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PubMed2001

Mechanisms of action of antimicrobials: focus on fluoroquinolones.

Five bacterial targets have been exploited in the development of antimicrobial drugs: cell wall synthesis, protein synthesis, ribonucleic acid synthesis, deoxyribonucleic acid (DNA) synthesis, and intermediary metabolism. Because resistance to drugs that interact with these targets is widespread, new antimicrobials and an understanding of their mechanisms of action are vital. The fluoroquinolones are the only direct inhibitors of DNA synthesis; by binding to the enzyme-DNA complex, they stabilize DNA strand breaks created by DNA gyrase and topoisomerase IV. Ternary complexes of drug, enzyme, and DNA block progress of the replication fork. Cytotoxicity of fluoroquinolones is likely a 2-step process involving (1) conversion of the topoisomerase-quinolone-DNA complex to an irreversible form and (2) generation of a double-strand break by denaturation of the topoisomerase. The molecular factors necessary for the transition from step 1 to step 2 remain unclear, but downstream pathways for cell death may overlap with those used by other bactericidal antimicrobials. Studies of fluoroquinolone-resistant mutants and purified topoisomerases indicate that many quinolones have differing activities against the two targets. Drugs with similar activities against both targets may prove less likely to select de novo resistance.

Anti-Infective Agents↗

PubMed2003

Werner protein stimulates topoisomerase I DNA relaxation activity.

Werner syndrome (WS) is a human premature aging disorder characterized by the early onset of age-related clinical features and an elevated incidence of cancer. The Werner protein (WRN) belongs to the RecQ family of DNA helicases and is required for the maintenance of genomic stability in human cells. Potential cooperation between RecQ helicases and topoisomerases in many aspects of DNA metabolism, such as the progression of replication forks, transcription, recombination, and repair, has been reported. Here, we show a physical and functional interaction between WRN and topoisomerase I (topo I). WRN colocalizes and interacts directly with topo I. WRN stimulates the ability of topo I to relax negatively supercoiled DNA and specifically stimulates the religation step of the relaxation reaction. Moreover, cell extracts from WS fibroblasts exhibit a decrease in the relaxation activity of negatively supercoiled DNA. We have identified two regions of WRN that mediate functional interaction with topo I, and they are located at the NH(2) and COOH termini of the WRN protein. In a reciprocal functional interaction, topo I inhibits the ATPase activity of WRN. Our data provide new insight into the interrelationship between RecQ helicases and topoisomerases in the maintenance of genomic integrity and prevention of tumorigenesis.

Adenosine Triphosphatases↗

PubMed2006

Smc5/6 is required for repair at collapsed replication forks.

In eukaryotes, three pairs of structural-maintenance-of-chromosome (SMC) proteins are found in conserved multisubunit protein complexes required for chromosomal organization. Cohesin, the Smc1/3 complex, mediates sister chromatid cohesion while two condensin complexes containing Smc2/4 facilitate chromosome condensation. Smc5/6 scaffolds an essential complex required for homologous recombination repair. We have examined the response of smc6 mutants to the inhibition of DNA replication. We define homologous recombination-dependent and -independent functions for Smc6 during replication inhibition and provide evidence for a Rad60-independent function within S phase, in addition to a Rad60-dependent function following S phase. Both genetic and physical data show that when forks collapse (i.e., are not stabilized by the Cds1Chk2 checkpoint), Smc6 is required for the effective repair of resulting lesions but not for the recruitment of recombination proteins. We further demonstrate that when the Rad60-dependent, post-S-phase Smc6 function is compromised, the resulting recombination-dependent DNA intermediates that accumulate following release from replication arrest are not recognized by the G2/M checkpoint.

Cell Cycle↗

PubMed2024

FANCJ DNA helicase is recruited to the replisome by AND-1 to ensure genome stability.

FANCJ, a DNA helicase linked to Fanconi anemia and frequently mutated in cancers, counteracts replication stress by dismantling unconventional DNA secondary structures (such as G-quadruplexes) that occur at the DNA replication fork in certain sequence contexts. However, how FANCJ is recruited to the replisome is unknown. Here, we report that FANCJ directly binds to AND-1 (the vertebrate ortholog of budding yeast Ctf4), a homo-trimeric protein adaptor that connects the CDC45/MCM2-7/GINS replicative DNA helicase with DNA polymerase α and several other factors at DNA replication forks. The interaction between FANCJ and AND-1 requires the integrity of an evolutionarily conserved Ctf4-interacting protein (CIP) box located between the FANCJ helicase motifs IV and V. Disruption of the CIP box significantly reduces FANCJ association with the replisome, causing enhanced DNA damage, decreased replication fork recovery and fork asymmetry in cells unchallenged or treated with Pyridostatin, a G-quadruplex-binder, or Mitomycin C, a DNA inter-strand cross-linking agent. Cancer-relevant FANCJ CIP box variants display reduced AND-1-binding and enhanced DNA damage, a finding that suggests their potential role in cancer predisposition.

Humans↗

PubMed2000

Initiation of DNA replication within oriP is dispensable for stable replication of the latent Epstein-Barr virus chromosome after infection of established cell lines.

The 165-kb circularized chromosome of Epstein-Barr virus (EBV) is replicated in latently infected cells once per cell cycle by host proteins during S phase. Replication initiates at multiple sites on latent EBV chromosomes, including within a 1.8-kb region called oriP, which can provide both replication and stabilization for recombinant plasmids in the presence of the EBV-encoded protein, EBNA-1. Replication initiates at or near the dyad symmetry component (DS) of oriP, which depends on multiple EBNA-1 binding sites for activity. To test the importance of the replication function of oriP, the DS was deleted from the viral genome. EBV mutants lacking the DS and carrying a selectable gene could establish latent infections in BL30 cells, in which circular, mutant viral chromosomes were stably maintained. Analysis of replication fork movement using two-dimensional gel electrophoresis showed that the deletion of the DS reduced the initiation events to an undetectable level within the oriP region so that this segment was replicated exclusively by forks entering the region from either direction. A significant slowing or stalling of replication forks that occurs normally at the approximate position of the DS was also eliminated by deletion of the DS. The results confirm the DS as both a replication origin and a place where replication forks pause. Since the replication function of oriP is dispensable at least in certain cell lines, the essential role of EBNA-1 for infection of these cell lines is likely to be that of stabilizing the EBV chromosome by associating with the 30-bp repeats of oriP. The results also imply that in established cell lines, the EBV chromosome can be efficiently replicated entirely from origins that are activated by cellular factors. Presumably, initiation of replication at the DS, mediated by EBNA-1, is important for the natural life cycle of EBV, perhaps in establishing latent infections of normal B cells.

Base Sequence↗

PubMed2005

Crosstalk between SUMO and ubiquitin on PCNA is mediated by recruitment of the helicase Srs2p.

Posttranslational modification of proliferating cell nuclear antigen (PCNA), an essential processivity clamp for DNA polymerases, by ubiquitin and SUMO contributes to the coordination of DNA replication, damage tolerance, and mutagenesis. Whereas ubiquitination in response to DNA damage promotes the bypass of replication-blocking lesions, sumoylation during S phase is damage independent. As both modifiers target the same site on PCNA, an antagonistic action of SUMO on ubiquitin-dependent DNA damage tolerance has been proposed. We now present evidence that the apparent negative effect of SUMO on lesion bypass is not due to competition with ubiquitination but is rather mediated by the helicase Srs2p, which affects genome stability by suppressing unscheduled homologous recombination. We show that Srs2p physically interacts with sumoylated PCNA, which contributes to the recruitment of the helicase to replication forks. Our findings suggest a mechanism by which SUMO and ubiquitin cooperatively control the choice of pathway for the processing of DNA lesions during replication.

Chromatin Immunoprecipitation↗

PubMed2003

Homologous recombination in Archaea: new Holliday junction helicases.

Homologous recombination (HR) maintains genome stability by promoting high fidelity DNA repair. Several recent reports have established that the primary function of HR enzymes is to underpin DNA replication, resetting forks that are blocked or collapsed at sites of DNA damage remote from replication origins. These functions are crucial to ensuring that genomes are transmitted successfully into subsequent generations of cells. Enzymes of HR have been unearthed in all three domains of life: bacteria, Archaea and eukarya. Helicases that specifically unwind branched DNA molecules are pivotal in linking HR and DNA replication in bacteria. However, knowledge of helicases with these functions in eukaryotes is vague and is wholly absent in Archaea. We are using the archaeal species Methanothermobacter thermautotrophicus to identify new DNA helicases of homologous recombination.

Archaea↗

PubMed2004

Role of replication and CpG methylation in fragile X syndrome CGG deletions in primate cells.

Instability of the fragile X CGG repeat involves both maternally derived expansions and deletions in the gametes of full-mutation males. It has also been suggested that the absence of aberrant CpG methylation may enhance repeat deletions through an unknown process. The effect of CGG tract length, DNA replication direction, location of replication initiation, and CpG methylation upon CGG stability were investigated using an SV40 primate replication system. Replication-dependant deletions with 53 CGG repeats were observed when replication was initiated proximal to the repeat, with CGG as the lagging-strand template. When we initiated replication further from the repeat, while maintaining CGG as the lagging-strand template or using CCG as the lagging-strand template, significant instability was not observed. CpG methylation of the unstable template stabilized the repeat, decreasing both the frequency and the magnitude of deletion events. Furthermore, CpG methylation slowed the efficiency of replication for all templates. Interestingly, replication forks displayed no evidence of a block at the CGG repeat tract, regardless of replication direction or CpG methylation status. Templates with 20 CGG repeats were stable under all circumstances. These results reveal that CGG deletions occur during replication and are sensitive to replication-fork dynamics, tract length, and CpG methylation.

Cell Culture Techniques↗

PubMed1999

Differences in induction of p53, p21WAF1 and apoptosis in relation to cell cycle phase of MCF-7 cells treated with camptothecin.

The DNA topoisomerase I (topI) inhibitor camptothecin (CPT), stabilizes so-called cleavable complexes which consist of topI covalently attached to 3' OH ends of DNA nicks. Collisions between the progressing DNA replication forks (occurring in S phase cells) or between the transcription driven RNA polymerase molecules (occurring in G1, S and G2 cells) and these complexes convert the latter into secondary DNA lesions which are unrepairable and lethal to the cell. Changes induced by CPT in the level of the tumor suppressor p53, cyclin-dependent kinase inhibitor p21WAF1 and proapoptotic protein Bax (all detected immunocytochemically), were measured separately in the nucleus and cytoplasm of individual human breast carcinoma MCF-7 cells by laser scanning cytometry (LSC) in relation to cell cycle position and induction of apoptosis. The initial transient cell arrest at the G1 checkpoint seen at 8-16 h of treatment with 0.15 microM CPT was accompanied by the rapid accumulation of p53 (preventable by cycloheximide) in the nucleus; the rise (>20-fold) in p53 was maximal for S phase cells. The magnitude of the nuclear p53 increase induced by CPT, at maximum, was 2-fold higher than that induced by the proteasome inhibitor N-acetyl-Leu-Leu-norleucinal (LLnL). While the accumulation of p53 was seen in all phases of the cycle, only G1 cells responded by induction ( approximately 60-fold increase) of p21WAF1. Inhibition of DNA replication by aphidicolin prevented the accumulation of p53 in S and G2/M but had no effect on its induction in G1 cells. Perturbation of cell progression through S phase was seen between 24-72 h of treatment, and it coincided with induction of Bax and apoptosis (both maximal in S phase cells). Thus, the changes observed in S phase cells (nuclear accumulation of p53 preventable by aphidicolin, induction of Bax, apoptosis), triggered by the collisions of DNA replication forks with the CPT-induced lesions, were distinct from the changes in G1 (nuclear p53 accumulation unaffected by aphidicolin, induction of p21WAF1) presumably triggered by collisions of RNA polymerase with the CPT-lesions. Great heterogeneity in expression of p53 and p21WAF1 of the G1 cell population in response to CPT was observed, which may reflect the intercellular variability in the rate of transcription (i.e., frequencies of collisions of RNA polymerase with the lesions). Thus, differences in the transcriptional activity of G1 cells may play a role in their sensitivity to CPT and similar topI inhibitors.

Apoptosis↗

PubMed2000

Allele-specific protein-DNA interactions between the single-stranded DNA-binding protein, ssA-TIBF, and DNA replication determinants in Tetrahymena.

Type I elements are multifunctional, cis-acting determinants that regulate the initiation of DNA replication, replication fork movement and transcription of the Tetrahymena thermophila rDNA minichromosome. Previous studies identified a protein, ssA-TIBF, that binds specifically to the A-rich strand of type I elements. Here, we examine interactions of ssA-TIBF with the wild-type C3 allele, and a natural variant, B rDNA, which manifests a defect in replication initiation and fork pausing. Purified ssA-TIBF is a homotetramer that binds one substrate molecule and contacts DNA via a single 24 kDa subunit. Both the A-rich and T-rich strands of type I elements are bound by ssA-TIBF, suggesting that this protein might stabilize replication origins in their unwound state. Nucleotides downstream of type I elements contribute to DNA binding, with the extent of DNA-protein contact being greater for wild-type C3 rDNA compared to B rDNA. Allele-specific protein-DNA contacts also occur within the conserved type I element itself. Despite these differences, the binding affinities of ssA-TIBF for C3 and B rDNA substrates are indistinguishable. Consequently, the mode of DNA binding must account for any role ssA-TIBF might play in the regulation of rDNA replication.

Alleles↗

PubMed2003

BRCA2-dependent and independent formation of RAD51 nuclear foci.

The formation of RAD51 foci in response to ionizing radiation (IR) represents an important step in the repair of DNA double-strand breaks. RAD51 foci also appear during S phase and are thought to be required for the restart of stalled or broken replication forks. The RAD51 recombinase interacts directly with the breast cancer-associated tumour suppressor BRCA2, an interaction that is required for normal recombination proficiency, radiation resistance and genome stability. In CAPAN-1 cells, which express a truncated form of BRCA2 that is cytoplasmic because of loss of the nuclear localization signal, the formation of IR-induced RAD51 foci is impaired. In this work, we show that S-phase RAD51 foci form normally in CAPAN-1 cells expressing truncated BRCA2. Moreover, we find that RAD51 specifically associates with chromatin at S phase in a reaction that is BRCA2-independent. The observed BRCA2-dependent and independent formation of RAD51 foci shows that intact BRCA2 is not required for RAD51 focus formation per se, leading us to suggest that S phase and IR-induced RAD51 foci assemble by distinct pathways with defined protein requirements.

Active Transport, Cell Nucleus↗

PubMed2005

Mrc1 is required for normal progression of replication forks throughout chromatin in S. cerevisiae.

Mrc1 associates with replication forks, where it transmits replication stress signals and is required for normal replisome pausing in response to nucleotide depletion. Mrc1 also plays a poorly understood role in DNA replication, which appears distinct from its role in checkpoint signaling. Here, we demonstrate that Mrc1 functions constitutively to promote normal replication fork progression. In mrc1Delta cells, replication forks proceed slowly throughout chromatin, rather than being specifically defective in pausing and progression through loci that impede fork progression. Analysis of genetic interactions with Rrm3, a DNA helicase required to resolve paused forks, indicates that Mrc1 checkpoint signaling is dispensable for the resolution of stalled replication forks and suggests that replication forks lacking Mrc1 create DNA damage that must be repaired by Rrm3. These findings elucidate a central role for Mrc1 in normal replisome function, which is distinct from its role as a checkpoint mediator, but nevertheless critical to genome stability.

Cell Cycle Proteins↗

PubMed2005

Current advances in unraveling the function of the Werner syndrome protein.

Werner syndrome (WS) is an autosomal recessive premature aging disease manifested by the mimicry of age-related phenotypes such as atherosclerosis, arteriosclerosis, cataracts, osteoporosis, soft tissue calcification, premature thinning, graying, and loss of hair, as well as a high incidence of some types of cancers. The gene product defective in WS, WRN, is a member of the RecQ family of DNA helicases that are widely distributed in nature and believed to play central roles in genomic stability of organisms ranging from prokaryotes to mammals. Interestingly, WRN is a bifunctional protein that is exceptional among RecQ helicases in that it also harbors an exonuclease activity. Furthermore, it preferentially operates on aberrant DNA structures believed to exist in vivo as intermediates in specific DNA transactions such as replication (forked DNA), recombination (Holliday junction, triplex and tetraplex DNA), and repair (partial duplex with single stranded bubble). In addition, WRN has been shown to physically and functionally interact with a variety of DNA-processing proteins, including those that are involved in resolving alternative DNA structures, repair DNA damage, and provide checkpoints for genomic stability. Despite significant research activity and considerable progress in understanding the biochemical and molecular genetic function of WRN, the in vivo molecular pathway(s) of WRN remain elusive. The following review focuses on the recent advances in the biochemistry of WRN and considers the putative in vivo functions of WRN in light of its many protein partners.

DNA Helicases↗

PubMed1993

DNA topoisomerase I and II as targets for rational design of new anticancer drugs.

BACKGROUND: Topoisomerase I and II (topo I and II) are enzymes which alter the topological state of DNA through DNA strand cleavage, strand passage and religation. They participate in most aspects of DNA metabolism and are therefore vital to the cell undergoing division. Only one form of topo I has been identified whereas two isoenzymes of topo II have been described: the alpha form (170 kDa protein) and beta form (180 kDa protein). Both topo II isoenzymes have distinct nuclear localisation, are regulated independently, differ in their responsiveness to inhibitors and are differentially expressed in drug resistant cell lines. RESULTS: Several clinically active anticancer drugs (e.g., doxorubicin, m-AMSA, VP-16 and camptothecins) poison these enzymes by stabilizing a putative reaction intermediate called the cleavable complex (cc) where the topoisomerase remains covalently attached to either one strand of DNA (topo I) or both strands of double helix (topo II) after strand cleavage. DNA cleavage sites appear unique for different classes of inhibitor, and are probably critical for defining cytotoxicity. Formation of the cc may cause cell death either by colliding with replication forks, by promoting illegitimate genomic-DNA recombination, by arresting cells in the G2-phase of the cell cycle or by inducing apoptosis. CONCLUSION: New classes of inhibitor have recently been described with novel mechanisms of action including compounds which do not stabilize cleavable complexes or bind significantly to DNA. These may prove to be more selective and less toxic. They may also avoid the possible problem of therapy-related leukemias associated with topo inhibitors which induce DNA cleavage and chromosomal aberrations.

Animals↗

PubMed1994

Initiation of DNA replication by simian virus 40 T antigen is inhibited by the p107 protein.

The p107 protein is related to the Rb protein by a 60-kDa region of homology called the pocket domain that binds cellular proteins such as E2F and cyclins A and E as well as the viral oncoproteins E1A, E7, and SV40 T antigen. The p107 and Rb proteins have both been implicated as negative regulators of cell growth. We have examined the effect of the unphosphorylated pocket domain of p107 on specific stages of the T antigen-mediated replication of SV40 DNA in vitro. The pocket domain inhibited replication by preventing the assembly of T antigen at the SV40 core origin DNA containing binding site II. However, both proteins formed a ternary complex with DNA containing T antigen-binding site I. The pocket domain of p107 did not inhibit the oligomerization of T antigen in the absence of DNA, and the p107 derivative is bound to the intermediates of this reaction. In the unwinding assay, once T antigen was preassembled as hexamers at the core origin, the pocket domain bound to and stabilized the complex, resulting in an increase in the yield of unwound product. Preformed T antigen hexamers complexed with the pocket domain bind to a synthetic replication fork, and this complex supported unwinding. Consistent with this, the p107 pocket domain had no effect on the helicase activity of T antigen in an assay using a partial duplex substrate. However, a complex containing p107 and T antigen assembled at the core origin did not support SV40 DNA replication in HeLa cell crude extracts or in the monopolymerase reaction. This inhibition is due to the inability of the complex to bind to DNA polymerase alpha, which is required for the initiation of DNA synthesis. The data suggest pathways by which the pocket domain of p107 can negatively regulate T antigen-mediated replication in vitro. The binding of T antigen to p107 is discussed with respect to its role in mitigating negative cell growth control, resulting in viral mediated transformation.

Animals↗

PubMed2006

Lysine 63-polyubiquitination guards against translesion synthesis-induced mutations.

Eukaryotic cells possess several mechanisms to protect the integrity of their DNA against damage. These include cell-cycle checkpoints, DNA-repair pathways, and also a distinct DNA damage-tolerance system that allows recovery of replication forks blocked at sites of DNA damage. In both humans and yeast, lesion bypass and restart of DNA synthesis can occur through an error-prone pathway activated following mono-ubiquitination of proliferating cell nuclear antigen (PCNA), a protein found at sites of replication, and recruitment of specialized translesion synthesis polymerases. In yeast, there is evidence for a second, error-free, pathway that requires modification of PCNA with non-proteolytic lysine 63-linked polyubiquitin (K63-polyUb) chains. Here we demonstrate that formation of K63-polyUb chains protects human cells against translesion synthesis-induced mutations by promoting recovery of blocked replication forks through an alternative error-free mechanism. Furthermore, we show that polyubiquitination of PCNA occurs in UV-irradiated human cells. Our findings indicate that K63-polyubiquitination guards against environmental carcinogenesis and contributes to genomic stability.

Carcinogens↗

PubMed2003

Dna2 helicase/nuclease causes replicative fork stalling and double-strand breaks in the ribosomal DNA of Saccharomyces cerevisiae.

We have proposed that faulty processing of arrested replication forks leads to increases in recombination and chromosome instability in Saccharomyces cerevisiae and contributes to the shortened lifespan of dna2 mutants. Now we use the ribosomal DNA locus, which is a good model for all stages of DNA replication, to test this hypothesis. We show directly that DNA replication pausing at the ribosomal DNA replication fork barrier (RFB) is accompanied by the occurrence of double-strand breaks near the RFB. Both pausing and breakage are elevated in the early aging, hypomorphic dna2-2 helicase mutant. Deletion of FOB1, encoding the fork barrier protein, suppresses the elevated pausing and DSB formation, and represses initiation at rDNA ARSs. The dna2-2 mutation is synthetically lethal with deltarrm3, encoding another DNA helicase involved in rDNA replication. It does not appear to be the case that the rDNA is the only determinant of genome stability during the yeast lifespan however since strains carrying deletion of all chromosomal rDNA but with all rDNA supplied on a plasmid, have decreased rather than increased lifespan. We conclude that the replication-associated defects that we can measure in the rDNA are symbolic of similar events occurring either stochastically throughout the genome or at other regions where replication forks move slowly or stall, such as telomeres, centromeres, or replication slow zones.

Adenosine Triphosphatases↗

PubMed2000

Werner's syndrome cell lines are hypersensitive to camptothecin-induced chromosomal damage.

Werner's syndrome (WS) is a recessive human genetic disorder associated with an elevated incidence of many types of cancer. The WS gene product, WRNp, belongs to the RecQ family of DNA helicases and is required for the maintenance of genomic stability in human cells. A possible interaction between helicases and topoisomerases that could co-operate in many aspects of DNA metabolism such as progression of the replication forks, recombination and repair has been recently suggested. In addition, sgs1 gene product in yeast, homologous to WS gene, has been shown to physically interact with topoisomerase types I and II. Earlier data from our laboratory suggested that WRN helicase might play a role in a G2 recombinational pathway of double strand breaks (DSBs) repair, co-operating with topoisomerase II. In this work, the effect of the topoisomerase I inhibitor camptothecin in WS cells has been investigated at the chromosomal level. The data from the present work suggest that the inhibition of topoisomerase I activity by camptothecin results in a higher induction of chromosomal damage in WS cell lines in the G2-phase and in the S-phase of the cell cycle compared to normal cells, perhaps associated with the defects in DNA replication synthesis.

Camptothecin↗