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DNA topoisomerase I: customs officer at the border between DNA and RNA worlds?

DNA topoisomerase I is required for the normal development of multicellular organisms, probably because it plays a role in controlling gene activity, in addition to its function in relieving tortional stress during DNA replication and transcription. The discovery of DNA topoisomerase I as a specific kinase that phosphorylates serine-arginine rich (SR) splicing factors may provide new insights into their precise function in regulating gene expression. It is clear that the splicing factors phosphorylated by DNA topoisomerase I can modulate gene expression by changing the splicing pattern of structural genes. Studies of the splicing mechanism suggest that the phosphorylation of serine residues of SR proteins contribute to their activity. As this phosphorylation can be accomplished by several kinases, it remains to be determined whether phosphorylation by DNA topoisomerase I protein kinase is the limiting step in regulating this process. The availability of specific inhibitors of DNA topoisomerase I, structurally related to the alkaloid camptothecin, have made it possible to address this question experimentally. These inhibitors, which hold great promise as antineoplastic drugs, lead to specific inhibition of SR protein phosphorylation in cultured cells. This observation will hopefully lead to improved understanding of the mechanism by which these drugs act at cellular level.

Animals↗

PubMed1999

Apoptosis, Bcl-2, and proliferating cell nuclear antigen in the failing human heart: observations made after implantation of left ventricular assist device.

BACKGROUND: Heart failure is characterized by progressive left ventricular remodeling, a complex process that results from cell growth and cell death. The quantitative contribution of apoptotic cells toward left ventricular remodeling has varied widely in tissue removed from cardiomyopathic hearts. Apoptosis has been responsive to angiotensin-converting enzyme inhibition in experimental heart failure, but the dynamics and responsiveness to chronic left ventricular unloading have not been studied. METHODS AND RESULTS: We studied 8 patients with severe heart failure before and after chronic left ventricular unloading with a left ventricular assist device (LVAD). Tissue from the left ventricular apex removed at the time of LVAD implantation was examined for apoptosis using the technique of terminal deoxynucleotidyl transferase deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) in 10 patients. These same hearts explanted at the time of cardiac transplantation were then examined for apoptosis after patients had been on the LVAD for 99 +/- 20 (SEM) days. An additional 10 patients with equally severe heart failure who underwent heart transplantation without the use of an LVAD served as controls. Eight hearts obtained at autopsy approximately 6 hours after death from patients who died of non-cardiovascular disease causes served as non-heart failure controls. Additionally, 6 hearts were examined by immunohistochemistry for the antiapoptotic protein, Bcl-2, and for the repair and/or proliferation marker, proliferating cell nuclear antigen (PCNA), before and after LVAD. Apoptosis was not detected in the tissue sections from the hearts of 8 patients at the time of LVAD implantation. Only 1 of these patients had limited apoptosis (< 1 apoptotic cell/1,000 myocytes) after LVAD insertion. Three of 10 patients with severe heart failure who did not receive an LVAD but underwent transplantation showed limited apoptosis (< 1 apoptotic cell/1,000 myocytes). Likewise, none of the control hearts from patients who died of noncardiovascular disease manifested apoptosis. Six of 6 patients overexpressed Bcl-2 at the time of LVAD insertion. In all these patients, Bcl-2 returned to negligible levels after chronic unloading of the heart. Likewise, PCNA was abundantly expressed in 5 of 6 failing hearts at the time of LVAD implantation and was reduced in 4 of 5 hearts after chronic unloading by LVAD. CONCLUSION: Apoptosis is a rare or inconsistent finding in the failing human heart. Overexpression of such indicators of cellular stress and DNA replication and/or repair as Bcl-2 and PNCA in heart failure may be altered by optimizing left ventricular loading conditions by such mechanical devices as the LVAD.

Adult↗

PubMed2006

Nucleolin inhibits Hdm2 by multiple pathways leading to p53 stabilization.

Nucleolin is a c-Myc-induced gene product with defined roles in ribosomal RNA processing and the inhibition of chromosomal DNA replication following stress. Here we find that changes in nucleolin protein levels in unstressed cells cause parallel changes in the amount of p53 protein. Alterations in p53 levels arise from nucleolin binding to the p53 antagonist Hdm2, resulting in the inhibition of both p53 ubiquitination and Hdm2 auto-ubiquitination. Nucleolin does not alter p53 ubiquitination by human papillomavirus E6, indicating that the effect is specific for Hdm2. Although the inhibition of ligase activity would be expected to stabilize Hdm2, we instead find that nucleolin also reduces Hdm2 protein levels, demonstrating that nucleolin inhibits Hdm2 using multiple mechanisms. Increases in nucleolin levels in unstressed cells led to higher expression of p21(cip1/waf1), a reduced rate of cellular proliferation, and an increase in apoptosis. Thus, nucleolin has a number of properties in common with the tumor suppressor ARF (alternate reading frame). We propose that nucleolin, like ARF, responds to hyperproliferative signals by upregulation of p53 through Hdm2 inhibition.

Blotting, Western↗

PubMed2005

YB-1 represses AP1-dependent gene transactivation and interacts with an AP-1 DNA sequence.

Involvement of the AP-1 (activator protein-1) transcription factor has been demonstrated previously in the regulation of cell proliferation and cell-cycle progression, in the control of cell migration, invasion and metastasis, and in signal transduction, stress responsiveness, DNA replication and DNA repair. YB-1 (Y-box-binding protein-1) has also been implicated in many of these processes. However, the mechanism by which YB-1 mediates these processes is poorly understood. In the present study, we report that overexpression of a transfected gene encoding YB-1 in human HeLa cervical carcinoma cells significantly represses the transactivation of a minimal AP-1 reporter construct in response to the tumour promoter PMA. YB-1 also represses mRNA expression and PMA-induced promoter transactivation of the endogenous AP-1 target gene encoding matrix metalloproteinase-12 (metalloelastase). YB-1 transrepression of both the minimal and matrix metalloproteinase-12 promoter reporter constructs is dependent on the AP-1 sequence. To identify new nuclear proteins that bind specifically to the AP-1 DNA-binding site, we devised a DNA-affinity-chromatography-based assay termed NAPSTER (nucleotide-affinity preincubation specificity test of recognition) and discovered a 49 kDa protein from human cancer cells that binds in a sequence-specific manner to the AP-1 DNA sequence. By tandem MS fragmentation sequencing analyses we determined that p49 is a YB-1. Immunoblotting of the NAPSTER-purified p49 protein using anti-YB-1 antibodies confirmed YB-1 binding to the AP-1 DNA sequence, as did gel mobility-supershift assays using YB-1 antibodies. This is the first report of YB-1 transrepression and interaction at the AP-1 DNA-binding site.

Base Sequence↗

PubMed1995

Isolation of mutants of Saccharomyces cerevisiae requiring DNA topoisomerase I.

Despite evidence that DNA topoisomerase I is required to relieve torsional stress during DNA replication and transcription, yeast strains with a top1 null mutation are viable and display no gross defects in DNA or RNA synthesis, possibly because other proteins provide overlapping functions. We isolated mutants whose inviablility or growth defect is relieved when TOP1 is expressed [trf mutants (topoisomerase one-requiring function)]. The TRF genes define at least four complementation groups. TRF3 is allelic to TOP2. TRF1 is allelic to HPR1, previously shown to be homologous to TOP1 over two short regions. TRF4 encodes a novel 584-amino acid protein with homology to the N-terminus of Saccharomyces cerevisiae topo I. Like top1 mutants, trf4 mutants have elevated rDNA recombination and fail to shut off RNA polymerase II transcription in stationary phase. trf4 null mutants are cs for viability, display reduced expression of GAL1 and Cell Cycle Box UAS::LacZ fusions, and are inviable in combination with trfI null mutants, indicating that both proteins may share a common function with DNA topoisomerase I. The existence of multiple TRF complementation groups suggests that not all biological functions of topo I can be carried out by topo II.

Alleles↗

PubMed2005

Mechanisms of common fragile site instability.

The study of common fragile sites has its roots in the early cytogenetic investigations of the fragile X syndrome. Long considered an interesting component of chromosome structure, common fragile sites have taken on novel significance as regions of the genome that are particularly sensitive to certain forms of replication stress, which are frequently rearranged in cancer cells. In recent years, much has been learned about the genomic structure at fragile sites and the cellular checkpoint functions that monitor their stability. Recent findings suggest that common fragile sites may serve as markers of chromosome damage caused by replication stress during early stages of tumorigenesis. Thus, the study of common fragile sites can provide insight not only into the nature of fragile sites, but also into the broader consequences of replication stress on DNA damage and cancer. However, despite recent advances, many questions remain regarding the normal functional significance of these conserved regions and the basis of their fragility.

Chromosome Aberrations↗

PubMed2005

CDK-dependent stabilization of Cdc6: linking growth and stress signals to activation of DNA replication.

Cyclin-dependent kinases (CDKs) play a crucial role in cell cycle progression by controlling the transition from G(1) phase into S phase where DNA is replicated. Key to this transition is the regulation of initiation of DNA replication at replication origins. CDKs are thought to regulate origins of replication both positively and negatively by phosphorylating replication proteins at origins. Several replication proteins that are potentially negatively regulated upon CDK phosphorylation have been identified. However, the mechanism by which CDKs activate replication is currently less well understood. New observations revealing that the initiation protein Cdc6 is stabilized by CDK2-dependent phosphorylation may give more insight in this process.

Cell Cycle Proteins↗

PubMed2025

Dihydroxyacetone decreases the dATP pool, inducing replication stress and genomic instability in BEAS-2B cells.

Dihydroxyacetone (DHA), found in sunless tanning products and electronic cigarette aerosol, induces cytotoxic and genotoxic effects in cells. Studies across skin and systemic models demonstrated that DHA induces cell cycle arrest and mitochondrial stress, but its impact on replication is unknown. We investigated DHA exposure effects on lung epithelial BEAS-2B cells to determine if it induces replication stress and genomic instability. Acute DHA exposure generated oxidative stress, triggering 53BP1 foci formation 24 and 48 h after exposure. Evaluation of DNA damage response showed increased levels of pChk2 and pP53, demonstrating activation of double-strand break response. Using a DNA fiber assay, we observed decreased replication fork progression, which coincided with increased micronuclei formation. Removal of DHA from cell media partially alleviated the replication stress, similar to the removal of hydroxyurea, suggesting a reversible effect. Given DHA's incorporation into glycolytic pathways and induction of mitochondrial stress, we examined its effects on nucleotide biosynthesis and pool composition. DHA exposure reduced ribonucleotide reductase (RRM1/2) expression and specifically depleted dATP pools after 48 h. We confirmed that dATP depletion drives replication stress by supplementing cells with adenine during DHA exposure, which decreased DNA lesions, reduced damage signaling, and restored replication. Adenine supplementation also partially rescued DHA-induced cytotoxicity and micronuclei formation. These data demonstrate that DHA-induced DNA damage and dATP pool depletion cause replication stress in BEAS-2B cells, providing new information on DHA's genotoxic mechanism. The inability of adenine to completely rescue micronuclei formation also suggests additional mechanisms of action that impact mitosis, requiring further investigation.

Humans↗

PubMed1984

Enzymology of DNA in replication in prokaryotes.

This review stresses recent developments in the in vitro study of DNA replication in prokaryotes. New insights into the enzymological mechanisms of initiation and elongation of leading and lagging strand DNA synthesis in ongoing studies are emphasized. Data from newly developed systems, such as those replicating oriC containing DNA or which are dependent on the lambda, O, and P proteins, are presented and the information compared to existing mechanisms. Evidence bearing on the coupling of DNA synthesis on both parental strands through protein-protein interactions and on the turnover of the elongation systems are analyzed. The structure of replication origins, and how their tertiary structure affects recognition and interaction with the various replication proteins is discussed.

Bacterial Proteins↗

PubMed2004

Choreography of the DNA damage response: spatiotemporal relationships among checkpoint and repair proteins.

DNA repair is an essential process for preserving genome integrity in all organisms. In eukaryotes, recombinational repair is choreographed by multiprotein complexes that are organized into centers (foci). Here, we analyze the cellular response to DNA double-strand breaks (DSBs) and replication stress in Saccharomyces cerevisiae. The Mre11 nuclease and the ATM-related Tel1 kinase are the first proteins detected at DSBs. Next, the Rfa1 single-strand DNA binding protein relocalizes to the break and recruits other key checkpoint proteins. Later and only in S and G2 phase, the homologous recombination machinery assembles at the site. Unlike the response to DSBs, Mre11 and recombination proteins are not recruited to hydroxyurea-stalled replication forks unless the forks collapse. The cellular response to DSBs and DNA replication stress is likely directed by the Mre11 complex detecting and processing DNA ends in conjunction with Sae2 and by RP-A recognizing single-stranded DNA and recruiting additional checkpoint and repair proteins.

Cell Cycle Proteins↗

PubMed2005

The fission yeast Crb2/Chk1 pathway coordinates the DNA damage and spindle checkpoint in response to replication stress induced by topoisomerase I inhibitor.

Living organisms experience constant threats that challenge their genome stability. The DNA damage checkpoint pathway coordinates cell cycle progression with DNA repair when DNA is damaged, thus ensuring faithful transmission of the genome. The spindle assembly checkpoint inhibits chromosome segregation until all chromosomes are properly attached to the spindle, ensuring accurate partition of the genetic material. Both the DNA damage and spindle checkpoint pathways participate in genome integrity. However, no clear connection between these two pathways has been described. Here, we analyze mutants in the BRCT domains of fission yeast Crb2, which mediates Chk1 activation, and provide evidence for a novel function of the Chk1 pathway. When the Crb2 mutants experience damaged replication forks upon inhibition of the religation activity of topoisomerase I, the Chk1 DNA damage pathway induces sustained activation of the spindle checkpoint, which in turn delays metaphase-to-anaphase transition in a Mad2-dependent fashion. This new pathway enhances cell survival and genome stability when cells undergo replicative stress in the absence of a proficient G(2)/M DNA damage checkpoint.

Alleles↗

PubMed1996

Differential response to UV stress and DNA damage during the yeast replicative life span.

The yeast Saccharomyces cerevisiae is mortal. Before they die, individual yeasts bud repeatedly producing a finite number of progeny, which have the capacity for a full life span. A feature of aging in many species is the waning of resistance to stress. To determine whether this is the case in yeast, we have examined the survival (viability) of age-synchronized populations of yeasts of various ages, spanning youth, midlife, and old age, after irradiation with ultraviolet light (UV). Resistance to UV was biphasic. There was an increase through midlife, followed by a precipitous decline. For comparison, another mutagenic agent, ethyl methanesulfonate (EMS), was tested in the same way. The response was very different. A uniphase decrease in resistance to this DNA-alkylating agent was found with a plateau later in life. The results argue that the increase in resistance to UV with age is an active process and not simply a monotonic age change. RAS2 is among the genes that determine yeast longevity. This gene is preferentially expressed in young cells and has a life span-extending effect on yeasts. One known function of RAS2 is to mount a protective response to irradiation by UV, which occurs independently of DNA damage. The distinction between UV and EMS found here is consistent with the notion that resistance to UV plays a role in yeast longevity in a manner not related to DNA damage. Furthermore, it suggests that RAS2 may participate in this response. We have found that RAS2 expression and UV resistance coincide in middle-aged yeasts bolstering this possibility. These data and the eclipse in activity of several longevity determining genes at midlife in yeasts also raise the possibility that active life maintenance processes function through this period, after which the organism operates on any remaining reserves until death.

DNA Damage↗

PubMed1998

The DNA replication and damage checkpoint pathways induce transcription by inhibition of the Crt1 repressor.

We have identified the yeast CRT1 gene as an effector of the DNA damage and replication checkpoint pathway. CRT1 encodes a DNA-binding protein that recruits the general repressors Ssn6 and Tup1 to the promoters of damage-inducible genes. Derepression of the Crt1 regulon suppresses the lethality of mec1 and rad53 null alleles and is essential for cell viability during replicative stress. In response to DNA damage and replication blocks, Crt1 becomes hyperphosphorylated and no longer binds DNA, resulting in transcriptional induction. CRT1 is autoregulated and is itself induced by DNA damage, indicating the existence of a negative feedback pathway that facilitates return to the repressed state after elimination of damage. The inhibition of an autoregulatory repressor in response to DNA damage is a strategy conserved throughout prokaryotic and eukaryotic evolution.

Animals↗

PubMed2006

SUMO-binding motifs mediate the Rad60-dependent response to replicative stress and self-association.

In fission yeast, the replication checkpoint is enforced by the kinase Cds1 (human Chk2), which regulates both cell cycle progression and DNA repair factors to ensure that the genome is faithfully duplicated prior to mitosis. Cds1 contains a forkhead-associated domain that mediates its interaction with phosphorylated residues in target proteins. One target of Cds1 is the essential nuclear protein Rad60, which contains the unique structural feature of tandem SUMO homology domains at its C terminus. Hypomorphic mutants of Rad60 cause profound defects in DNA repair and replication stress tolerance. To explore the physiological significance of the Cds1-Rad60 interaction, we have examined the phosphorylation of Rad60 by Cds1 in vitro and the in vivo phosphorylation of Rad60 in response to replication blocks. We find that the N terminus but not the SUMO-like domain of Rad60 is phosphorylated in both conditions. Three important Rad60 phosphorylation sites were identified: Thr(72), Ser(32), and Ser(34). Rad60 Thr(72) mediates the Cds1-Rad60 interaction and is required for the Cds1-dependent phosphorylation of Rad60 in response to replication arrest. Phosphorylation of Rad60 Ser(32) and Ser(34) in a putative SUMO-binding motif is critical for the survival of replication stress. In addition, mutation of Rad60 Ser(32) and Ser(34) to alanine is lethal in cells deleted for the RecQ DNA helicase Rqh1. Finally, we find that Rad60 self-associates via its C-terminal SUMO-like domain and putative SUMO-binding motifs.

Amino Acid Motifs↗

PubMed2004

Human Claspin works with BRCA1 to both positively and negatively regulate cell proliferation.

Claspin is a homolog of Mrc1, a checkpoint protein required for the DNA replication checkpoint in yeast. In Xenopus, phosphorylated Claspin binds to xChk1 and regulates xChk1 activation in response to replication stress. In this study, we have shown that the human homolog of Claspin is required for resistance to multiple forms of genotoxic stress including UV, IR, and hydroxyurea. Phosphorylation of Claspin was found to depend on the ataxia telangiectasia mutated-Rad3 related (ATR) pathway. DNA damage induces the formation of a complex between Claspin and BRCA1, a second regulator of Chk1 activation. Claspin was found to control BRCA1 phosphorylation on serine 1524, a site whose phosphorylation is controlled by the ATR pathway. These results are consistent with a model in which ATR regulates Claspin phosphorylation in response to DNA damage and replication stress resulting in recruitment and phosphorylation of BRCA1. BRCA1 and Claspin then function to activate the tumor suppressor Chk1. Unexpectedly, we found that Claspin has a second, positive role in control of the cell cycle as Claspin overexpression increased cell proliferation. These results suggest that Claspin has properties of both a tumor suppressor and an oncogene.

Adaptor Proteins, Signal Transducing↗

PubMed2026

Anoxia tolerant DNA replication is supported by ATR kinase in the annual killifish Austrofundulus limnaeus.

Hypoxia and anoxia suppress cell proliferation due to an increase in replication stress and activation of DNA damage checkpoints. Embryos of the annual killifish Austrofundulus limnaeus tolerate prolonged anoxia, indicating improved genomic stability under oxygen starvation. We investigated the cell cycle regulation of the anoxia tolerant killifish embryonic cell line PSU-AL-WS40NE during anoxia. Live-cell imaging confirms continued proliferation of WS40NE cells for the first 24 h of anoxia with minimal cell death. Fluorescence imaging shows that cells accumulate in G1 after the first day in anoxia with a rapid entry into S phase upon reoxygenation. Pharmacological inhibition shows a reliance on ataxia telangiectasia and Rad3 related (ATR) signaling, suggesting that increased γH2AX levels are driven by replication stress instead of DNA damage. This conclusion is supported by a lack of induction of a G2 checkpoint, suggesting minimal DNA damage during anoxic exposure. Maintaining cellular proliferation during anoxia and accumulating cells in the G1 phase for extended anoxic exposure is likely one way that embryos of the killifish can survive prolonged anoxia, which provides insight into mechanisms that enable cells to proliferate under metabolic stress.

Animals↗

PubMed2004

Replication protein A (RPA) phosphorylation prevents RPA association with replication centers.

Mammalian replication protein A (RPA) undergoes DNA damage-dependent phosphorylation at numerous sites on the N terminus of the RPA2 subunit. To understand the functional significance of RPA phosphorylation, we expressed RPA2 variants in which the phosphorylation sites were converted to aspartate (RPA2(D)) or alanine (RPA2(A)). Although RPA2(D) was incorporated into RPA heterotrimers and supported simian virus 40 DNA replication in vitro, the RPA2(D) mutant was selectively unable to associate with replication centers in vivo. In cells containing greatly reduced levels of endogenous RPA2, RPA2(D) again did not localize to replication sites, indicating that the defect in supporting chromosomal DNA replication is not due to competition with the wild-type protein. Use of phosphospecific antibodies demonstrated that endogenous hyperphosphorylated RPA behaves similarly to RPA2(D). In contrast, under DNA damage or replication stress conditions, RPA2(D), like RPA2(A) and wild-type RPA2, was competent to associate with DNA damage foci as determined by colocalization with gamma-H2AX. We conclude that RPA2 phosphorylation prevents RPA association with replication centers in vivo and potentially serves as a marker for sites of DNA damage.

Animals↗

PubMed2006

Destruction of Claspin by SCFbetaTrCP restrains Chk1 activation and facilitates recovery from genotoxic stress.

We show that Claspin, an adaptor protein required for Chk1 activation, becomes degraded at the onset of mitosis. Claspin degradation was triggered by its interaction with, and ubiquitylation by, the SCFbetaTrCP ubiquitin ligase. This interaction was phosphorylation dependent and required the activity of the Plk1 kinase and the integrity of a betaTrCP recognition motif (phosphodegron) in the N terminus of Claspin. Uncoupling of Claspin from betaTrCP by mutating the conserved serines in Claspin's phosphodegron or by knocking down betaTrCP stabilized Claspin in mitosis, impaired Chk1 dephosphorylation, and delayed G2/M transition during recovery from cell cycle arrest imposed by DNA damage or replication stress. Moreover, the inability to degrade Claspin allowed partial reactivation of Chk1 in cells exposed to DNA damage after passing the G2/M transition. Our data suggest that degradation of Claspin facilitates timely reversal of the checkpoint response and delineates the period permissive for Chk1 activation during cell cycle progression.

Adaptor Proteins, Signal Transducing↗