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PubMed2005

Proliferating cell nuclear antigen recruits cyclin-dependent kinase inhibitor Xic1 to DNA and couples its proteolysis to DNA polymerase switching.

The Xenopus cyclin-dependent kinase (CDK) inhibitor, p27(Xic1) (Xic1), binds to CDK2-cyclins and proliferating cell nuclear antigen (PCNA), inhibits DNA synthesis in Xenopus extracts, and is targeted for ubiquitin-mediated proteolysis. Previous studies suggest that Xic1 ubiquitination and degradation are coupled to the initiation of DNA replication, but the precise timing and molecular mechanism of Xic1 proteolysis has not been determined. Here we demonstrate that Xic1 proteolysis is temporally restricted to late replication initiation following the requirements for DNA polymerase alpha-primase, replication factor C, and PCNA. Our studies also indicate that Xic1 degradation is absolutely dependent upon the binding of Xic1 to PCNA in both Xenopus egg and gastrulation stage extracts. Additionally, extracts depleted of PCNA do not support Xic1 proteolysis. Importantly, while the addition of recombinant wild-type PCNA alone restores Xic1 degradation, the addition of a PCNA mutant defective for trimer formation does not restore Xic1 proteolysis in PCNA-depleted extracts, suggesting Xic1 proteolysis requires both PCNA binding to Xic1 and the ability of PCNA to be loaded onto primed DNA by replication factor C. Taken together, our studies suggest that Xic1 is targeted for ubiquitination and degradation during DNA polymerase switching through its interaction with PCNA at a site of initiation.

Amino Acid Sequence↗
PubMed1986

5-Azacytidine-induced conversion to cadmium resistance correlates with early S phase replication of inactive metallothionein genes in synchronized CHO cells.

Previous studies have shown both hypermethylation and late replication of DNA sequences to be associated with gene inactivity. To determine whether there is a causal relationship between patterns of DNA methylation and replication timing during S phase, we have examined the timing of replication of the inactive, hypermethylated metallothionein (MT) I and II genes in synchronized, cadmium-sensitive (Cds) CHO cells. The time of S-phase replication of the MT genes was ascertained by determining the period of S phase wherein cadmium-resistant (Cdr) cells could be induced with highest frequency by pulse treatment of synchronized Cds cells with the hypomethylating drug 5-azacytidine (5-aza-CR), and by analyzing Southern blots of density fractionated DNAs isolated from synchronized cells pulse-labeled with BrdU during different intervals after release from hydroxyurea blockade. Southern filter hybridization analyses demonstrated replication of both MTI and II gene sequences within the first half of S phase. Consistent with this result, phenotypic conversion of Cds to Cdr was maximal immediately after hydroxyurea release and decreased abruptly within three hours. The replication of inactive hypermethylated MT genes in early S phase argues that transcriptional inactivity and gene-specific hypermethylation are not sufficient conditions for late DNA replication.

Animals↗
PubMed1976

Cutaneous chemical carcinogenesis: past, present, and future.

Skin tumors chemically induced in mice have provided an important experimental model for studying carcinogenesis and for bioassaying carcinogenic agents. The information obtained from this model suggests that the events leading to tumor formation can be divided into at least two stages, initiation and promotion. A single small dose of carinogen produces initiation which appears to be irreversible. These initiating agents may have to be metabolically activated and can interact with cellular macromolecules. The extent to which they bind to DNA correlates well with their carcinogenicity. Increased DNA replication at the time of or during the first day after these agents have been applied appears to enhance carcinogenesis. Unlike initiation, promotion appears to be reversible and the promoting agents must be applied repeatedly before tumors are formed. Promoters interact with membranes, stimulate and alter genetic expression, and increase the rate of cell proliferation. The knowledge gained from these studies in mouse skin has immeasurably helped the entire field of chemical carcinogenesis. But efforts to determine the cellular and molecular mechanisms involved in the carcinogenic process, particularly in the skin, have been hampered by the difficulties of working on whole animals and by the special problems associated with the biologic and biochemical methods required for this target organ. Such problems, however, can be solved by the use of cell cultures of mouse epidermis which can metabolize and bind carcinogens just as is done in vivo. The fact that epidermal cells in vitro proliferate synchronously should facilitate the study of the relation between the cell cycle and carcinogenesis. These cells repair chemically induced DNA damage by at least two mechanisms, excision repair and base-specific repair. When epidermal cells in vitro are exposed to promoting agents, a proliferative response analogous to that in vivo is elicited, apparently mediated through control of polyamine metabolism. Neoplastic transformation has been induced in these cultures by known skin carcinogens.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2004

CTCF elements direct allele-specific undermethylation at the imprinted H19 locus.

The H19 imprinted gene locus is regulated by an upstream 2 kb imprinting control region (ICR) that influences allele-specific expression, DNA methylation, and replication timing. This ICR becomes de novo methylated during late spermatogenesis in the male but emerges from oogenesis in an unmethylated form, and this allele-specific pattern is then maintained throughout early development and in all tissues of the mouse. We have used a genetic approach involving transfection into embryonic stem (ES) cells in order to decipher how the maternal allele is protected from de novo methylation at the time of implantation. Our studies show that CCCTC binding factor (CTCF) boundary elements within the ICR have the ability to prevent de novo methylation on the maternal allele. Since CTCF does not recognize its binding sequence when methylated, this reaction does not occur on the paternal allele, thus preserving the gamete-derived, allele-specific pattern. These results suggest that CTCF may play a general role in the maintenance of differential methylation patterns in vivo.

Alleles↗
PubMed1983

DNA sequences showing a delay in cytosine methylation after replication. Time course of methylation in synchronized mammalian cell populations and relationship to DNAase I sensitive domains.

We have shown that in several mammalian cell lines a minor fraction of cytosine methylation is delayed for up to several hours after strand synthesis and that different methylases performed the immediate and the delayed classes of DNA methylation. To investigate the time course of this delayed methylation we have used three different cell lines, two of human and one of hamster origin. These were synchronized by two different methods: mitotic detachment and double hydroxyurea blocks. A uniform picture was obtained with all three cell lines. Delayed methylation of early replicating sequences occurred while cells were still in mid-S-phase, with the maximum rate of delayed methylation occurring in cells in the second half of S and in G2. Delayed methylation seems to be complete before cells entered the next G1-phase. Limited DNAase I hydrolysis of cell nuclei was used to test whether the delay in methylation in some DNA sequences was due to high levels of transcriptional activity. However, DNA sequences exhibiting delayed methylation showed no preferential concentration in or exclusion from DNAase I hypersensitive regions.

Animals↗
PubMed1989

Evidence for a ligation step in the DNA replication of the autonomous parvovirus minute virus of mice.

Newly replicated DNA of the autonomous parvovirus minute virus of mice was pulse-labeled with 32PO4 during the time of maximal viral DNA replication in highly synchronized A9 cells. The subsequent processing of viral DNA-protein complexes was monitored during a chase period with no label. Several distinct classes of duplex replicative-form and progeny single-stranded DNA molecules were characterized and found to accumulate at different times during infection. Analysis of the terminal structures associated with these various forms provided new insights into the mechanism by which viral DNA replicates and, in particular, suggested that interstrand ligation occurs during this process.

Animals↗
PubMed2001

The cyclin-dependent kinase inhibitor p21 protects the lung from oxidative stress.

The lung is a major target tissue for oxidative stress, including hyperoxia used to relieve tissue hypoxia. Unfortunately, severe hyperoxia damages DNA, inhibits proliferation, and kills cells, resulting in morbidity and mortality. Although hyperoxia induces the tumor suppressor p53 and its downstream target, the cyclin-dependent kinase inhibitor p21(Cip1/WAF1/Sdi1) (p21), their role in pulmonary injury remains unknown. Using p53- and p21-deficient mice we demonstrate that hyperoxia induces p21 in the absence of p53, suggesting that previous conclusions that p53 does not modify hyperoxic lung injury cannot be extrapolated to p21. In fact, mean survival of p21-deficient mice decreased by 40% and was associated with terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling staining of alveolar debris, indicative of DNA fragmentation and cell death. Ultrastructural analyses revealed that alveolar endothelial and type I epithelial cells died rapidly by necrosis. Although hyperoxia decreased DNA replication in p21-wild-type lungs, it had no effect on replication in p21-deficient lungs. Our findings suggest that p21 protects the lung from oxidative stress, in part, by inhibiting DNA replication and thereby allowing additional time to repair damaged DNA. Our findings have implications for patients suffering from the toxic effects of supplemental oxygen therapies.

Animals↗
PubMed1977

A protective effect of caffeine on drosophila larvae treated with methyl methanesulfonate.

Lethality induced in larval populations of Drosophila melanogaster was recorded after treatment with (1) caffeine, (2) MMS or (3) caffeine plus MMS. The mixture of caffeine plus MMS was less toxic than expected from the effects observed after treatment with either substance individually. It is postulated that in the combined treatment the caffeine, by inhibiting semiconservative DNA replication, allows for some additional time for repair of alkylated DNA by a repair pathway which is not sensitive to caffeine, possibly excision repair.

Animals↗
PubMed1986

Oncogenes and the mammalian X chromosome.

None of the up to now localized and expressed oncogenes maps to the mammalian X chromosome. This fact is discussed in the light of a trans-acting regulation mechanism for oncogenes. Such a specific regulation mechanism is demonstrated here for a qualitative change--i.e., varying timing of DNA-replication--at the putative c-myc gene locus in band 15E of murine T-cell leukemia. In intraspecific hybrids between tumor and non-tumor cells this qualitative change spreads over to all chromosomes 15 of the same cell, irrespective of their origin. This effect is thought to reflect a binary trans-acting regulation mechanism between homologous chromosomal loci. In the past specific chromosomal aberrations have been described in various tumors but none of these aberrations involve the X chromosome. For the mammalian X chromosome where there is usually only one gene copy per cell active the described kind of binary trans-acting regulation between homologous gene loci is rendered impossible.

Animals↗
PubMed1978

Decrease of DNA synthesis in amniotic fluid cells during the middle part of S-phase revealed by differential chromosome staining after incorporation of BrdU.

The time sequence of DNA replication in partially synchronized human amniotic fluid cells has been analysed, employing BrdU incorporation techniques.--Regardless of the interval between removal of the methotrexate/uridine block and addition of BrdU during S-phase, the treatment results in an R-type replication pattern. Conversely, replacement of BrdU containing medium by another one with thymidine yields G-type replication patterns. A thymidine pulse during the first 4 h of S-phase results in R-type replication patterns; from 7--10 h after block removal it produces G-type pattern. In between, only faint red staining dots can be found indicating a marked decrease of replicational activity during the middle part of the S-phase.

Amniotic Fluid↗
PubMed2003

Efficient replication of cloned African cassava mosaic virus in cassava leaf disks.

A transient viral replication assay for cloned African cassava mosaic virus (ACMV) was developed using cassava leaf disks. TMS60444 leaf disks were transfected using biolistic-mediated inoculation with ACMV clones pKACMVA and pKACMVB, which originate from West Kenya ACMV isolate 844 (ACMV-KE). Viral DNA synthesized de novo was monitored by Southern hybridization with an AV1 DNA probe. By using the methylation-sensitive restriction enzymes DpnI and MboI, it was possible to distinguish between the input DNA (dam-methylated) and the de novo synthesized viral DNA (not methylated). Different media used for pre- and post-culture of inoculated leaf disks significantly affected the efficiency of viral DNA accumulation. Without pre-culture, replicated viral DNA was not detectable. Culture time in optimized medium also affected the accumulation of nascent viral DNA, and the best results were obtained after 4 days pre-culture on CIM medium followed by 4-6 days post-culture in SH medium. Time-course analysis showed that viral DNA replication can persist for 5-6 days post-inoculation. Our results also confirmed that DNA B of ACMV could assist the accumulation of viral DNA in the leaf disks. The novel protocol described here has also been used successfully with other cassava cultivars (MCol22, MCol1505, TME282 and TMS92/0326) and ACMV clones from the ACMV Nigeria isolate (ACMV-NOg).

Base Sequence↗
PubMed1985

Properties of adenoviral DNA bound to the nuclear matrix.

The association of adenoviral DNA with the high salt (2 M NaCl) resistant nuclear fraction, termed the nuclear matrix, has been investigated in HeLa cells at different times after infection with adenovirus type 5. When nuclear matrices were prepared in the absence of exogenously added nucleases, Ad5 DNA was quantitively associated with the matrix throughout the infection period (0-24 h). Moreover, early in infection (0-10 h) Ad5 DNA was severalfold enriched in DNase I digested nuclear matrices (8-15% of total nuclear DNA) compared to the high salt soluble chromatin fraction (85-90% of total nuclear DNA). At later times after infection, progressively more Ad5 DNA appeared in the chromatin fraction until, at 24 h, the nuclear matrix was strikingly depleted in Ad5 DNA. A large proportion of the Ad5 DNA in nuclear matrices prepared early in infection, e.g., 4 h, was full length in size. At later times (12-24 h) most of the viral DNA was fragmented to a size equivalent to total matrix DNA (100-1000 base pairs). The apparent switch of the matrix-associated viral DNA from a relatively DNase I resistant to sensitive state was initiated approximately at the time when viral DNA replication began (12 h). Since no discrete portion of the Ad5 genome was significantly enriched at the sites of attachment to the nuclear matrix throughout the infection period, the switch in DNase I sensitivity is not mediated by a change in DNA sequence attachment to the matrix.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenoviruses, Human↗
PubMed2002

Chk1 is activated transiently and targets Cdc25A for degradation at the Xenopus midblastula transition.

In Xenopus embryos, cell cycle elongation and degradation of Cdc25A (a Cdk2 Tyr15 phosphatase) occur naturally at the midblastula transition (MBT), at which time a physiological DNA replication checkpoint is thought to be activated by the exponentially increased nucleo-cytoplasmic ratio. Here we show that the checkpoint kinase Chk1, but not Cds1 (Chk2), is activated transiently at the MBT in a maternal/zygotic gene product-regulated manner and is essential for cell cycle elongation and Cdc25A degradation at this transition. A constitutively active form of Chk1 can phosphorylate Cdc25A in vitro and can target it rapidly for degradation in pre-MBT embryos. Intriguingly, for this degradation, however, Cdc25A also requires a prior Chk1-independent phosphorylation at Ser73. Ectopically expressed human Cdc25A can be degraded in the same way as Xenopus Cdc25A. Finally, Cdc25A degradation at the MBT is a prerequisite for cell viability at later stages. Thus, the physiological replication checkpoint is activated transiently at the MBT by developmental cues, and activated Chk1, only together with an unknown kinase, targets Cdc25A for degradation to ensure later development.

Animals↗
PubMed2002

Complex transcriptional circuitry at the G1/S transition in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, SBF (Swi4-Swi6 cell cycle box binding factor) and MBF (MluI binding factor) are the major transcription factors regulating the START of the cell cycle, a time just before DNA replication, bud growth initiation, and spindle pole body (SPB) duplication. These two factors bind to the promoters of 235 genes, but bind less than a quarter of the promoters upstream of genes with peak transcript levels at the G1 phase of the cell cycle. Several functional categories, which are known to be crucial for G1/S events, such as SPB duplication/migration and DNA synthesis, are under-represented in the list of SBF and MBF gene targets. SBF binds the promoters of several other transcription factors, including HCM1, PLM2, POG1, TOS4, TOS8, TYE7, YAP5, YHP1, and YOX1. Here, we demonstrate that these factors are targets of SBF using an independent assay. To further elucidate the transcriptional circuitry that regulates the G1-to-S-phase progression, these factors were epitope-tagged and their binding targets were identified by chIp-chip analysis. These factors bind the promoters of genes with roles in G1/S events including DNA replication, bud growth, and spindle pole complex formation, as well as the general activities of mitochondrial function, transcription, and protein synthesis. Although functional overlap exists between these factors and MBF and SBF, each of these factors has distinct functional roles. Most of these factors bind the promoters of other transcription factors known to be cell cycle regulated or known to be important for cell cycle progression and differentiation processes indicating that a complex network of transcription factors coordinates the diverse activities that initiate a new cell cycle.

Binding Sites↗
PubMed1995

Dynamics of Nuclear DNA Quantities during Zygote Development in Barley.

Quantities of DNA were estimated in the nuclei of mechanically isolated egg and zygote protoplasts in two cultivars of barley using 4[prime],6-diamidino-2-phenylindole staining and microfluorometry. Unlike many previous studies on DNA amounts within the sex cells of flowering plants, we obtained consistent and unambiguous results indicating that the egg and sperm nuclei are at the 1C DNA level (basic haploid amount) at the time of karyogamy. Karyogamy was initiated within 60 min postpollination, and the male chromatin became completely integrated into the egg nucleus within 6 to 7 hr postpollination (hpp). Zygotic nuclear DNA levels began to increase at ~9 to 12 hpp in cultivar Alexis and at 12 to 15 hpp in cultivar Igri. The 4C DNA complement was reached in most zygotes by 22 to 26 hpp in cultivar Alexis and by 23 to 29 hpp in cultivar Igri. These data are fundamental to a better understanding of fertilization and zygote maturation in flowering plants. They are also relevant to studies in which the timing of zygotic DNA replication is of interest, such as ongoing investigations on genetic transformations in barley using the microinjection technique.

Journal Article↗
PubMed1985

Cytotoxic and mutagenic effects of specific carcinogen-DNA adducts in diploid human fibroblasts.

A comparison of the cytotoxicity and mutagenicity of a series of carcinogens in normal diploid human fibroblasts and in cells deficient in one or more DNA repair processes has provided insight into the specific DNA adduct(s) responsible for these biological effects. The carcinogens tested include ultraviolet radiation; reactive derivatives of structurally related aromatic amides; metabolites of benzo(a)pyrene; the simple alkylating agents N-methyl-N'-nitro-N-nitrosoguanidine and N-ethyl-N-nitrosourea; and aflatoxin B1 dichloride, a model for the reactive 2,3-epoxide of aflatoxin B1. Exponentially growing cells were exposed to agents and assayed for mutations (induction of 6-thioguanine resistance) and cell killing (loss of colony-forming ability). Cells deficient in repair of particular DNA adducts or lesions proved more sensitive to the agent causing those lesions than did normally repairing cells. Many of the carcinogens were compared for their mutagenic and/or cytotoxic effect, not only as a function of dose administered, but also as a function of the initial number of adducts or photoproducts induced in DNA and the number remaining at critical times posttreatment. Density-inhibited cultures were exposed to cytotoxic and mutagenic doses. Immediately after treatment, or after various lengths of time in confluence, the cells were harvested and analyzed for the number of lesions remaining in DNA. A portion was plated at lower densities and assayed for mutations and/or for survival. In several instances, the adducts were analyzed by high-pressure liquid chromatography. As an alternative approach, cells were synchronized and treated at various times prior to the onset of DNA synthesis and analyzed for survival and/or the frequency of mutations. The results demonstrated a high correlation between the number of DNA lesions remaining unexcised at the time the DNA was replicated and the frequency of mutations induced. Comparative studies of the frequency of UV-induced transformation (to anchorage independence) of normal and repair-deficient cells showed this also to be true for transformation.

Carcinogens↗
PubMed1995

The centromere: kinetochore complex.

At the meta-anaphase transition the centromeres in a genome separate in non-random sequential manner. This sequential separation depends upon the timing of replication of DNA located in the pericentric and centromeric region. Cells in long term cultures as well as some newborn humans carry dicentric chromosomes. The inactive centromeres in these dicentric chromosomes do not show any sequence of separation. Whether or not a dicentric chromosome would segregate equationally depends upon if only one centromere binds to microtubules or both are functional. In man and other higher apes, a 171 base pair long DNA repeat (the alphoid sequence) is present on all centromeres. In mouse, the minor satellite fraction is said to constitute the centromere. These two DNAs also carry a 17 bp long sequence, the CENP-B 'box' to which the CENP-B antigen is bound. Other species-eg, rat, pig, fish, Chinese hamster-exhibit still different sequences at the centromere and do not carry the CENP-B 'box' even though the antigen is ubiquitously present in all species. It is not clear why so many diverse sequences constitute the centromere when all centromeres look alike and perform the same function. I propose that the primary constriction owes its property not necessarily to its DNA composition but to some stereophysical property, eg the curvature and that the region is held together till late metaphase-anaphase due to a specific proteinaceous factor. The mammalian centromeres bind a complex of several proteins dubbed as CENtromere Proteins (CENP's). This complex, however, is not what constitutes the trilamellar kinetochore structure as see under the electron microscope.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Host cell variations resulting from F plasmid-controlled replication of the Escherichia coli chromosome.

Cell size and DNA concentration were measured in Escherichia coli K-12 ET64. This strain carries a dnaA (Ts) mutation that has been suppressed by the insertion of the F plasmid into the chromosome. ET64 can grow in a balanced steady state of exponential growth at the restrictive temperature for its dnaA allele (39 degrees C), in which chromosome replication is controlled by the F plasmid, and at the permissive temperature (30 degrees C), in which chromosome replication is controlled by dnaA-oriC. When cells grown at the indicated temperatures were compared, it was observed that at 39 degrees C, the cell mass increased and the amount of cellular DNA decreased slightly; therefore, the DNA concentration was strongly reduced. These changes can neither be explained by the reduction of the generation time (which is only 10-15%) nor from observed changes in the replication time and in the time between DNA synthesis termination and cell division. Variations were mainly due to the increase in cell mass per origin of replication, at initiation, in cells grown at 39 degrees C. Control of chromosome replication by the F plasmid appears to be the reason for the increase in the initiation mass. Other possible causes, such as the modification of growth temperature, the generation time, or both, were discarded. These observations suggest that at one growth rate, the F plasmid replicates at a particular cell mass to F particle number ratio, and that this ratio is higher than the cell mass to oriC ratio at the initiation of chromosome replication. This fact might be significant to coordinate the replication of two different replicons in the same cell.

Bacterial Proteins↗