scholarly journals Mechanism of replication-coupled DNA-protein crosslink proteolysis by SPRTN and the proteasome

2018 ◽  
Author(s):  
Alan Gao ◽  
Nicolai B. Larsen ◽  
Justin L. Sparks ◽  
Irene Gallina ◽  
Matthias Mann ◽  
...  
Keyword(s):  
Dna Replication ◽  
S Phase ◽  
Dna Lesions ◽  
List Type ◽  
Genomic Integrity ◽  
Dna Metabolism ◽  
Single Stranded Dna ◽  
Egg Extracts ◽  
Protein Crosslinks ◽  
Bulky Dna Lesions

SummaryDNA-protein crosslinks (DPCs) are bulky DNA lesions that interfere with DNA metabolism and therefore threaten genomic integrity. Recent studies implicate the metalloprotease SPRTN in S-phase removal of DPCs, but how SPRTN activity is coupled to DNA replication is unknown. Using Xenopus egg extracts that recapitulate replication-coupled DPC proteolysis, we show that DPCs can be degraded by SPRTN or the proteasome, which act as independent DPC proteases. Proteasome recruitment requires DPC polyubiquitylation, which is triggered by single-stranded DNA, a byproduct of DNA replication. In contrast, SPRTN-mediated DPC degradation is independent of DPC polyubiquitylation but requires polymerase extension of a nascent strand to the lesion. Thus, SPRTN and proteasome activities are coupled to DNA replication by distinct mechanisms and together promote replication across immovable protein barriers.HighlightsThe proteasome, in addition to SPRTN, degrades DPCs during DNA replicationProteasome-dependent DPC degradation requires DPC ubiquitylationDPC ubiquitylation is triggered by ssDNA and does not require the replisomeSPRTN-dependent DPC degradation is a post-replicative process

scholarly journals DNA–protein crosslink proteases in genome stability

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Annamaria Ruggiano ◽  
Kristijan Ramadan
Keyword(s):  
Dna Replication ◽  
Cancer Therapy ◽  
Genome Stability ◽  
Dna Lesions ◽  
Protein Component ◽  
Human Diseases ◽  
Direct Link ◽  
The Past ◽  
Protein Crosslinks ◽  
Bulky Dna Lesions

AbstractProteins covalently attached to DNA, also known as DNA–protein crosslinks (DPCs), are common and bulky DNA lesions that interfere with DNA replication, repair, transcription and recombination. Research in the past several years indicates that cells possess dedicated enzymes, known as DPC proteases, which digest the protein component of a DPC. Interestingly, DPC proteases also play a role in proteolysis beside DPC repair, such as in degrading excess histones during DNA replication or controlling DNA replication checkpoints. Here, we discuss the importance of DPC proteases in DNA replication, genome stability and their direct link to human diseases and cancer therapy.

scholarly journals DNA- and DNA-Protein-Crosslink Repair in Plants

2019 ◽  
Vol 20 (17) ◽  
pp. 4304 ◽  
Author(s):  
Janina Enderle ◽  
Annika Dorn ◽  
Holger Puchta
Keyword(s):  
S Phase ◽  
Dna Lesions ◽  
Replication Forks ◽  
Genomic Integrity ◽  
Major Threat ◽  
Dna Crosslinks ◽  
Hereditary Disorders ◽  
Toxic Potential ◽  
Protein Crosslinks ◽  
Crosslink Repair

DNA-crosslinks are one of the most severe types of DNA lesions. Crosslinks (CLs) can be subdivided into DNA-intrastrand CLs, DNA-interstrand CLs (ICLs) and DNA-protein crosslinks (DPCs), and arise by various exogenous and endogenous sources. If left unrepaired before the cell enters S-phase, ICLs and DPCs pose a major threat to genomic integrity by blocking replication. In order to prevent the collapse of replication forks and impairment of cell division, complex repair pathways have emerged. In mammals, ICLs are repaired by the so-called Fanconi anemia (FA) pathway, which includes 22 different FANC genes, while in plants only a few of these genes are conserved. In this context, two pathways of ICL repair have been defined, each requiring the interaction of a helicase (FANCJB/RTEL1) and a nuclease (FAN1/MUS81). Moreover, homologous recombination (HR) as well as postreplicative repair factors are also involved. Although DPCs possess a comparable toxic potential to cells, it has only recently been shown that at least three parallel pathways for DPC repair exist in plants, defined by the protease WSS1A, the endonuclease MUS81 and tyrosyl-DNA phosphodiesterase 1 (TDP1). The importance of crosslink repair processes are highlighted by the fact that deficiencies in the respective pathways are associated with diverse hereditary disorders.

scholarly journals Determination of initiation of DNA replication before and after nuclear formation in Xenopus egg cell free extracts.

1993 ◽  
Vol 123 (6) ◽  
pp. 1321-1331 ◽  
Author(s):  
Y Kubota ◽  
H Takisawa
Keyword(s):  
Dna Replication ◽  
Specific Activity ◽  
S Phase ◽  
Egg Cell ◽  
Sperm Dna ◽  
Before And After ◽  
M Phase ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Initiation Of Dna Replication

Xenopus egg extracts prepared before and after egg activation retain M- and S-phase specific activity, respectively. Staurosporine, a potent inhibitor of protein kinase, converted M-phase extracts into interphase-like extracts that were capable of forming nuclei upon the addition of sperm DNA. The nuclei formed in the staurosporine treated M-phase extract were incapable of replicating DNA, and they were unable to initiate replication upon the addition of S-phase extracts. Furthermore, replication was inhibited when the staurosporine-treated M-phase extract was added in excess to the staurosporine-treated S-phase extract before the addition of DNA. The membrane-depleted S-phase extract supported neither nuclear formation nor replication; however, preincubation of sperm DNA with these extracts allowed them to form replication-competent nuclei upon the addition of excess staurosporine-treated M-phase extract. These results demonstrate that positive factors in the S-phase extracts determined the initiation of DNA replication before nuclear formation, although these factors were unable to initiate replication after nuclear formation.

scholarly journals Both cyclin A and cyclin E have S-phase promoting (SPF) activity in Xenopus egg extracts

1996 ◽  
Vol 109 (6) ◽  
pp. 1555-1563 ◽  
Author(s):  
U.P. Strausfeld ◽  
M. Howell ◽  
P. Descombes ◽  
S. Chevalier ◽  
R.E. Rempel ◽  
...  
Keyword(s):  
Dna Replication ◽  
Cyclin E ◽  
Cyclin A ◽  
Early Embryo ◽  
S Phase ◽  
Chromosomal Dna ◽  
Cyclin E1 ◽  
Egg Extracts ◽  
High Concentrations ◽  
Xenopus Egg Extracts

Extracts of activated Xenopus eggs in which protein synthesis has been inhibited support a single round of chromosomal DNA replication. Affinity-depletion of cyclin dependent kinases (Cdks) from these extracts blocks the initiation of DNA replication. We define ‘S-phase promoting factor’ (SPF) as the Cdk activity required for DNA replication in these Cdk-depleted extracts. Recombinant cyclins A and E, but not cyclin B, showed significant SPF activity. High concentrations of cyclin A promoted entry into mitosis, which inhibited DNA replication. In contrast, high concentrations of cyclin E1 promoted neither nuclear envelope disassembly nor full chromosome condensation. In the early embryo cyclin E1 complexes exclusively with Cdk2 and cyclin A is complexed predominantly with Cdc2; only later in development does cyclin A associate with Cdk2. We show that baculovirus-produced complexes of cyclin A-Cd2, cyclin A-Cdk2 and cyclin E-Cdk2 could each provide SPF activity. These results suggest that although in the early Xenopus embryo cyclin E1-Cdk2 is sufficient to support entry into S-phase, cyclin A-Cdc2 provides a significant additional quantity of SPF as its levels rise during S phase.

scholarly journals Mitotic CDK promotes replisome disassembly, fork breakage, and complex DNA rearrangements

2018 ◽  
Author(s):  
Lin Deng ◽  
R. Alex. Wu ◽  
Olga V. Kochenova ◽  
David Pellman ◽  
Johannes C. Walter
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Chromosomal Rearrangements ◽  
Cyclin B1 ◽  
Template Switching ◽  
List Type ◽  
End Joining ◽  
Dna Rearrangements ◽  
Egg Extracts ◽  
The Impact

SUMMARYDNA replication errors generate complex chromosomal rearrangements and thereby contribute to tumorigenesis and other human diseases. Although the events that trigger these errors are not well understood, one candidate is mitotic entry before the completion of DNA replication. To address the impact of mitosis on DNA replication, we employed Xenopus egg extracts. When mitotic CDK (Cyclin B1-CDK1) is used to drive these extracts into mitosis, the E3 ubiquitin ligase TRAIP promotes ubiquitylation of the replicative CMG (CDC45/MCM2–7/GINS) helicase at stalled forks and at forks that have completed DNA synthesis. In both cases, ubiquitylation is followed by CMG extraction from chromatin by the CDC48/p97 ATPase. At stalled forks, CMG removal results in fork breakage and complex end joining events involving deletions and template-switching. Our results identify TRAIP-dependent replisome disassembly as a novel trigger of replication fork collapse and propose it underlies complex DNA rearrangements in mitosis.HIGHLIGHTSTRAIP-dependent MCM7 ubiquitylation removes all CMGs from chromatin in mitosisCMG unloading from stalled forks causes replication fork breakageReplication fork breakage in mitosis causes complex rearrangementsNew model of replication fork collapse

scholarly journals Transcription activity contributes to the firing of non-constitutive origins in African trypanosomes helping to maintain robustness in S-phase duration

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Marcelo S. da Silva ◽  
Gustavo R. Cayres-Silva ◽  
Marcela O. Vitarelli ◽  
Paula A. Marin ◽  
Priscila M. Hiraiwa ◽  
...  
Keyword(s):  
Dna Replication ◽  
Mechanistic Model ◽  
S Phase ◽  
Dna Lesions ◽  
Phase Duration ◽  
African Trypanosomes ◽  
Transcription Activity ◽  
Dna And Rna ◽  
Polycistronic Transcription ◽  
Dna Combing

AbstractThe co-synthesis of DNA and RNA potentially generates conflicts between replication and transcription, which can lead to genomic instability. In trypanosomatids, eukaryotic parasites that perform polycistronic transcription, this phenomenon and its consequences are still little studied. Here, we showed that the number of constitutive origins mapped in the Trypanosoma brucei genome is less than the minimum required to complete replication within S-phase duration. By the development of a mechanistic model of DNA replication considering replication-transcription conflicts and using immunofluorescence assays and DNA combing approaches, we demonstrated that the activation of non-constitutive (backup) origins are indispensable for replication to be completed within S-phase period. Together, our findings suggest that transcription activity during S phase generates R-loops, which contributes to the emergence of DNA lesions, leading to the firing of backup origins that help maintain robustness in S-phase duration. The usage of this increased pool of origins, contributing to the maintenance of DNA replication, seems to be of paramount importance for the survival of this parasite that affects million people around the world.

scholarly journals An ATR and CHK1 kinase signaling mechanism that limits origin firing during unperturbed DNA replication

2019 ◽  
Vol 116 (27) ◽  
pp. 13374-13383 ◽  
Author(s):  
Tatiana N. Moiseeva ◽  
Yandong Yin ◽  
Michael J. Calderon ◽  
Chenao Qian ◽  
Sandra Schamus-Haynes ◽  
...  
Keyword(s):  
Dna Replication ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
S Phase ◽  
Dna Lesions ◽  
Dependent Manner ◽  
Kinase Signaling ◽  
Origin Firing ◽  
Atr Kinase ◽  
Chk1 Kinase

DNA damage-induced signaling by ATR and CHK1 inhibits DNA replication, stabilizes stalled and collapsed replication forks, and mediates the repair of multiple classes of DNA lesions. We and others have shown that ATR kinase inhibitors, three of which are currently undergoing clinical trials, induce excessive origin firing during unperturbed DNA replication, indicating that ATR kinase activity limits replication initiation in the absence of damage. However, the origins impacted and the underlying mechanism(s) have not been described. Here, we show that unperturbed DNA replication is associated with a low level of ATR and CHK1 kinase signaling and that inhibition of this signaling induces dormant origin firing at sites of ongoing replication throughout the S phase. We show that ATR and CHK1 kinase inhibitors induce RIF1 Ser2205 phosphorylation in a CDK1-dependent manner, which disrupts an interaction between RIF1 and PP1 phosphatase. Thus, ATR and CHK1 signaling suppresses CDK1 kinase activity throughout the S phase and stabilizes an interaction between RIF1 and PP1 in replicating cells. PP1 dephosphorylates key CDC7 and CDK2 kinase substrates to inhibit the assembly and activation of the replicative helicase. This mechanism limits origin firing during unperturbed DNA replication in human cells.

scholarly journals Differential requirements for DNA replication in the activation of mitotic checkpoints in Saccharomyces cerevisiae.

1997 ◽  
Vol 17 (6) ◽  
pp. 3315-3322 ◽  
Author(s):  
P A Tavormina ◽  
Y Wang ◽  
D J Burke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Cell Division ◽  
Dna Replication ◽  
Gamma Irradiation ◽  
Chromosome Segregation ◽  
Mitotic Checkpoint ◽  
S Phase ◽  
Temperature Sensitive ◽  
Dna Metabolism

Checkpoints prevent inaccurate chromosome segregation by inhibiting cell division when errors in mitotic processes are encountered. We used a temperature-sensitive mutation, dbf4, to examine the requirement for DNA replication in establishing mitotic checkpoint arrest. We used gamma-irradiation to induce DNA damage and hydroxyurea to limit deoxyribonucleotides in cells deprived of DBF4 function to investigate the requirement for DNA replication in DNA-responsive checkpoints. In the absence of DNA replication, mitosis was not inhibited by these treatments, which normally activate the DNA damage and DNA replication checkpoints. Our results support a model that indicates that the assembly of replication structures is critical for cells to respond to defects in DNA metabolism. We show that activating the spindle checkpoint with nocodazole does not require prior progression through S phase but does require a stable kinetochore.

scholarly journals Direct regulation of Treslin by cyclin-dependent kinase is essential for the onset of DNA replication

2011 ◽  
Vol 193 (6) ◽  
pp. 995-1007 ◽  
Author(s):  
Akiko Kumagai ◽  
Anna Shevchenko ◽  
Andrej Shevchenko ◽  
William G. Dunphy
Keyword(s):  
Dna Replication ◽  
Deoxyribonucleic Acid ◽  
S Phase ◽  
Human Cells ◽  
Target Sequence ◽  
Cyclin Dependent Kinase ◽  
Functional Importance ◽  
Interacting Protein ◽  
Egg Extracts

Treslin, a TopBP1-interacting protein, is necessary for deoxyribonucleic acid (DNA) replication in vertebrates. Association between Treslin and TopBP1 requires cyclin-dependent kinase (Cdk) activity in Xenopus laevis egg extracts. We investigated the mechanism and functional importance of Cdk for this interaction using both X. laevis egg extracts and human cells. We found that Treslin also associated with TopBP1 in a Cdk-regulated manner in human cells and that Treslin was phosphorylated within a conserved Cdk consensus target sequence (on S976 in X. laevis and S1000 in humans). Recombinant human Cdk2–cyclin E also phosphorylated this residue of Treslin in vitro very effectively. Moreover, a mutant of Treslin that cannot undergo phosphorylation on this site showed significantly diminished binding to TopBP1. Finally, human cells harboring this mutant were severely deficient in DNA replication. Collectively, these results indicate that Cdk-mediated phosphorylation of Treslin during S phase is necessary for both its effective association with TopBP1 and its ability to promote DNA replication in human cells.

Distinct roles of cdk2 and cdc2 in RP-A phosphorylation during the cell cycle

1993 ◽  
Vol 106 (3) ◽  
pp. 983-994 ◽  
Author(s):  
F. Fang ◽  
J.W. Newport
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Dna Binding ◽  
Dna Binding Protein ◽  
Model System ◽  
S Phase ◽  
Chromosomal Dna ◽  
Cdc2 Kinase ◽  
Single Stranded Dna

RP-A is a single-stranded DNA-binding protein, which has been shown to be required for DNA replication using an SV40 model system. The protein has also been shown to be phosphorylated at the G1-S phase transition. Using Xenopus cell-free extracts we have investigated the role of RP-A in nuclear replication and characterized the kinases and conditions that lead to phosphorylation of RP-A during the cell cycle. By immunodepleting RP-A from Xenopus extracts we have shown that RP-A is essential for replication of chromosomal DNA. Our results show that, during S phase, only that RP-A which is associated with nuclei is phosphorylated. Furthermore our results indicate that during S phase RP-A is only phosphorylated when associated with single-stranded DNA. By immunodepleting cdk2 kinase we show that cdk2 kinase is required for the observed phosphorylation of RP-A in nuclei during S phase. However, using purified cdk2 kinase and RP-A we are unable to detect a direct phosphorylation of RP-A by cdk2 kinase. This observation suggests that phosphorylation of DNA-bound RP-A at S phase is carried out by a kinase distinct from cdk2. Consistent with this we find that when single-stranded DNA is added to S phase extracts depleted of cdk2 kinase, RP-A is phosphorylated. Together these results suggest that cdk2 kinase participates in the activation of DNA replication at a stage prior to the binding of RP-A to the initiation complex. In addition to RP-A phosphorylation in S phase, we have also found that at the onset of mitosis RP-A is quantitatively phosphorylated and that phosphorylation is directly mediated by cdc2 kinase. However, at this time during the cell cycle, cdc2-dependent phosphorylation of RP-A is independent of DNA binding. These observations further demonstrate the distinctions between cdk2 and cdc2 kinases.

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