p53 Response to DNA Damage in S-phase

Objective: To overcome the disadvantages of cisplatin, numerous platinum (Pt) complexes have been prepared. However, the anticancer activity and mechanism of Pt(II) complexed with 2-benzoylpyridine [Pt(II)- Bpy]: [PtCl2(DMSO)L] (DMSO = dimethyl sulfoxide, L = 2-benzoylpyridine) in cancer cells remain unknown. Methods: Pt(II)-Bpy was synthesized and characterized by spectrum analysis. Its anticancer activity and underlying mechanisms were demonstrated at the cellular, molecular, and in vivo levels. Results: Pt(II)-Bpy inhibited tumor cell growth, especially HepG2 human liver cancer cells, with a halfmaximal inhibitory concentration of 9.8±0.5μM, but with low toxicity in HL-7702 normal liver cells. Pt(II)- Bpy induced DNA damage, which was demonstrated through a marked increase in the expression of cleavedpoly (ADP ribose) polymerase (PARP) and gamma-H2A histone family member X and a decrease in PARP expression. The interaction of Pt(II)-Bpy with DNA at the molecular level was most likely through an intercalation mechanism, which might be evidence of DNA damage. Pt(II)-Bpy initiated cell cycle arrest at the S phase in HepG2 cells. It also caused severe loss of the mitochondrial membrane potential; a decrease in the expression of caspase-9 and caspase-3; an increase in reactive oxygen species levels; the release of cytochrome c and apoptotic protease activation factor; and the activation of caspase-9 and caspase-3 in HepG2 cells, which in turn resulted in apoptosis. Meanwhile, changes in p53 and related proteins were observed including the upregulation of p53, the phosphorylation of p53, p21, B-cell lymphoma-2-associated X protein, and NOXA; and the downregulation of B-cell lymphoma 2. Moreover, Pt(II)-Bpy displayed marked inhibitory effects on tumor growth in the HepG2 nude mouse model. Conclusion: Pt(II)-Bpy is a potential candidate for cancer chemotherapy.

Download Full-text

Mutations in SID2, a Novel Gene in Saccharomyces cerevisiae, Cause Synthetic Lethality With sic1 Deletion and May Cause a Defect During S Phase

Genetics ◽

10.1093/genetics/159.1.17 ◽

2001 ◽

Vol 159 (1) ◽

pp. 17-33

Author(s):

Matthew D Jacobson ◽

Claudia X Muñoz ◽

Kirstin S Knox ◽

Beth E Williams ◽

Lenette L Lu ◽

...

Keyword(s):

Dna Damage ◽

Synthetic Lethality ◽

S Phase ◽

Temperature Sensitive ◽

Replication Forks ◽

Novel Gene ◽

Mutant Strains ◽

Single Nucleus ◽

2C Dna Content ◽

Slow Growing

Abstract SIC1 encodes a nonessential B-type cyclin/CDK inhibitor that functions at the G1/S transition and the exit from mitosis. To understand more completely the regulation of these transitions, mutations causing synthetic lethality with sic1Δ were isolated. In this screen, we identified a novel gene, SID2, which encodes an essential protein that appears to be required for DNA replication or repair. sid2-1 sic1Δ strains and sid2-21 temperature-sensitive strains arrest preanaphase as large-budded cells with a single nucleus, a short spindle, and an ~2C DNA content. RAD9, which is necessary for the DNA damage checkpoint, is required for the preanaphase arrest of sid2-1 sic1Δ cells. Analysis of chromosomes in mutant sid2-21 cells by field inversion gel electrophoresis suggests the presence of replication forks and bubbles at the arrest. Deleting the two S phase cyclins, CLB5 and CLB6, substantially suppresses the sid2-1 sic1Δ inviability, while stabilizing Clb5 protein exacerbates the defects of sid2-1 sic1Δ cells. In synchronized sid2-1 mutant strains, the onset of replication appears normal, but completion of DNA synthesis is delayed. sid2-1 mutants are sensitive to hydroxyurea indicating that sid2-1 cells may suffer DNA damage that, when combined with additional insult, leads to a decrease in viability. Consistent with this hypothesis, sid2-1 rad9 cells are dead or very slow growing even when SIC1 is expressed.

Download Full-text

Regulation of DNA Replication Licensing and Re-Replication by Cdt1

International Journal of Molecular Sciences ◽

10.3390/ijms22105195 ◽

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.

Download Full-text

Combined Inactivation of Pocket Proteins and APC/CCdh1 by Cdk4/6 Controls Recovery from DNA Damage in G1 Phase

Cells ◽

10.3390/cells10030550 ◽

2021 ◽

Vol 10 (3) ◽

pp. 550

Author(s):

Indra A. Shaltiel ◽

Alba Llopis ◽

Melinda Aprelia ◽

Rob Klompmaker ◽

Apostolos Menegakis ◽

...

Keyword(s):

Dna Damage ◽

Normal Cell ◽

S Phase ◽

G1 Phase ◽

Anaphase Promoting Complex ◽

Inhibitory Effects ◽

Pocket Proteins ◽

Cyclin Dependent Kinases ◽

Independent Functions ◽

Phase Entry

Most Cyclin-dependent kinases (Cdks) are redundant for normal cell division. Here we tested whether these redundancies are maintained during cell cycle recovery after a DNA damage-induced arrest in G1. Using non-transformed RPE-1 cells, we find that while Cdk4 and Cdk6 act redundantly during normal S-phase entry, they both become essential for S-phase entry after DNA damage in G1. We show that this is due to a greater overall dependency for Cdk4/6 activity, rather than to independent functions of either kinase. In addition, we show that inactivation of pocket proteins is sufficient to overcome the inhibitory effects of complete Cdk4/6 inhibition in otherwise unperturbed cells, but that this cannot revert the effects of Cdk4/6 inhibition in DNA damaged cultures. Indeed, we could confirm that, in addition to inactivation of pocket proteins, Cdh1-dependent anaphase-promoting complex/cyclosome (APC/CCdh1) activity needs to be inhibited to promote S-phase entry in damaged cultures. Collectively, our data indicate that DNA damage in G1 creates a unique situation where high levels of Cdk4/6 activity are required to inactivate pocket proteins and APC/CCdh1 to promote the transition from G1 to S phase.

Download Full-text

Cell cycle inertia underlies a bifurcation in cell fates after DNA damage

Science Advances ◽

10.1126/sciadv.abe3882 ◽

2021 ◽

Vol 7 (3) ◽

pp. eabe3882

Author(s):

Jenny F. Nathans ◽

James A. Cornwell ◽

Marwa M. Afifi ◽

Debasish Paul ◽

Steven D. Cappell

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Cell Tracking ◽

Time Lapse ◽

S Phase ◽

Cdk Inhibitor ◽

Cyclin Dependent Kinase ◽

Cell Fates ◽

Damage Response ◽

Time Lapse Imaging

The G1-S checkpoint is thought to prevent cells with damaged DNA from entering S phase and replicating their DNA and efficiently arrests cells at the G1-S transition. Here, using time-lapse imaging and single-cell tracking, we instead find that DNA damage leads to highly variable and divergent fate outcomes. Contrary to the textbook model that cells arrest at the G1-S transition, cells triggering the DNA damage checkpoint in G1 phase route back to quiescence, and this cellular rerouting can be initiated at any point in G1 phase. Furthermore, we find that most of the cells receiving damage in G1 phase actually fail to arrest and proceed through the G1-S transition due to persistent cyclin-dependent kinase (CDK) activity in the interval between DNA damage and induction of the CDK inhibitor p21. These observations necessitate a revised model of DNA damage response in G1 phase and indicate that cells have a G1 checkpoint.

Download Full-text

Excess ribonucleotide reductase R2 subunits coordinate the S phase checkpoint to facilitate DNA damage repair and recovery from replication stress

Biochemical Pharmacology ◽

10.1016/j.bcp.2006.11.014 ◽

2007 ◽

Vol 73 (6) ◽

pp. 760-772 ◽

Cited By ~ 25

Author(s):

Z. Ping Lin ◽

Michael F. Belcourt ◽

Rocco Carbone ◽

Jana S. Eaton ◽

Philip G. Penketh ◽

...

Keyword(s):

Dna Damage ◽

Ribonucleotide Reductase ◽

Dna Damage Repair ◽

Replication Stress ◽

S Phase ◽

Damage Repair ◽

Ribonucleotide Reductase R2 ◽

S Phase Checkpoint

Download Full-text

Distinct Targets of the Eco1 Acetyltransferase Modulate Cohesion in S Phase and in Response to DNA Damage

Molecular Cell ◽

10.1016/j.molcel.2009.04.008 ◽

2009 ◽

Vol 34 (3) ◽

pp. 311-321 ◽

Cited By ~ 97

Author(s):

Jill M. Heidinger-Pauli ◽

Elçin Ünal ◽

Douglas Koshland

Keyword(s):

Dna Damage ◽

S Phase

Download Full-text

SPK1 is an essential S-phase-specific gene of Saccharomyces cerevisiae that encodes a nuclear serine/threonine/tyrosine kinase

Molecular and Cellular Biology ◽

10.1128/mcb.13.9.5829-5842.1993 ◽

1993 ◽

Vol 13 (9) ◽

pp. 5829-5842

Author(s):

P Zheng ◽

D S Fay ◽

J Burton ◽

H Xiao ◽

J L Pinkham ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Dna Damage ◽

Dna Synthesis ◽

Genomic Library ◽

Excision Repair ◽

Low Frequency ◽

S Phase ◽

Upstream Region ◽

Specific Gene

SPK1 was originally discovered in an immunoscreen for tyrosine-protein kinases in Saccharomyces cerevisiae. We have used biochemical and genetic techniques to investigate the function of this gene and its encoded protein. Hybridization of an SPK1 probe to an ordered genomic library showed that SPK1 is adjacent to PEP4 (chromosome XVI L). Sporulation of spk1/+ heterozygotes gave rise to spk1 spores that grew into microcolonies but could not be further propagated. These colonies were greatly enriched for budded cells, especially those with large buds. Similarly, eviction of CEN plasmids bearing SPK1 from cells with a chromosomal SPK1 disruption yielded viable cells with only low frequency. Spk1 protein was identified by immunoprecipitation and immunoblotting. It was associated with protein-Ser, Thr, and Tyr kinase activity in immune complex kinase assays. Spk1 was localized to the nucleus by immunofluorescence. The nucleotide sequence of the SPK1 5' noncoding region revealed that SPK1 contains two MluI cell cycle box elements. These elements confer S-phase-specific transcription to many genes involved in DNA synthesis. Northern (RNA) blotting of synchronized cells verified that the SPK1 transcript is coregulated with other MluI box-regulated genes. The SPK1 upstream region also includes a domain highly homologous to sequences involved in induction of RAD2 and other excision repair genes by agents that induce DNA damage. spk1 strains were hypersensitive to UV irradiation. Taken together, these findings indicate that SPK1 is a dual-specificity (Ser/Thr and Tyr) protein kinase that is essential for viability. The cell cycle-dependent transcription, presence of DNA damage-related sequences, requirement for UV resistance, and nuclear localization of Spk1 all link this gene to a crucial S-phase-specific role, probably as a positive regulator of DNA synthesis.

Download Full-text