Stages Of DNA Replication Research Articles

The Herpes Simplex Viruses Utilize a Recombination‐Dependent Replication Mechanism to Replicate Viral Genomes

Although it has been known for decades that HSV replication results in the formation head‐to‐tail concatermers, the mechanism by which concatemers form is not understood. One long‐standing model involves circularization of incoming viral genomes followed by rolling circle replication. However, several lines of evidence suggest that HSV DNA replication may be more complex, relying instead on a recombination‐dependent replication mechanism. The HSV single‐strand DNA binding protein (SSB), ICP8, is the central player in all stages of DNA replication. Additionally, ICP8 can promote efficient annealing of complementary ssDNA and is thus considered to be a single strand annealing protein (SSAP). HSV encodes a conserved alkaline nuclease (UL12) that interacts with ICP8 to form a complex that is reminiscent of evolutionarily conserved phage recombinases, such as lambda red a/ß. We hypothesize that ICP8 and UL12 interact to promote recombination‐dependent DNA replication to generate head‐to‐tail concatemers necessary for the production of infectious virus. Due to the multifunctional nature of ICP8, it has been difficult to tea1se out the role of ICP8 as a SSAP from the various roles it plays during viral replication as a classical SSB. We have recently identified a novel ICP8 mutant, Q706A/F707A, that lacks annealing activity but retains many of the other functions characteristic of classical SSBs. Interestingly, this mutant is unable to synthesize viral DNA, indicating that the annealing function of ICP8 is essential for viral DNA synthesis (Weerasooriya, DiScipio et al. PNAS 2018)Annealing is a complex process requiring many types of molecular interactions. We hypothesize that it involves both ICP8‐DNA and ICP8‐ICP8 interactions as well as conformational changes induced by the recognition of complementary DNA. Based on their proximity to Q706A/F707A, we suggest that annealing may be mediated by a cluster of conserved residues (residues 701 to 719) in the shoulder domain of ICP8. We have identified five conserved arginine residues (R701, R704, R712, R713 and R714) in this region. Alanine mutagenesis of these residues showed that all mutants, except R704A are unable to complement the growth of a null mutant suggesting that these residues are important for viral growth. Biochemical characterization of purified mutant and WT proteins will be presented along with models for how ICP8 promotes efficient annealing during DNA synthesis.Support or Funding InformationNIH funding AI069136 and AI021747 to S.K.W and F30AI143125 to K.A.D

Photoreactive DNA as a Tool to Study Replication Protein A Functioning in DNA Replication and Repair.

Replication protein A (RPA), eukaryotic single-stranded DNA-binding protein, is a key player in multiple processes of DNA metabolism including DNA replication, recombination and DNA repair. Human RPA composed of subunits of 70-, 32- and 14-kDa binds ssDNA with high affinity and interacts specifically with multiple proteins. The RPA heterotrimer binds ssDNA in several modes, with occlusion lengths of 8-10, 13-22 and 30 nucleotides corresponding to global, transitional and elongated conformations of protein. Varying the structure of photoreactive DNA, the intermediates of different stages of DNA replication or DNA repair were designed and applied to identify positioning of the RPA subunits on the specific DNA structures. Using this approach, RPA interactions with various types of DNA structures attributed to replication and DNA repair intermediates were examined. This review is dedicated to blessed memory of Prof. Alain Favre who contributed to the development of photoreactive nucleotide derivatives and their application for the study of protein-nucleic acids interactions.

Herpes simplex virus 1 ICP8 mutant lacking annealing activity is deficient for viral DNA replication

Most DNA viruses that use recombination-dependent mechanisms to replicate their DNA encode a single-strand annealing protein (SSAP). The herpes simplex virus (HSV) single-strand DNA binding protein (SSB), ICP8, is the central player in all stages of DNA replication. ICP8 is a classical replicative SSB and interacts physically and/or functionally with the other viral replication proteins. Additionally, ICP8 can promote efficient annealing of complementary ssDNA and is thus considered to be a member of the SSAP family. The role of annealing during HSV infection has been difficult to assess in part, because it has not been possible to distinguish between the role of ICP8 as an SSAP from its role as a replicative SSB during viral replication. In this paper, we have characterized an ICP8 mutant, Q706A/F707A (QF), that lacks annealing activity but retains many other functions characteristic of replicative SSBs. Like WT ICP8, the QF mutant protein forms filaments in vitro, binds ssDNA cooperatively, and stimulates the activities of other replication proteins including the viral polymerase, helicase-primase complex, and the origin binding protein. Interestingly, the QF mutant does not complement an ICP8-null virus for viral growth, replication compartment formation, or DNA replication. Thus, we have been able to separate the activities of ICP8 as a replicative SSB from its annealing activity. Taken together, our data indicate that the annealing activity of ICP8 is essential for viral DNA replication in the context of infection and support the notion that HSV-1 uses recombination-dependent mechanisms during DNA replication.

Mechanism of Subunit Coordination of an AAA + Hexameric Molecular Nanomachine

Simian virus 40 large tumor antigen (LT) is both a potent oncogenic protein and an efficient hexameric nanomachine that harnesses the energy from ATP binding/hydrolysis to melt origin DNA and unwind replication forks. However, how the six subunits of the helicase motor coordinate during ATP hydrolysis and DNA unwinding/translocation is unresolved. Here we investigated the subunit coordination mechanisms “binomial distribution mutant doping” experiments in the presence of various DNA substrates. For ATP hydrolysis, we observed multiple coordination modes, ranging from random and semi-random, and semi-coordinated modes, depending on which type of DNA is present. For DNA unwinding, however, the results indicated a fully-coordinated mode for the natural origin-containing duplex DNA, but a semi-coordinated mode for a pre-existing fork-DNA, providing direct evidence for LT to use potentially different mechanisms to unwind the two types of substrates. The results of this study provide insights into DNA translocation and unwinding mechanisms for LT hexameric biomotor. From the Clinical EditorThe study describes the subunit coordination of simian virus 40 large tumor antigen (LT) showing that multiple mechanisms exist that handle the specific needs of different stages of DNA replication.

Mcm8 and Mcm9 form a dimeric complex in Xenopus laevis egg extract that is not essential for DNA replication initiation

Hexameric complexes of the six related Mcm2–7 proteins form the core of the replicative helicase. Two other proteins, Mcm8 and Mcm9, with significant homology to Mcm2–7 were first shown to play distinct roles during DNA replication in Xenopus laevis egg extract. Recent work has revealed that Mcm8 and 9 form a complex that plays a role during homologous recombination in human, chicken and mouse cells. We have therefore re-examined the behavior of the Xenopus homologs of these proteins. We show that Mcm8 and Mcm9 form a dimeric complex in Xenopus egg extract. They both associate with chromatin at later stages of DNA replication, and this association is stimulated by DNA damage, suggesting that their function is analogous to the one described in higher eukaryotes. In contrast to previous reports, we do not find Mcm9 essential for loading of Mcm2–7 complex onto chromatin during origin licensing nor detect its interaction with Cdt1 origin licensing factor. Altogether, we conclude that the role Mcm8 and Mcm9 play in Xenopus egg extract is not different from recent findings in higher eukaryotes, consistent with an evolutionary conservation of their function.

Enhanced Sensitivity of Nucleoli in Human Proliferating Cells to Inhibition of Protein Synthesis with Anisomycin

We describe the reaction of nuclei in cultured human cells from different tissues to inhibition of total protein synthesis with anisomycin - ribotoxin, which is now considered as a potential antitumor drug. It was shown that nucleoli in sensitive cells demonstrate typical reaction: under the action of the inhibitor, labile nucleolar protein, a component of RNA polymerase I transcription complex (previously called A3 antigen), rapidly migrates from the nucleolus to numerous discrete foci in the nucleoplasm. These changes are specific for translation suppression and are not induced by other influences on the cells. Migration of A3 antigen into the nucleoplasm manifests primarily in cells at the stage of DNA replication and is absent in resting cells. These results suggest that localization of A3 antigen can be a marker of artificial suppression of translation in proliferating human cells in vitro.

New model for assembly dynamics of bacterial tubulin in relation to the stages of DNA replication

How living cells receive their genome through cell division has been one of the important questions of biology. In prokaryotes, cell division starts with formation of a ring-shaped microtubule-like structure, FtsZ-ring, at the potential division site. All the previous models suggested that FtsZ-ring is formed coupling to termination or far after initiation of DNA replication. In contrast, we demonstrated that a close communication with DNA replication is maintained throughout the cell cycle. FtsZ starts to assemble to the cell center coupling to initiation of DNA replication, and stabilizes as FtsZ-ring at its termination, but does not constrict before separation of nucleoids. This combination of a positive and a negative control would guarantee that a successful replication event would inevitably induce one cell division such that each of the daughter cells would receive one and only one daughter nucleoid.

Replication protein A is an independent prognostic indicator with potential therapeutic implications in colon cancer

AbstractReplication protein A (RPA), a component of the origin recognition complex, is required for stabilization of single-stranded DNA at early and later stages of DNA replication being thus critical for eukaryotic DNA replication. Experimental studies in colon cancer cell lines have shown that RPA protein may be the target of cytotoxins designed to inhibit cellular proliferation. This is the first study to investigate the expression of RPA1 and RPA2 subunits of RPA protein and assess their prognostic value in colon cancer patients. We analyzed immunohistochemically the expression of RPA1 and RPA2 proteins in a series of 130 colon cancer resection specimens in relation to conventional clinicopathological parameters and patients' survival. Statistical significant positive associations emerged between: (a) RPA1 and RPA2 protein expressions (P=0.0001), (b) RPA1 and RPA2 labelling indices (LIs) and advanced stage of the disease (P=0.001 and 0.003, respectively), (c) RPA1 and RPA2 LIs and the presence of lymph node metastasis (P=0.002 and 0.004, respectively), (d) RPA1 LI and the number of infiltrated lymph nodes (P=0.021), (e) RPA2 LI and histological grade of carcinomas (P=0.05). Moreover, a statistical significant higher RPA1 LI was observed in the metastatic sites compared to the original ones (P=0.012). RPA1 and RPA2 protein expression associated with adverse patients' outcome in both univariate (log rank test: P<0.00001 and 0.00001, respectively) and multivariate (Cox model: P=0.092 and 0.0001, respectively) statistical analysis. Statistical significant differences according to the expression of RPA1 and RPA2 proteins were also noticed in the survival of stage II (P<0.00001 and 0.0016, respectively) and stage III (P=0.0029 and 0.0079, respectively) patients. In conclusion, RPA1 and RPA2 proteins appear to be useful prognostic indicators in colon cancer patients and attractive therapeutic targets for regulation by tumor suppressors or other proteins involved in the control of cell proliferation.

Apoptotic and necrotic effects of hexanedione derivatives on the human neuroblastoma line SK-N-SH

The potential cytotoxicity of two hexanedione food additives (2,3 and 3,4 isomers) was evaluated in comparison with the neurotoxic hexane metabolite 2,5-hexanedione in the human SK-N-SH neuroblastoma line using the MTT assay to indicate mitochondrial dehydrogenase activity and flow cytometry to monitor the cell cycle over 48 h. The IC 50s of the 2,3-hexanedione (3.3 ± 0.1 mM) and 3,4-hexanedione (3.5 ± 0.1 mM), indicated that the sensitivity of the cells was approximately seven-fold greater to these toxins compared with the 2,5 derivative (IC 50 of 22.4 ± 0.2 mM). Comparison between the respective IC 50s of the 2,3-hexanedione and 3,4-hexanedione revealed no difference between the two isomers in terms of their effects on MTT turnover. With flow cytometry analysis, all three hexanediones showed increases in apoptosis within their respective concentration ranges of toxicity shown previously by MTT. In the presence of 2,5-hexanedione, between 8.5 and 17 mM concentrations, there was a significant increase in apoptotic nucleoids which was accompanied by a significant fall in the percentage of nucleoids in the G0/G1 phase (72.4 ± 0.3–45.3 ± 0.6%,), and a rise in the numbers of cells in the G2/M phase. This is likely to indicate growth arrest at cell cycle G2/M checkpoint in response to toxin damage. G2/M accumulation was also shown with 3,4 and 2,3 HD, which was maximal at much lower concentrations (approximately 4 and 3 mM, respectively). Arrest at G1 and G2/M phase is indicative of inhibition of the cell cycle at the stages of DNA replication and chromosome segregation, respectively. It was also apparent that flow cytometry, rather than the MTT assay, did distinguish between the effects of the α-diketones 2,3-hexanedione and 3,4-hexanedione on the cell cycle. At a concentration of 5.8 mM 3,4-hexanedione, the percentage of apoptotic nucleoids was 10.9 ± 0.8% whilst apoptosis induced by 3,4-hexanedione had already reached a maximal level of 60.4 ± 0.5%. In summary, flow cytometry indicated that the 3,4-hexanedione derivative was more toxic than its 2,3 isomer and that both food additives caused interruption in the neuroblastoma cell cycle and further investigation may be required to assess if these α-diketones present in diets pose any possible risks to human health.

Differential expression of members of the E2F family of transcription factors in rodent testes

BackgroundThe E2F family of transcription factors is required for the activation or repression of differentially expressed gene programs during the cell cycle in normal and abnormal development of tissues. We previously determined that members of the retinoblastoma protein family that interacts with the E2F family are differentially expressed and localized in almost all the different cell types and tissues of the testis and in response to known endocrine disruptors. In this study, the cell-specific and stage-specific expression of members of the E2F proteins has been elucidated.MethodsWe used immunohistochemical (IHC) analysis of tissue sections and Western blot analysis of proteins, from whole testis and microdissected stages of seminiferous tubules to study the differential expression of the E2F proteins.ResultsFor most of the five E2F family members studied, the localizations appear conserved in the two most commonly studied rodent models, mice and rats, with some notable differences. Comparisons between wild type and E2F-1 knockout mice revealed that the level of E2F-1 protein is stage-specific and most abundant in leptotene to early pachytene spermatocytes of stages IX to XI of mouse while strong staining of E2F-1 in some cells close to the basal lamina of rat tubules suggest that it may also be expressed in undifferentiated spermatogonia. The age-dependent development of a Sertoli-cell-only phenotype in seminiferous tubules of E2F-1 knockout males corroborates this, and indicates that E2F-1 is required for spermatogonial stem cell renewal. Interestingly, E2F-3 appears in both terminally differentiated Sertoli cells, as well as spermatogonial cells in the differentiative pathway, while the remaining member of the activating E2Fs, E2F-2 is most concentrated in spermatocytes of mid to late prophase of meiosis. Comparisons between wildtype and E2F-4 knockout mice demonstrated that the level of E2F-4 protein displays a distinct profile of stage-specificity compared to E2F-1, which is probably related to its prevalence and role in Sertoli cells. IHC of rat testis indicates that localization of E2F-5 is distinct from that of E2F-4 and overlaps those of E2F-1 and E2F-2.ConclusionThe E2F-1 represents the subfamily of transcription factors required during stages of DNA replication and gene expression for development of germ cells and the E2F-4 represents the subfamily of transcription factors that help maintain gene expression for a terminally differentiated state within the testis.

Photoaffinity Labeling Technique for Studying DNA Replication and DNA Repair&amp;#x2020;

The photoaffinity labeling technique is based on UV-light induced crosslinking of proteins to photoreactive DNA. Photoreactive groups can be introduced at different points of DNA (base or phosphate) either at the ends or in inner positions of the DNA chain by combination of enzymatic and chemical synthesis. Varying the structure of photoreactive DNA one can design the intermediates of different stages of DNA replication or DNA repair and apply them to identify the proteins crosslinked to specific positions of the DNA chain in single- or double-stranded DNA, partial DNA duplexes, gapped or nicked duplexes or DNA carrying flap structures. A wide range of base-substituted dNTP analogs containing photoreactive groups of different photoreactivity and spacers of various lengths has been synthesized and characterized. Photoreactive dNTP analogs have been shown to be effective substrates of viral, bacterial and eukaryotic DNA polymerases. The efficiencies of crosslinking of DNA bearing various photoreactive dNMP to protein and DNA targets were estimated. New approaches for enzymatic introduction of photoreactive groups onto the 5'-end of oligonucleotides and into the inner positions of DNA chain have been elaborated. Photoreactive DNAs have been successfully used to study enzymes and protein factors of DNA replication and repair in reconstituted systems and cellular/nuclear extracts. Photoaffinity labeling technique was shown to be a prominent tool of proteomics to elucidate structural and functional aspects of protein-DNA interactions. It can be also applied for identification of proteins including unknown ones, which interact with specific DNA intermediates in cellular/nuclear extracts.

Interaction of replication protein A with photoreactive DNA structures.

A new photoreactive oligonucleotide derivative was synthesized with a perfluoroarylazido group attached to the 2'-position of the ribose fragment of the 5'-terminal nucleotide. Using this conjugate, photoreactive DNA duplexes were produced which contained single-stranded regions of different length, single-stranded breaks (nicks), and also ds duplex with a photoreactive group inside one of the chains. These structures imitate DNA intermediates generated at different stages of DNA replication and repair. The interaction of replication protein A (RPA) with the resulting DNA structures was studied using photoaffinity modification and gel retardation assay. Independently of the DNA structure, only the large subunit of RPA (p70) was crosslinked to photoreactive DNAs, and the intensity of its labeling increased with decrease in the size of the single-stranded region and was maximal in the case of the nick-containing DNA structure. By gel retardation, the most effective binding of RPA to this structure was shown, whereas the complexing of RPA with DNA containing the unmodified nick and also with the full duplex containing the photoreactive group inside the chain was significantly less effective. The data suggest that RPA should be sensitive to such damages in the double-stranded DNA structure.

Cdc7 kinase complex: a key regulator in the initiation of DNA replication.

DNA replication results from the action of a staged set of highly regulated processes. Among the stages of DNA replication, initiation is the key point at which all the G1 regulatory signals culminate. Cdc7 kinase is the critical regulator for the ultimate firing of the origins of initiation. Cdc7, originally identified in budding yeast and later in higher eukaryotes, forms a complex with a Dbf4-related regulatory subunit to generate an active kinase. Genetic evidence in mammals demonstrates essential roles for Cdc7 in mammalian DNA replication. Mini-chromosome maintenance protein (MCM) is the major physiological target of Cdc7. Genetic studies in yeasts indicate additional roles of Cdc7 in meiosis, checkpoint responses, maintenance of chromosome structures, and repair. The interplay between Cdc7 and Cdk, another kinase essential for the S phase, is also discussed.

The human autoantigen MCP1 is required during early stages of DNA replication.

Metaphase chromosome protein 1 (MCP1) is a nuclear autoantigen that is associated with condensed chromosomes throughout mitosis. During interphase, this antigen shows a speckle distribution in the nucleus, excluding the nucleolus. Additionally, MCP1 binds tightly to the scaffold/matrix component of nuclei and isolated chromosomes. In order to determine the in-vivo localization of the antigen, we have expressed MCP1 fused to EGFP in tissue culture cells. The results demonstrate that MCP1 is located in the nucleus during interphase and during mitosis associates tightly to condensed chromosomes. Furthermore, microinjection of specific antibody confirms these results. We have used a specific monoclonal antibody (mAb 402) against MCP1 to assess the function of this antigen during cell cycle progression. HeLa and Ptk-2 cells that were microinjected into the nucleus and/or cytoplasm at G1/S and very early S phase were not able to progress and complete DNA replication. However, injection of mAb 402 at mid or late S phase does not prevent completion of DNA replication and subsequent progression into mitosis. Microinjection of mAb 402 in Ptk-2 cells synchronized in mitosis did not interfere with progression of mitosis and cells divided. Our results suggest that MCP1 is required at the G1/S transition and during early S phase.

H2O2-induced apoptotic death in serum-deprived cultures of oligodendroglia origin is linked to cell differentiation.

When deprived of serum, oligodendroglialike (OLN 93) cells grown on poly-L-lysine-coated culture dishes cease to proliferate after 3 days and morphologically extend many fibers resembling morphologically differentiated, immature oligodendrocytes. At this time no cell death is apparent unless serum deprivation is extended for a period longer than 1 week. After 3 days in serum-deprived medium, treatment of cells with 1 mM H2O2 for 30 min facilitates apoptotic cell death, even when serum is added during the recovery period. Both serum-deprived, differentiated cells, and proliferating cells, respond to H2O2 by an initial growth arrest followed by growth resumption after 48 hr. However proliferating cells show resistance to the apoptotic effect of H2O2. This is correlated with growth arrest in the S phase at different stages of DNA replication, as well as with different timing of induced p21Waf1 expression. Thus, cells grown in serum, express elevated p21Waf1 protein levels after 4 hr, whereas serum-deprived, differentiated cells, only after 24 hr. The mRNA levels of p21Waf1 follow a similar timed pattern. Hence p21Waf1 may protect OLN 93 cells against the genotoxic effect of H2O2. The data suggest an intimate relationship between G1-arrest, morphological differentiation, and H2O2-mediated apoptosis.

Relationship between viral DNA synthesis and virion envelopment in hepatitis B viruses

While the intracellular pool of encapsidated hepatitis B viral DNA contains genomes in all stages of DNA replication, serum-derived virions contain predominantly mature, partially duplex, circular DNA genomes. To account for this finding, Summers and Mason proposed in 1982 that virion envelopment is somehow linked to the state of genomic maturation (J. Summers and W.S. Mason, Cell 29:403-415, 1982). Core gene mutations with phenotypes consistent with this concept have previously been identified in the duck hepatitis B virus (DHBV). Here we show that DHBV polymerase mutants with altered DNA synthesis also display defects in envelopment, and we provide quantitative estimates of the magnitude of the preference for the envelopment of mature DNA. In cells transfected with wild-type DHBV DNA, immature minus-strand DNA represents 18% of the intracellular pool but only 4% of extracellular virion DNA. A point mutation in the C-terminal domain of the polymerase strongly and selectively impairs plus-strand synthesis; in this mutant, the ratio of immature to mature DNA in the intracellular pool rises to 6:1 but is reduced to 1.5:1 in released virions. A missense mutation in the polymerase active site inactivates all viral DNA synthesis but still allows efficient RNA encapsidation; in this mutant, no detectable viral nucleic acid is enveloped and released. Thus, viral DNA synthesis is absolutely required for envelopment and export, and a strong further bias exists in favor of the export of genomes that have completed minus-strand synthesis and at least initiated plus-strand synthesis. These results imply that events within the interior of the nucleocapsid can powerfully influence its interactions with external viral envelope glycoproteins.

The levels of ubiquitinated histone H2A are highly upregulated in transformed human cells: Partial colocalization of uH2A clusters and PCNA/cyclin foci in a fraction of cells in S-phase

The levels and distribution of ubiquitinated histone H2A (uH2A) have been studied in normal and transformed human cells using a monoclonal antibody (mAb E6C5) that reacts specifically with this ubiquitin conjugate as determined by two-dimensional gel western blotting and microsequencing. Immunoblotting experiments demonstrated that the levels of the protein are highly upregulated in SV40-transformed human fibroblasts (WI-38 SV40) and keratinocytes (K14) relative to their normal counterparts, a finding that was further confirmed by indirect immunofluorescence studies of formaldehyde/Triton X-100-treated cells, which showed that about 97% of the transformed cells and 26% of the normal populations reacted with the antibody to yield a fine granular nuclear staining associated with the chromatin. Transformed cells contained in addition clusters of uH2A that were quite abundant and that showed variable size, shape and distribution even within a single cell line. The clusters, which were rare in normal cells, did not colocalize with other known nuclear antigens and may correspond to novel nuclear domains where ubiquitination/deubiquitination takes place. Electron microscopic immunocytochemistry of K14 cells confirmed the existence of the clusters. Double immunofluorescence studies of K14 keratinocytes with proliferating cell nuclear antigen (PCNA)/cyclin antibodies, which react with the nuclei of cells engaged in DNA replication, showed partial colocalization of PCNA/cyclin foci and large uH2A clusters in about 14% of the S-phase cells, and these corresponded mainly to late S-phase cells. Inhibition of DNA replication with hydroxyurea resulted in an overall increase in the intensity of the uH2A staining as well as in a more clear colocalization of uH2A clusters and PCNA/cyclin foci. Taken together, the results support the contention that uH2A plays a role at some stage of DNA replication.

108 Cyclocreatine (1-carboxymethyl-2-iminoimidazolidine) inhibits HCMV at or near the stage of DNA replication: L. J. Hansen 1, J. W. Lillie 1, D. L. Barnard 2, J. H. Huffman 2, S. Rook 1, G. Mulamba 1, R. W. Sidwell 2, and R. Kaddurah-Daouk 1. 1AMIRA, Inc., Cambridge, MA, USA; 2Utah State University, Institute for Antiviral Research, Logan, Utah, USA

108 Cyclocreatine (1-carboxymethyl-2-iminoimidazolidine) inhibits HCMV at or near the stage of DNA replication: L. J. Hansen 1, J. W. Lillie 1, D. L. Barnard 2, J. H. Huffman 2, S. Rook 1, G. Mulamba 1, R. W. Sidwell 2, and R. Kaddurah-Daouk 1. 1AMIRA, Inc., Cambridge, MA, USA; 2Utah State University, Institute for Antiviral Research, Logan, Utah, USA

Dideoxynucleoside triphosphates inhibit a late stage of SV40 DNA replication in vitro.

The role of DNA polymerases in the replication of SV40 DNA was studied using a T-antigen-dependent assay supplemented with a human KB cell extract. Inhibition of DNA polymerase alpha by addition of aphidicolin or monoclonal antibodies prevented DNA synthesis, confirming the requirement for this enzyme in replication. The replication process was unaffected by ddTTP at a concentration (5 microM) inhibitory to DNA polymerases beta and gamma, however, higher concentrations of ddTTP (200 microM) caused an apparent accumulation of relaxed circular plasmid with a concomitant decrease in DNA synthesis. An analysis of this replication intermediate indicated that it was formed during the replication reaction and that the replicative cycle was nearly complete. A kinetic study of ddTTP inhibition strongly suggested DNA polymerase epsilon (PCNA-independent DNA polymerase delta) was the target of the inhibitor and that this enzyme functions during the final stages of DNA replication.

Characterization of Escherichia coli mutants with altered ploidy

We describe the isolation and characterization of new mutants in the cell cycle of Escherichia coli. The mutants were selected as gain of function mutants that are able to maintain more than the normal number of chromosomes. Our increased ploidy mutants were isolated as resistant to camphor vapours, which selects for cells with more chromosomes than normal. The mutants (called mbr for moth-ball-resistant) map to four chromosomal locations: mbrA at 68 min; mbrB at 88.5 min; mbrC at 89.5 min; and mbrD at 90 min. To investigate the nature of these cell cycle mutants, we have coupled them with defects in recA, to test for induction of the SOS response, and dam, to determine if methylation is required for mbr function. Based on the results of these and other tests, we have made a preliminary placement of the mbr mutants within the context of the cell cycle. mbrA mutations appear to be defective in the coupling of the DNA replication cycle to the cell division cycle, and as such, may define a new link between the two processes. mbrB does not seem to be able to coordinate the cell cycle and the growth rate of the cell. mbrC appears to be defective in partitioning of chromosomes. mbrD, which may be allelic to rpoB (a subunit of RNA polymerase), appears to be defective in either chromosomal partitioning of the later stages of DNA replication.