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PubMed1996

Inhibition of topoisomerase II by ICRF-193 prevents efficient replication of herpes simplex virus type 1.

Cellular topoisomerase II is specifically inactivated by the drug ICRF-193. This compound turns topoisomerase II into a closed clamp that is unable to cleave DNA. We have investigated the effects of this inhibitor on the replication of herpes simplex virus type 1. We show that ICRF-193 at low multiplicities of infection dramatically inhibits viral DNA synthesis and the production of infectious virus. The inhibition is less efficient at high multiplicities of infection. In addition, inhibition of viral DNA synthesis was observed only when ICRF-193 was present during the first 4 h of the infectious cycle. The transient replication of plasmids containing a herpes simplex virus type 1 origin of DNA replication, oriS, was affected by ICRF-193 in the same way. In contrast, neither cellular DNA synthesis nor replication of plasmids containing a simian virus 40 origin of DNA replication was inhibited. The observed effect on herpes simplex virus DNA replication was not caused by a decreased transcription of replication genes inasmuch as the levels of UL8, UL9, UL29, and UL30 rmRNAs were unaffected by the drug. These results suggest that topoisomerase II plays a vital role during the replication of herpes simplex virus type 1 DNA. We speculate that topoisomerase II is involved in the decatenation of newly synthesized daughter molecules.

Animals↗
PubMed1997

Bovine papillomavirus type 1 DNA replication: the transcriptional activator E2 acts in vitro as a specificity factor.

We previously devised cell-free conditions supporting efficient replication of bovine papillomavirus type 1 (BPV1) DNA (C. Bonne-Andréa, S. Santucci, and P. Clertant, J. Virol. 69:3201-3205, 1995): the use of highly active preparations of viral initiator protein E1, together with extract from a particular cell source, allowed the synthesis of complete DNA circles through successive rounds of replication; this occurred in the absence of the viral transcriptional activator E2, required in vivo for the replication of viral genomes. We now report that adding E2 to cell-free assays produced only slight effects both on the yield of E1-dependent DNA synthesis and on the quality of newly made DNA molecules when a template carrying a wild-type BPV1 replication origin (ori) was used. The performance of mouse cell extracts, unable to sustain efficient BPV1 DNA replication in the presence of E1 only, was likewise not improved by the addition of E2. In a proper in vitro environment, E1 is thus fully capable of efficiently initiating viral DNA synthesis by itself, an activity which is not enhanced by interaction with E2. An important effect, however, was detected: E2 totally suppressed the nonspecific replication of ori-defective DNA templates, otherwise observed in high E1 concentrations. We examined the requirements for building a minimal DNA sequence behaving in vitro as a specific ori sequence under stringent recognition conditions, i.e., in the presence of both E1 and E2. Only two elements, the 18-bp E1 binding palindrome and an AT-rich sequence, were required in cis to allow specific cell-free DNA replication; there seemed to be no need for an E2 binding site to ensure discrimination between specific ori templates and other DNA molecules, even in the presence of E2. This suggests that during the initiation of BPV1 DNA replication, at least in vitro, E2 acts as a specificity factor restricting the action of E1 to a defined ori sequence; this function, likely not demanding the direct binding of E2 to cognate DNA sites, might primarily involve protein-protein interactions.

Animals↗
PubMed1995

Initiation of latent DNA replication in the Epstein-Barr virus genome can occur at sites other than the genetically defined origin.

Our laboratory has previously shown that replication of a small plasmid, p174, containing the genetically defined Epstein-Barr virus (EBV) latent origin of replication, oriP, initiates within oriP at or near a dyad symmetry (DS) element and terminates specifically at a family of repeated sequences (FR), also located within oriP. We describe here an analysis of the replication of intact approximately 170-kb EBV genomes in four latently infected cell lines that uses two-dimensional gel replicon mapping. Initiation was detected at oriP in all EBV genomes examined; however, some replication forks appear to originate from alternative initiation sites. In addition, pausing of replication forks was observed at the two clusters of EBV nuclear antigen 1 binding sites within oriP and at or near two highly expressed viral genes 0.5 to 1 kb upstream of oriP, the EBV-encoded RNA (EBER) genes. In the Raji EBV genome, the relative abundance of these stalled forks and the direction in which they are stalled indicate that most replication forks originate upstream of oriP. We thus searched for additional initiation sites in the Raji EBV and found that the majority of initiation events were distributed over a broad region to the left of oriP. This delocalized pattern of initiation resembles initiation of replication in several well-characterized mammalian chromosomal loci and is the first described for any viral genome. EBV thus provides a unique model system with which to investigate factors influencing the selection of replication initiation and termination sites in mammalian cells.

Antigens, Viral↗
PubMed1999

Human topoisomerase I promotes initiation of simian virus 40 DNA replication in vitro.

Addition of purified human topoisomerase I (topo I) to simian virus 40 T antigen-driven in vitro DNA replication reactions performed with topo I-deficient extracts results in a greater than 10-fold stimulation of completed molecules as well as a more than 3-fold enhancement of overall DNA replication. To further characterize this stimulation, we first demonstrate that bovine topo I but not Escherichia coli topo I can also enhance DNA replication. By using several human topo I mutants, we show that a catalytically active form of topo I is required. To delineate whether topo I influences the initiation or the elongation step of replication, we performed delayed pulse, pulse-chase, and delayed pulse-chase experiments. The results illustrate that topo I cannot promote the completion of partially replicated molecules but is needed from the beginning of the reaction to initiate replication. Competitive inhibition experiments with the topo I binding T antigen fragment 1-246T and a catalytically inactive topo I mutant suggest that part of topo I's stimulation of replication is mediated through a direct interaction with T antigen. Collectively, our data indicate that topo I enhances the synthesis of fully replicated DNA molecules by forming essential interactions with T antigen and stimulating initiation.

Animals↗
PubMed1999

The linking regions of EBNA1 are essential for its support of replication and transcription.

The ability of distant cis-acting DNA elements to interact functionally has been proposed to be mediated by the interaction of proteins associated site specifically with those cis-acting elements. We have found that the DNA-linking regions of EBNA1 are essential for its contribution to both replication and transcription. The synthesis of plasmids containing the Epstein-Barr virus (EBV) origin of plasmid replication (oriP) can be mediated entirely by the cellular machinery; however, the replicated molecules are lost rapidly from proliferating cells. When EBNA1 is provided in trans, plasmids containing oriP (oriP plasmids) are synthesized during repeated S phases, and the newly formed daughter molecules are precisely segregated to the daughter cells. The contribution(s) of EBNA1 to the stable replication of oriP plasmids is therefore likely to be postsynthetic. In latently infected cells, EBNA1 also regulates the expression of multiple EBV promoters located as many as 10 kbp away. EBNA1 supports replication and transcription through binding to oriP; both the ability of EBNA1 to bind to DNA and the integrity of its binding sites in oriP are required. However, DNA binding by EBNA1 is not sufficient to support replication or transcription, indicating that an additional activity (or activities) is required. EBNA1 links DNAs to which it binds and can form a loop between the two subelements of oriP, the family of repeats and the region of dyad symmetry, each of which contains multiple binding sites for EBNA1. We have constructed a set of derivatives of EBNA1 which contain both, one, or neither of its linking regions in various contexts. Analyses of these derivatives demonstrate that the linking regions of EBNA1 are essential for its support of replication and transcription and that the ability of derivatives of EBNA1 to link DNAs correlates strongly with their support of these activities in cells. These findings indicate that protein-protein associations of the linking regions of EBNA1 underlie its long-range contributions to replication and transcription.

Cell Line↗
PubMed2003

The papillomavirus E8-E2C protein represses DNA replication from extrachromosomal origins.

Carcinogenic DNA viruses such as high-risk human papillomaviruses (HPV) and Epstein-Barr-Virus (EBV) replicate during persistent infections as low-copy-number plasmids. EBV DNA replication is restricted by host cell replication licensing mechanisms. In contrast, copy number control of HPV genomes is not under cellular control but involves the viral sequence-specific DNA-binding E2 activator and E8-E2C repressor proteins. Analysis of HPV31 mutant genomes revealed that residues outside of the DNA-binding/dimerization domain of E8-E2C limit viral DNA replication, indicating that binding site competition or heterodimerization among E2 and E8-E2C proteins does not contribute to copy number control. Domain swap experiments demonstrated that the amino-terminal 21 amino acids of E8-E2C represent a novel, transferable DNA replication repressor domain, whose activity requires conserved lysine and tryptophan residues. Furthermore, E8-E2C (1-21)-GAL4 fusion proteins inhibited the replication of the plasmid origin of replication of EBV, suggesting that E8-E2C functions as a general replication repressor of extrachromosomal origins. This finding could be important for the development of novel therapies against persistent DNA tumor virus infections.

Base Sequence↗
PubMed2000

Decreased cellular activity and replicative capacity of osteoblastic cells isolated from the periarticular bone of rheumatoid arthritis patients compared with osteoarthritis patients.

OBJECTIVE: Periarticular osteopenia is frequently observed in rheumatoid arthritis (RA). Bone loss has been considered to be at least partly due to inadequate bone formation, which in turn, is largely dependent on the number of osteoblasts and the osteoblastic activity. Normal human somatic cells undergo a finite number of cell divisions and ultimately enter a nondividing state called replicative senescence. It has been proposed that the telomere, the terminal sequence of chromosomes, is the mitotic clock that triggers senescence. In the present study, we sought to clarify the relationship between periarticular osteopenia and osteoblast replicative senescence in RA. METHODS: We examined age-related changes in cellular activity (alkaline phosphatase activity, osteocalcin and C-terminal type I procollagen secretion, and cAMP response to parathyroid hormone), replicative capacity, and senescent cell expression in osteoblasts from periarticular bone samples obtained from 15 patients with RA and 15 age-matched patients with osteoarthritis (OA). Cellular replicative capacity was analyzed by the mean telomere length and in vitro remaining replicative lifespan of the cells. RESULTS: In both OA and RA groups, the cell proliferation rate, the levels of osteoblastic markers, mean telomere length, and replicative lifespan in osteoblastic cells gradually decreased with the increasing age of the donor. The percentage of senescent osteoblastic cells in the periarticular bone increased with age in both groups, and the rate of expression of senescent cells was higher in RA patients than in age-matched OA patients. The osteoblastic activities and replicative capacity of osteoblastic cells from RA patients were lower than those from OA patients at any donor age. The age-related decreases in the osteoblastic activity and replicative capacity of osteoblastic cells from periarticular bone were greater in RA patients than in OA patients. CONCLUSION: Our results suggest that osteoblast replicative senescence in periarticular bones occurs more rapidly with aging in RA than in OA patients and contributes to periarticular osteopenia in RA.

Aged↗
PubMed1995

Quantification of particle sizes with metal replication under standard freeze-etching conditions: a gold ball standard for calibrating shadow widths was used to measure freeze-etched globular proteins.

The real size of platinum-carbon (Pt-C) replicated particles is not directly equivalent to either its metal-coated diameter or its shadow width. This paper describes two indirect methods, shadow widths and coated particle diameters, for determining a particle's actual size beneath a Pt-C replication film. Both produce equivalent measurements using the same standardized conditions: 2.3 nm Pt-C films deposited at a 45 degree angle on an approximately -100 degrees C surface in a 10(-6) torr vacuum. For the first method, gold balls nucleated in a partial pressure of helium and deposited on flat indirect carbon films (root mean square roughness of 0.8 nm) on 400 mesh grids were used as test particles for calibrating shadow widths as a function of particle size. The gold ball test specimens were replicated, and a distribution of Pt-C shadow widths orthogonal to the Pt-C deposition direction was measured and averaged for gold balls 1.5 +/- 0.25 nm, 2.0 +/- 0.25 nm, etc. The diameter of each gold ball was measured within the Pt-C film along with its shadow width because the Pt-C did not obscure or adhere well to the gold. The shadow width distributions for each gold size do not differ significantly from log normal. Two proteins, the lactose repressor and the mitochondrial ATPase, F1, were also used as replication test objects. Negative staining of both proteins was conducted to measure their average diameters. In the second method, a distribution of Pt-C-coated lac repressor diameters perpendicular to the shadow direction was measured. The Pt-C film thickness measured on the quartz crystal monitor was subtracted from the average metal-coated protein diameter to obtain the lac repressor's diameter. The Pt-C-coated particle diameter distributions also did not differ significantly from log normal. While doing this work it was discovered that outgassing the Pt-C electron gun greatly affected Pt-C film granularity: 19 sec produced a high contrast, granular Pt-C film, whereas 120 sec yielded a low contrast, less granular Pt-C film. Both gold balls and protein particles were subjected in separate experiments to either 19 or 120 sec of outgassing of the Pt-C gun prior to Pt-C replication. Outgassing had a profound effect on the average size of the Pt-C shadow widths on both gold and protein particles. The Pt-C gun outgassing procedure also determined the smallest replicated particle that could be resolved. The frequency of some smaller gold ball sizes detected after replication was reduced disproportionately with 19 sec vs. 120 sec outgassing. However, Pt-C gun outgassing did not affect the average measured diameter of the Pt-C-coated protein particles. The "geometric assumption" that each metal-coated particle creates a shadow width the same size as the metal-coated particle diameter was tested using a globular protein. Pt-C replication of protein particles at a 45 degree and 20 degree angle could not confirm the geometric assumption because an average shadow width was always significantly larger than its average Pt-C-coated particle diameter. A model for how the large shadow widths are formed is presented. Gold balls were also replicated at a 45 degree angle with current high resolution conditions at a substrate temperature of -185 degrees C, and the results of these replicas were compared to the results reported here at approximately -100 degrees C.

Calibration↗
PubMed1999

Heterogeneity in intracellular replication and cytopathogenicity of Legionella pneumophila and Legionella micdadei in mammalian and protozoan cells.

In contrast to Legionella pneumophila, little is known about the pathogenesis of other legionellae species that are capable of causing Legionnaires' disease. In this report, we contrast L. pneumophila and L. micdadei for their cytopathogenicity and intracellular replication within mammalian and protozoan cells. We show by transmission electron microscopy that L. micdadei replicates within an endoplasmic reticulum (RER)-free phagosome within human macrophages, alveolar epithelial cells, and within the protozoan Hartmannella vermiformis. In contrast, L. pneumophila replicates within a RER-surrounded phagosome within the same host cells. In contrast to replication of L. pneumophila within Acanthamoebae polyphaga, L. micdadei does not replicate within this protozoan host. Despite the prolific intracellular replication, L. micdadei is less cytopathogenic to all host cells than L. pneumophila. Since both species replicate intracellularly to a similar level, we have examined whether the reduced cytopathogenicity of L. micdadei is due to a reduced capacity to induce apoptosis or pore formation-mediated necrosis, both of which contribute to killing of the host cell by L. pneumophila. The data show that both species induced apoptosis-mediated killing of mammalian cells to a similar level. In contrast to L. pneumophila, expression of the pore-forming toxin by L. micdadei and its necrotic effect on macrophages and alveolar epithelial cells is undetectable. This has been further confirmed showing that L. micdadei is completely defective in contact-dependent haemolysis of RBCs, an activity mediated by the pore-forming toxin. Finally, in contrast to L. pneumophila, there was no significant intrapulmonary replication of L. micdadei in the A/J mice animal model. Our data show dramatic differences between L. pneumophila and L. micdadei in intracellular replication, cytopathogenicity, and infectivity to mammalian and protozoan cells.

Acanthamoeba↗
PubMed2001

Deletion and substitution analysis defines regions and residues within the phosphoprotein of bovine respiratory syncytial virus that affect transcription, RNA replication, and interaction with the nucleoprotein.

The phosphoprotein (P) of bovine respiratory syncytial virus (BRSV) is a multifunctional protein that plays a central role in transcription and replication of the viral genomic RNA. To investigate the domains and specific residues involved in different activities of the P protein, we generated a total of 22 deletion and 17 point mutants of the P protein. These mutants were characterized using an intracellular BRSV-CAT minigenome replication system for the ability to (1) direct minigenome transcription, (2) direct minigenome replication, and (3) form complexes with nucleocapsid protein (N) and large polymerase protein (L). These studies revealed that all the regions of P protein except amino acids 41-80 are essential for minigenome transcription and replication. Interestingly, amino acids 41-60 appeared to contain sequences that negatively regulate transcription and replication. Analysis of the N- or C-terminal ends indicated that deletion of up to 3 amino acids from the N- or C-terminus completely ablated the replication, while leaving substantial residual transcription. Single amino acid substitutions within the N-terminal 4 or C-terminal 13 amino acids showed that substitution at position 2, 4, 234, 236, 238, 240, or 241 was highly inhibitory to both transcription and replication, whereas substitution at position 3 was highly inhibitory to replication while leaving substantial residual transcription. Substitution of serine residues at the C-terminus indicated that loss of phosphorylation sites did not appear to have any effect on transcription and replication. Coimmunoprecipitation of P-N and P-L complexes with P-specific antiserum revealed that substitution mutations at the N- or C-terminus did not affect binding to N and L proteins, except that substitution mutation at C-terminus position 234, 236, 238, 240, or 241 affected binding to N protein by 10-fold.

Amino Acid Substitution↗
PubMed1978

The replication of kinetoplast DNA networks in Crithidia fasciculata.

Kinetoplast DNA from the mitochondria of Crithidia is in the form of a two-dimensional network of thousands of minicircles each containing about 2.5 kb, and a small number of maxicircles each containing about 40 kb. Fractionation of kinetoplast DNA by equilibrium centrifugation in a CsCl-propidium diiodide gradient resolves it into three types of networks. Form I networks band at high density and contain minicircles which are covalently closed; form II networks band at low density and contain minicircles which are nicked or gapped; and replicating networks band at intermediate density and contain some minicircles of each type. Form I networks contain about 5000 minicircles; form II networks contain about 11,000; and replicating networks contain an intermediate number. When cells are pulse-labeled with 3H-thymidine, radioactivity in mitochondrial DNA is preferentially incorporated into replicating networks, but after a chase it appears first in form II networks and finally in form I. Examination of replicating networks by electron microscopy in the presence of ethidium bromide reveals that minicircles in the central region of the network are twisted and therefore covalently closed, whereas those in the peripheral region are not twisted and therefore must be nicked or gapped. The pulse-label is incorporated into the nicked or gapped minicircles of the replicating networks. These results indicate that replication of form I networks begins in peripheral minicircles and that progeny minicircles remain nicked or gapped. As replication proceeds, the size of the network increases, and the peripheral zone of nicked or gapped minicircles enlarges. Finally, when all minicircles have replicated, the network, now form II is double the size of form I and contains only nicked or gapped minicircles. The final step in replication presumably includes both the cleavage of the network into two form I species and the covalent closure of all the minicircles.

Animals↗
PubMed2006

Identification of putative DnaN-binding motifs in plasmid replication initiation proteins.

Recently the plasmid RK2 replication initiation protein, TrfA, has been shown to bind to the beta subunit of DNA Polymerase III (DnaN) via a short pentapeptide with the consensus QL[S/D]LF. A second consensus peptide, the hexapeptide QLxLxL, has also been demonstrated to mediate binding to DnaN. Here we describe the results of a comprehensive survey of replication initiation proteins encoded by bacterial plasmids to identify putative DnaN-binding sites. Both pentapeptide and hexapeptide motifs have been identified in a number of families of replication initiation proteins. The distribution of sites is sporadic and closely related families of proteins may differ in the presence, location, or type of putative DnaN-binding motif. Neither motif has been identified in replication initiation proteins encoded by plasmids that replicate via rolling circles or strand displacement. The results suggest that the recruitment of DnaN to the origin of replication of a replisome by plasmid replication initiation proteins is not generally required for plasmid replication, but that in some cases it may be beneficial for efficiency of replication initiation.

Amino Acid Motifs↗
PubMed2001

Telomere shortening and decline in replicative potential as a function of donor age in human adrenocortical cells.

Telomere shortening is the cause of replicative senescence of mammalian cells in culture and may be a cause of cellular aging in vivo. Some tissues clearly show telomere shortening during aging in humans, but the relationship between replication history and telomere length is obscured by complex relationships between stem cells and more differentiated cell types. Previous experiments on the adrenal cortex and human adrenocortical cells in culture indicate that the proliferative biology of this tissue is relatively simple; cell division occurs continuously throughout life, without evidence for a distinct stem cell compartment. In this tissue we investigated the relationship between telomere biology and replicative senescence by measuring replicative capacity and telomere length as a function of donor age. Cells cultured from adrenal tissue from donors of different ages showed a strong age-related decline in total replicative capacity, falling from about 50 population doublings for fetal cells to an almost total lack of division in culture for cells from older donors. Telomere restriction fragment (TRF) length was analyzed in the same sets of cells and decreased from a value of about 12 kb in fetal cells to approximately 7 kb in cells from older donors. The latter value is consistent with that in fibroblasts which have reached replicative senescence. Furthermore, there was a good correlation in individual donor samples between TRF length and replicative capacity in culture. To confirm the relationship between telomere length, telomerase, and replicative capacity, we measured telomere length in cells before and after infection with a retrovirus encoding hTERT, the catalytic component of human telomerase. The adult adrenal cortex does not have telomerase activity; cells after transduction with the hTERT retrovirus had high telomerase activity. Whereas control cells underwent a replication-dependent shortening in telomeres during long-term growth in culture, hTERT-modified cells maintained telomere length and are probably immortalized. Symmetric cell division in human adrenocortical cells, occurring slowly over the life span, is associated with progressive telomere shortening and may result in proliferative defects in vivo in old age, which could partly account for the age-related changes in the structure and function of the human adrenal cortex.

Adolescent↗
PubMed2003

Evidence for preferential mismatch repair of lagging strand DNA replication errors in yeast.

Duplex DNA is replicated in the 5'-3' direction by coordinated copying of leading and lagging strand templates with somewhat different proteins and mechanics, providing the potential for differences in the fidelity of replication of the two strands. We previously showed that in Saccharomyces cerevisiae, active replication origins establish a strand bias in the rate of base substitutions resulting from replication of unrepaired 8-oxo-guanine (GO) in DNA. Lower mutagenesis was associated with replicating lagging strand templates. Here, we test the hypothesis that this bias is due to more efficient repair of lagging stand mismatches by measuring mutation rates in ogg1 strains with a reporter allele in two orientations at loci on opposite sides of a replication origin on chromosome III. We compare a MMR-proficient strain to strains deleted for the MMR genes MSH2, MSH6, MLH1, or EXOI. Loss of MMR reduces the strand bias by preferentially increasing mutagenesis for lagging strand replication. We conclude that GO-A mismatches generated during lagging strand replication are more efficiently repaired. This is consistent with the hypothesis that 5' ends of Okazaki fragments and PCNA, present at high density during lagging strand replication, are used as strand discrimination signals for mismatch repair in vivo.

Base Pair Mismatch↗
PubMed1998

Vertical Pt-C replication for TEM, a revolution in imaging non-periodic macromolecules, biological gels and low-density polymer networks.

Vertical replication for TEM is ideal for studying non-periodic specimens from 0.7 to 3 nm, a resolution mid-range difficult to attain by any other technique. This paper discusses the importance of vertical replication, its methods and hardware for high-resolution TEM. Evidence from diverse published research will demonstrate vertical replication's versatility in imaging the molecular level normally unattainable in freeze-dried polymers, polyethylene tribological wear on surfaces, low-density polymer networks or biological gels. Vertical platinum-carbon (Pt-C) replication minimizes the horizontal movement of Pt-C on a surface. Surface objects are symmetrically enlarged by a vertically deposited Pt-C film. To estimate real size in replicas, 16-25 particles or filaments need to be measured in calibrated transmission electron microscopy (TEM) images and reduced by a value less than the Pt-C film thickness measured with a quartz monitor. Continuous, vertically deposited Pt-C films are formed on mica at a deposition thickness of around 1.0 nm and on silver at a thickness of 0.4-0.5 nm. The distance between helical turns in poly(1-tetradecene sulfone) of 0.7 nm is the highest resolution achieved with vertical replication. Two polysulfones freeze-dried and vertically replicated on mica contained structures are predicted by indirect physical chemical methods to be present in solution. Polymer chains are fully Pt-C coated, with no uncoated gaps along chains. Some side-chains on the extended non-helical poly(1-tetradecene sulfone) are also detected. To estimate the real chain width, polymer chains measured in images are reduced by the Pt-C film thickness minus 0.5 nm. The polymer chain widths estimated from molecular models are in the same range of widths as those measured using the image size correction method. Also, it is possible to distinguish random coil proteins (chain width of around 0.5 nm) from an alpha-helix (chain diameter of about 1 nm) in vertically replicated samples on silver substrates. In the future, subnanometer resolutions below 0.7 nm should be possible. The resolution of vertical replication depends on the thickness of a continuous, amorphous Pt-C film. That thin, continuous 0.4-0.5 nm Pt-C films on silver substrates can be made suggests that a point-to-point resolution limit of around 0.28 nm in TEM may ultimately be approachable with replication.

Amyloid beta-Peptides↗
PubMed2003

Novel immunocompetent murine tumor models for the assessment of replication-competent oncolytic adenovirus efficacy.

Oncolytic replication-selective adenoviruses constitute a rapidly expanding experimental approach to the treatment of cancer. However, due to the lack of an immunocompetent and replication-competent efficacy model, the role of the host immune response and viral E3 immunoregulatory genes remained unknown. We screened nine murine carcinoma lines for adenovirus (Ad5) uptake, gene expression, replication, and cytopathic effects. In seven of these murine cell lines the infectability and cytopathic effects were similar to those seen with human carcinoma lines. Surprisingly, productive viral replication was demonstrated in several lines; replication varied from levels similar to those for some human carcinoma lines (e.g., CMT-64) to very low levels. Seven of these lines were grown as subcutaneous xenografts in immunocompetent mice and were subsequently injected directly with Ad5, saline, or a replication-deficient control adenovirus particle to assess intratumoral viral gene expression, replication, and antitumoral effects. E1A, coat protein expression, and cytopathic effects were documented in five xenografts; Ad5 replication was demonstrated in CMT-64 and JC xenografts. Ad5 demonstrated significant efficacy compared to saline and nonreplicating control Ad particles in both replication-permissive xenografts (CMT-64, JC) and poorly permissive tumors (CMT-93); efficacy against CMT-93 tumors was significantly greater in immunocompetent mice compared to athymic mice. These murine tumor xenograft models have potential for elucidating viral and host immune mechanisms involved in oncolytic adenovirus antitumoral effects.

Adenoviridae↗
PubMed2026

Cohesin reshapes replication fork contacts to aid fork slowing and reversal.

DNA replication forks can be challenged by cancer chemotherapeutic treatments, leading to accumulation of single-stranded DNA and slowdown of DNA synthesis. The marked plasticity of replication forks under replication stress ensures fork stability, damage tolerance and complete genome duplication1. Initiation and progression of replication forks occur in a three-dimensionally organized genome. DNA loop extrusion by the cohesin complex organizes the genome2 and regulates the initiation and positioning of DNA replication origins3,4. Although transient interaction of sister forks was recently reported during unperturbed replication5, the functional relevance of fork contacts during replication stress and the role of cohesin in this context remain unknown. Here we show that cohesin-mediated loop extrusion rearranges nascent DNA contacts at stressed replication forks to promote genome stability. Using auxin-inducible degron6, separation-of-function mutants7-9 and a newly developed Micro-C-based technique to capture chromatin contacts at nascent DNA (Repli-C), we found that loop-extruding cohesin accumulates at stalled replication forks, limiting sister-fork coupling in favour of inter-replicon contacts. This process promotes active fork slowing and reversal by preventing PRIMPOL action on single-stranded DNA1. These findings show that the replication stress response is not merely an accumulation of individual regulatory events, but is topologically integrated across the genome through cohesin loop extrusion. While providing a new function for loop-extruding cohesin, our results indicate the potential impact on cancer therapy of frequent cohesin mutations in tumours10.

Journal Article↗
PubMed1980

Asynchronous replication of heterochromatin in maize.

Replication patterns of four classes of heterochromatin in the chromosomes of maize were analyzed. Centromeric heterochromatin, distal heterochromatin of the B chromosomes, and knob heterochromatin all have asynchronous late replication, knob heterochromatin being the last chromosomal DNA to complete replication. Nucleolus organizer heterochromatin replicates during the period of euchromatin DNA replication. The proportion of the S period involved in asynchronous late replication is directly dependent on the amount of heterochromatin in the nucleus. Both additional knob and B-chromosome heterochromatin participate in this effect. Each additional B chromosome produces an increment in the proportion of nuclei with asynchronous replication; in nuclei with 12 B chromosomes, the proportion of the S phase showing asynchronous late replication is more than 3 times greater than that in nuclei without B chromosomes. The data are consistent with the hypothesis that delayed DNA replication of knob heterochromatin is responsible, in some genotypes, for the loss of chromosome segments and for B-chromosome nondisjunction in the second pollen grain mitosis.

Journal Article↗