and the inner nuclear membrane protein lamina-associated polypeptide (LAP) 2β, and a role of this association in initiation of DNA replication. Precipitation of GST–LAP2β fusion proteins and overlays of immobilized HA95 indicate that a first HA95-binding region lies within amino acids 137–242 of LAP2β. A second domain sufficient to bind HA95 colocalizes with the lamin B–binding domain of LAP2β at residues 299–373. HA95–LAP2β interaction is not required for NE formation. However, disruption of the association of HA95 with the NH2-terminal HA95-binding domain of LAP2β abolishes the initiation, but not elongation, of DNA replication in purified G1 phase nuclei incubated in S-phase extract. Inhibition of replication initiation correlates with proteasome-mediated proteolysis of Cdc6, a component of the prereplication complex. Rescue of Cdc6 degradation with proteasome inhibitors restores replication. We propose that an interaction of LAP2β, or LAP2 proteins, with HA95 is involved in the control of initiation of DNA replication.

Initiation of polyoma virus origin-dependent DNA replication through STAT5 activation by human granulocyte-macrophage colony-stimulating factor

Blood ◽  
2001 ◽  
Vol 97 (5) ◽  
pp. 1266-1273 ◽  
Author(s):  
Sumiko Watanabe ◽  
Rong Zeng ◽  
Yutaka Aoki ◽  
Tohru Itoh ◽  
Ken-ichi Arai
Keyword(s):  
Dna Replication ◽  
Transcriptional Activation ◽  
Tandem Repeats ◽  
T Antigen ◽  
Replication Initiation ◽  
Macrophage Colony Stimulating Factor ◽  
Initiation Of Dna Replication ◽  
Macrophage Colony ◽  
Stimulating Factor

Several lines of evidence indicate that transcriptional activation is coupled with DNA replication initiation, but the nature of initiation of DNA replication in mammalian cells is unclear. Polyoma virus replicon is an excellent system to analyze the initiation of DNA replication in murine cells because its replication requires an enhancer, and all components of replication machinery, except for DNA helicase large T antigen, are supplied by host cells. This system was used to examine the role of signal transducer and activator of transcription (STAT5) in replication initiation of polyoma replicon in the mouse lymphoid cell line BA/F3. The plasmid with tandem repeats of consensus STAT5 binding sites followed by polyoma replication origin was replicated by stimulation with human granulocyte-macrophage colony-stimulating factor (hGM-CSF) in the presence of polyoma large T antigen in BA/F3 cells. Mutation analysis of the hGM-CSF receptor β subunit revealed that only the box1 region is essential, and the C-terminal tyrosine residues are dispensable for the activity. Addition of the tyrosine kinase inhibitor genistein suppressed this replication without affecting transcriptional activation of STAT5. Because deletion analysis of STAT5 indicates the importance of the C-terminal transcriptional activation domain of STAT5 for the initiation of replication, the role of this region in the activation of replication was examined with a GAL4–STAT5 fusion protein. GAL4–STAT5 activated replication of the plasmid containing tandem repeats of GAL4 binding sites and polyoma replication origin in BA/F3 cells. Mutation analysis of GAL4–STAT5 indicated that multiple serine residues coordinately have a role in activating replication. This is the first direct evidence indicating the potential involvement of STAT5 in replication.

scholarly journals Inhibition of Human Chk1 Causes Increased Initiation of DNA Replication, Phosphorylation of ATR Targets, and DNA Breakage

2005 ◽  
Vol 25 (9) ◽  
pp. 3553-3562 ◽  
Author(s):  
Randi G. Syljuåsen ◽  
Claus Storgaard Sørensen ◽  
Lasse Tengbjerg Hansen ◽  
Kasper Fugger ◽  
Cecilia Lundin ◽  
...  
Keyword(s):  
Dna Replication ◽  
Genome Stability ◽  
Protein A ◽  
S Phase ◽  
Replication Initiation ◽  
Dna Breakage ◽  
Histone H2ax ◽  
Initiation Of Dna Replication ◽  
Dna Strand ◽  
Interfering Rna

ABSTRACT Human checkpoint kinase 1 (Chk1) is an essential kinase required to preserve genome stability. Here, we show that Chk1 inhibition by two distinct drugs, UCN-01 and CEP-3891, or by Chk1 small interfering RNA (siRNA) leads to phosphorylation of ATR targets. Chk1-inhibition triggered rapid, pan-nuclear phosphorylation of histone H2AX, p53, Smc1, replication protein A, and Chk1 itself in human S-phase cells. These phosphorylations were inhibited by ATR siRNA and caffeine, but they occurred independently of ATM. Chk1 inhibition also caused an increased initiation of DNA replication, which was accompanied by increased amounts of nonextractable RPA protein, formation of single-stranded DNA, and induction of DNA strand breaks. Moreover, these responses were prevented by siRNA-mediated downregulation of Cdk2 or the replication initiation protein Cdc45, or by addition of the CDK inhibitor roscovitine. We propose that Chk1 is required during normal S phase to avoid aberrantly increased initiation of DNA replication, thereby protecting against DNA breakage. These results may help explain why Chk1 is an essential kinase and should be taken into account when drugs to inhibit this kinase are considered for use in cancer treatment.

scholarly journals Cell cycle-dependent phosphorylation and dephosphorylation of the yeast DNA polymerase alpha-primase B subunit.

1995 ◽  
Vol 15 (2) ◽  
pp. 883-891 ◽  
Author(s):  
M Foiani ◽  
G Liberi ◽  
G Lucchini ◽  
P Plevani
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Posttranslational Modifications ◽  
S Phase ◽  
Dna Polymerase Alpha ◽  
B Subunit ◽  
Initiation Of Dna Replication ◽  
Cycle Dependent

The yeast DNA polymerase alpha-primase B subunit functions in initiation of DNA replication. This protein is present in two forms, of 86 and 91 kDa, and the p91 polypeptide results from cell cycle-regulated phosphorylation of p86. The B subunit present in G1 arises by dephosphorylation of p91 while cells are exiting from mitosis, becomes phosphorylated in early S phase, and is competent and sufficient to initiate DNA replication. The B subunit transiently synthesized as a consequence of periodic transcription of the POL12 gene is phosphorylated no earlier than G2. Phosphorylation of the B subunit does not require execution of the CDC7-dependent step and ongoing DNA synthesis. We suggest that posttranslational modifications of the B subunit might modulate the role of DNA polymerase alpha-primase in DNA replication.

scholarly journals Cell Cycle Control of Cdc7p Kinase Activity through Regulation of Dbf4p Stability

1999 ◽  
Vol 19 (7) ◽  
pp. 4888-4896 ◽  
Author(s):  
Guy Oshiro ◽  
Julia C. Owens ◽  
Yiqun Shellman ◽  
Robert A. Sclafani ◽  
Joachim J. Li
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Dna Replication ◽  
Kinase Activity ◽  
Cell Cycle Control ◽  
S Phase ◽  
Regulatory Subunit ◽  
Anaphase Promoting Complex ◽  
Initiation Of Dna Replication

ABSTRACT In Saccharomyces cerevisiae, the heteromeric kinase complex Cdc7p-Dbf4p plays a pivotal role at replication origins in triggering the initiation of DNA replication during the S phase. We have assayed the kinase activity of endogenous levels of Cdc7p kinase by using a likely physiological target, Mcm2p, as a substrate. Using this assay, we have confirmed that Cdc7p kinase activity fluctuates during the cell cycle; it is low in the G1 phase, rises as cells enter the S phase, and remains high until cells complete mitosis. These changes in kinase activity cannot be accounted for by changes in the levels of the catalytic subunit Cdc7p, as these levels are constant during the cell cycle. However, the fluctuations in kinase activity do correlate with levels of the regulatory subunit Dbf4p. The regulation of Dbf4p levels can be attributed in part to increased degradation of the protein in G1 cells. This G1-phase instability is cdc16 dependent, suggesting a role of the anaphase-promoting complex in the turnover of Dbf4p. Overexpression of Dbf4p in the G1 phase can partially overcome this elevated turnover and lead to an increase in Cdc7p kinase activity. Thus, the regulation of Dbf4p levels through the control of Dbf4p degradation has an important role in the regulation of Cdc7p kinase activity during the cell cycle.

scholarly journals Multiple pathways can bypass the essential role of fission yeast Hsk1 kinase in DNA replication initiation

2011 ◽  
Vol 195 (3) ◽  
pp. 387-401 ◽  
Author(s):  
Seiji Matsumoto ◽  
Motoshi Hayano ◽  
Yutaka Kanoh ◽  
Hisao Masai
Keyword(s):  
Dna Replication ◽  
Fission Yeast ◽  
Replication Fork ◽  
Essential Role ◽  
Replication Initiation ◽  
Origin Firing ◽  
Physiological Conditions ◽  
Dna Replication Initiation ◽  
Initiation Of Dna Replication

Cdc7/Hsk1 is a conserved kinase required for initiation of DNA replication that potentially regulates timing and locations of replication origin firing. Here, we show that viability of fission yeast hsk1Δ cells can be restored by loss of mrc1, which is required for maintenance of replication fork integrity, by cds1Δ, or by a checkpoint-deficient mutant of mrc1. In these mutants, normally inactive origins are activated in the presence of hydroxyurea and binding of Cdc45 to MCM is stimulated. mrc1Δ bypasses hsk1Δ more efficiently because of its checkpoint-independent inhibitory functions. Unexpectedly, hsk1Δ is viable at 37°C. More DNA is synthesized, and some dormant origins fire in the presence of hydroxyurea at 37°C. Furthermore, hsk1Δ bypass strains grow poorly at 25°C compared with higher temperatures. Our results show that Hsk1 functions for DNA replication can be bypassed by different genetic backgrounds as well as under varied physiological conditions, providing additional evidence for plasticity of the replication program in eukaryotes.

A Novel Fluorescence-Based Screen for Inhibitors of the Initiation of DNA Replication in Bacteria

2019 ◽  
Vol 16 (3) ◽  
pp. 272-277 ◽  
Author(s):  
Rasmus N. Klitgaard ◽  
Anders Løbner-Olesen
Keyword(s):  
Dna Replication ◽  
High Throughput ◽  
Cyclic Peptide ◽  
Replication Initiation ◽  
False Positive Result ◽  
Over Expression ◽  
Initiator Protein ◽  
Dna Replication Initiation ◽  
Cellular Processes ◽  
Initiation Of Dna Replication

Background:One of many strategies to overcome antibiotic resistance is the discovery of compounds targeting cellular processes, which have not yet been exploited.Materials and Methods:Using various genetic tools, we constructed a novel high throughput, cellbased, fluorescence screen for inhibitors of chromosome replication initiation in bacteria.Results:The screen was validated by expression of an intra-cellular cyclic peptide interfering with the initiator protein DnaA and by over-expression of the negative initiation regulator SeqA. We also demonstrated that neither tetracycline nor ciprofloxacin triggers a false positive result. Finally, 400 extracts isolated mainly from filamentous actinomycetes were subjected to the screen.Conclusion:We concluded that the presented screen is applicable for identifying putative inhibitors of DNA replication initiation in a high throughput setup.

scholarly journals Regulation of DNA Replication Licensing and Re-Replication by Cdt1

2021 ◽  
Vol 22 (10) ◽  
pp. 5195
Author(s):  
Hui Zhang
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Replication ◽  
Genome Stability ◽  
E3 Ligase ◽  
S Phase ◽  
Replication Initiation ◽  
Nuclear Antigen ◽  
Repair Synthesis ◽  
Ubiquitin E3 Ligase

In eukaryotic cells, DNA replication licensing is precisely regulated to ensure that the initiation of genomic DNA replication in S phase occurs once and only once for each mitotic cell division. A key regulatory mechanism by which DNA re-replication is suppressed is the S phase-dependent proteolysis of Cdt1, an essential replication protein for licensing DNA replication origins by loading the Mcm2-7 replication helicase for DNA duplication in S phase. Cdt1 degradation is mediated by CRL4Cdt2 ubiquitin E3 ligase, which further requires Cdt1 binding to proliferating cell nuclear antigen (PCNA) through a PIP box domain in Cdt1 during DNA synthesis. Recent studies found that Cdt2, the specific subunit of CRL4Cdt2 ubiquitin E3 ligase that targets Cdt1 for degradation, also contains an evolutionarily conserved PIP box-like domain that mediates the interaction with PCNA. These findings suggest that the initiation and elongation of DNA replication or DNA damage-induced repair synthesis provide a novel mechanism by which Cdt1 and CRL4Cdt2 are both recruited onto the trimeric PCNA clamp encircling the replicating DNA strands to promote the interaction between Cdt1 and CRL4Cdt2. The proximity of PCNA-bound Cdt1 to CRL4Cdt2 facilitates the destruction of Cdt1 in response to DNA damage or after DNA replication initiation to prevent DNA re-replication in the cell cycle. CRL4Cdt2 ubiquitin E3 ligase may also regulate the degradation of other PIP box-containing proteins, such as CDK inhibitor p21 and histone methylase Set8, to regulate DNA replication licensing, cell cycle progression, DNA repair, and genome stability by directly interacting with PCNA during DNA replication and repair synthesis.

scholarly journals Role of Papillomavirus E1 Initiator Dimerization in DNA Replication

2005 ◽  
Vol 79 (13) ◽  
pp. 8661-8664 ◽  
Author(s):  
Stephen Schuck ◽  
Arne Stenlund
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Severe Effect ◽  
Origin Of Dna Replication ◽  
Initiation Of Dna Replication

ABSTRACT Viral initiator proteins are polypeptides that form oligomeric complexes on the origin of DNA replication (ori). These complexes carry out a multitude of functions related to initiation of DNA replication, and although many of these functions have been characterized biochemically, little is understood about how the complexes are assembled. Here we demonstrate that loss of one particular interaction, the dimerization between E1 DNA binding domains, has a severe effect on DNA replication in vivo but has surprisingly modest effects on most individual biochemical activities in vitro. We conclude that the dimer interaction is primarily required for initial recognition of ori.

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