Non-specific termination of simian virus 40 DNA replication.

Axial X-ray diffraction patterns have been studied from relaxed, contracted and rigor vertebrate striated muscles at different sarcomere lengths to determine which features of the patterns depend on the interaction of actin and myosin. The intensity of the myosin layer lines in a live, relaxed muscle is sometimes less in a stretched muscle than in the muscle at rest-length; the intensity depends not only on the sarcomere length but on the time that has elapsed since dissection of the muscle. The movement of cross-bridges giving rise to these intensity changes are not caused solely by the withdrawal of actin from the A-band.When a muscle contracts or passes into rigor many changes occur that are independent of the sarcomere length: the myosin layer lines decrease in intensity to about 30% of their initial value when the muscle contracts, and disappear completely when the muscle passes into rigor. Both in contracting and rigor muscles at all sarcomere lengths the spacings of the meridional reflections at 143 Å and 72 Å are 1% greater than from a live relaxed muscle at rest-length. It is deduced that the initial movement of cross-bridges from their positions in resting muscle does not depend on the interaction of each cross-bridge with actin, but on a conformational change in the backbone of the myosin filament: occurring as a result of activation. The possibility is discussed that the conformational change occurs because the myosin filament, like the actin filament, has an activation control mechanism. Finally, all the X-ray diffraction patterns are interpreted on a model in which the myosin filament can exist in one of two possible states: a relaxed state which gives a diffraction pattern with strong myosin layer lines and an axial spacing of 143.4 Å, and an activated state which gives no layer lines but a meridional spacing of 144.8 Å.     

Primer-DNA formation during simian virus 40 DNA replication in vitro.

Studies of simian virus 40 (SV40) DNA replication in vitro have identified a small (approximately 30-nucleotide) RNA-DNA hybrid species termed primer-DNA. Initial experiments indicated that T antigen and the polymerase alpha-primase complex are required to form primer-DNA. Proliferating cell nuclear antigen, and presumably proliferating cell nuclear antigen-dependent polymerases, is not needed to form this species. Herein, we present an investigation of the stages at which primer-DNA functions during SV40 DNA replication in vitro. Hybridization studies indicate that primer-DNA is initially formed in the origin region and is subsequently synthesized in regions distal to the origin. At all time points, primer-DNA is synthesized from templates for lagging-strand DNA replication. These studies indicate that primer-DNA functions during both initiation and elongation stages of SV40 DNA synthesis. Results of additional experiments suggesting a precursor-product relationship between formation of primer-DNA and Okazaki fragments are presented.     

DNA replication of SV40-infected cells. VII. Formation of SV40 catenated and circular dimers

The binding of methyl isonitrile (CH₃Nandz.tbnd;C) to hemoglobin β chains has been studied by measuring the ¹H nuclear magnetic resonance transverse relaxation times for methyl isonitrile as a function of protein concentration, temperature and ¹⁴N decoupling. Binding of methyl isonitrile both at the heme iron and at a non-specific site (or sites) has an effect upon the measured nuclear spin relaxation times. The results yield a value of 57 ± 12 seconds^?1 (20 °C) for the “off” rate constant K_?1 for specific binding and an Arrhenius activation energy for k_?1 of 14 ± 3 kcal mol^?1.     

TIMELESS‐TIPIN and UBXN‐3 promote replisome disassembly during DNA replication termination in Caenorhabditis elegans

The eukaryotic replisome is rapidly disassembled during DNA replication termination. In metazoa, the cullin‐RING ubiquitin ligase CUL‐2^LRR‐1 drives ubiquitylation of the CMG helicase, leading to replisome disassembly by the p97/CDC‐48 “unfoldase”. Here, we combine in vitro reconstitution with in vivo studies in Caenorhabditis elegans embryos, to show that the replisome‐associated TIMELESS‐TIPIN complex is required for CUL‐2^LRR‐1 recruitment and efficient CMG helicase ubiquitylation. Aided by TIMELESS‐TIPIN, CUL‐2^LRR‐1 directs a suite of ubiquitylation enzymes to ubiquitylate the MCM‐7 subunit of CMG. Subsequently, the UBXN‐3 adaptor protein directly stimulates the disassembly of ubiquitylated CMG by CDC‐48_UFD‐1_NPL‐4. We show that UBXN‐3 is important in vivo for replisome disassembly in the absence of TIMELESS‐TIPIN. Correspondingly, co‐depletion of UBXN‐3 and TIMELESS causes profound synthetic lethality. Since the human orthologue of UBXN‐3, FAF1, is a candidate tumour suppressor, these findings suggest that manipulation of CMG disassembly might be applicable to future strategies for treating human cancer.     

Differential Tus-Ter binding and lock formation: implications for DNA replication termination in Escherichia coli

In E. coli, DNA replication termination occurs at Ter sites and is mediated by Tus. Two clusters of five Ter sites are located on each side of the terminus region and constrain replication forks in a polar manner. The polarity is due to the formation of the Tus-Ter-lock intermediate. Recently, it has been shown that DnaB helicase which unwinds DNA at the replication fork is preferentially stopped at the non-permissive face of a Tus-Ter complex without formation of the Tus-Ter-lock and that fork pausing efficiency is sequence dependent, raising two essential questions: Does the affinity of Tus for the different Ter sites correlate with fork pausing efficiency? Is formation of the Tus-Ter-lock the key factor in fork pausing? The combined use of surface plasmon resonance and GFP-Basta showed that Tus binds strongly to TerA-E and G, moderately to TerH-J and weakly to TerF. Out of these ten Ter sites only two, TerF and H, were not able to form significant Tus-Ter-locks. Finally, Tus's resistance to dissociation from Ter sites and the strength of the Tus-Ter-locks correlate with the differences in fork pausing efficiency observed for the different Ter sites by Duggin and Bell (2009).     

On the termination of the DNA replication cycle in Escherichia coli.

The initiation of the DNA replication cycle in Escherichia coli requires protein synthesis. Marunouchi &; Messer (1973) have hypothesized that an additional protein synthesis step is required for the replication of the terminal segment of the chromosome, and that replication of this segment is a prerequisite for subsequent cell division. We have not confirmed the existence of a unique terminal segment using a protocol designed to label the hypothesized segment with [³H]dThd². Our protocol avoids the increased incorporation of [³H]dThd into DNA caused by abrupt increases in temperature, a complication implicit in the technique of Marunouchi &; Messer (1973).Treatment with nalidixic acid (an inhibitor of semiconservative DNA synthesis) in sufficient concentration to prevent replication of the postulated terminal segment prevents cell division but also causes loss of viability. This makes it difficult to correlate the effect of nalidixic acid on cell division with DNA synthesis inhibition alone.     

The role of topoisomerase IV in partitioning bacterial replicons and the structure of catenated intermediates in DNA replication.

Mutants in bacterial topoisomerase (topo) IV are deficient in chromosomal partitioning. To investigate the basis of this phenotype, we examined plasmid DNA topology in conditionally lethal topo IV mutants. We found that dimeric catenated plasmids accumulated in vivo after topo IV inhibition. The catenanes were supercoiled, contained from 2 to > 32 nodes, and were the products of DNA synthesis. Electron microscopy and recombination tests proved that the catenanes have the unique structure predicted for replication intermediates. These data provide strong evidence for a model in which unlinking of the double helix can occur in two stages during DNA replication and for the critical role of topo IV in the second stage. The interlocks in the catenanes appear to be sequestered from DNA gyrase, perhaps by compartmentalization in an enzyme complex dedicated to partitioning.     

Detection of template strand switching during initiation and termination of DNA replication of porcine circovirus

Nucleotide substitution mutagenesis was conducted to investigate the importance of the inverted repeats (palindrome) at the origin of DNA replication (Ori) of porcine circovirus type 1 (PCV1). Viral genomes with engineered mutations on either arm or both arms of the palindrome were not impaired in protein synthesis and yielded infectious progeny viruses with restored or new palindromes. Thus, a flanking palindrome at the Ori was not essential for initiation of DNA replication, but one was generated inevitably at termination. Among the 26 viruses recovered, 16 showed evidence of template strand switching, from minus-strand genome DNA to palindromic strand DNA, during biosynthesis of the Ori. Here I propose a novel rolling-circle "melting-pot" model for PCV1 DNA replication. In this model, the replicator Rep protein complex binds, destabilizes, and nicks the Ori sequence to initiate leading-strand DNA synthesis. All four strands of the destabilized inverted repeats exist in a "melted" configuration, and the minus-strand viral genome and a palindromic strand are available as templates, simultaneously, during initiation or termination of DNA replication. Inherent in this model is a "gene correction" or "terminal repeat correction" mechanism that can restore mutilated inverted-repeat sequences to a palindrome at the Ori of circular DNAs or at the termini of circularized linear DNAs. Potentially, the melted state of the inverted repeats increases the rate of noncomplementary or illegitimate nucleotide incorporation into the palindrome. Thus, this melting-pot model provides insight into the mechanisms of DNA replication, gene correction, and illegitimate recombination at the Ori of PCV1, and it may be applicable to the replication of other circular DNA molecules.     

Dynamics of termination during in vitro replication of ultraviolet-irradiated DNA with DNA polymerase III holoenzyme of Escherichia coli

During in vitro replication of UV-irradiated single-stranded DNA with Escherichia coli DNA polymerase III holoenzyme termination frequently occurs at pyrimidine photodimers. The termination stage is dynamic and characterized by at least three different events: repeated dissociation-reinitiation cycles of the polymerase at the blocked termini; extensive hydrolysis of ATP to ADP and inorganic phosphate; turnover of dNTPs into dNMP. The reinitiation events are nonproductive and are not followed by further elongation. The turnover of dNTPs into dNMPs is likely to result from repeated cycles of insertion of dNMP residues opposite the blocking lesions followed by their excision by the 3'----5' exonucleolytic activity of the polymerase. Although all dNTPs are turned over, there is a preference for dATP, indicating that DNA polymerase III holoenzyme has a preference for inserting a dAMP residue opposite blocking pyrimidine photodimers. We suggest that the inability of the polymerase to bypass photodimers during termination is due to the formation of defective initiation-like complexes with reduced stability at the blocked termini.     

PriA and phage T4 gp59: factors that promote DNA replication on forked DNA substrates microreview

The initiation of DNA synthesis on forked DNA templates is a vital process in the replication and maintenance of cellular chromosomes. Two proteins that promote replisome assembly on DNA forks have so far been identified. In phage T4 development the gene 59 protein (gp59) assembles replisomes at D-loops, the sites of homologous strand exchange. Bacterial PriA protein plays an analogous function, most probably restarting replication after replication fork arrest with the aid of homologous recombination proteins, and PriA is also required for phage Mu replication by transposition. Gp59 and PriA exhibit similar DNA fork binding activities, but PriA also has a 3' to 5' helicase activity that can promote duplex opening for replisome assembly. The helicase activity allows PriA's repertoire of templates to be more diverse than that of gp59. It may give PriA the versatility to restart DNA replication without recombination on arrested replication forks that lack appropriate duplex openings.     

Novel role for Cdc14 sequestration: Cdc14 dephosphorylates factors that promote DNA replication

The phosphatase Cdc14 is required for mitotic exit in budding yeast. Cdc14 promotes Cdk1 inactivation by targeting proteins that, when dephosphorylated, trigger degradation of mitotic cyclins and accumulation of the Cdk1 inhibitor, Sic1. Cdc14 is sequestered in the nucleolus during most of the cell cycle but is released into the nucleus and cytoplasm during anaphase. When Cdc14 is not properly sequestered in the nucleolus, expression of the S-phase cyclin Clb5 is required for viability, suggesting that the antagonizing activity of Clb5-dependent Cdk1 specifically is necessary when Cdc14 is delocalized. We show that delocalization of Cdc14 combined with loss of Clb5 causes defects in DNA replication. When Cdc14 is not sequestered, it efficiently dephosphorylates a subset of Cdk1 substrates including the replication factors, Sld2 and Dpb2. Mutations causing Cdc14 mislocalization interact genetically with mutations affecting the function of DNA polymerase epsilon and the S-phase checkpoint protein Mec1. Our findings suggest that Cdc14 is retained in the nucleolus to support a favorable kinase/phosphatase balance while cells are replicating their DNA, in addition to the established role of Cdc14 sequestration in coordinating nuclear segregation with mitotic exit.     

Two complementary strand-specific termination sites for adenovirus DNA replication

Adenovirus type 2 DNA, specifically labeled at the termini for DNA replication, was prepared by isolation of viral DNA molecules which were completed during short pulses with ³H-thymidine. The distribution of radioactivity in the two complementary strands at the termini for DNA replication was determined by liquid phase hybridization and gel electrophoresis. At the right-hand terminus, nearly all radioactivity was found in the viral h strand, whereas at the left-hand terminus, most radioactivity was confined to the viral I strand. The results suggest that both molecular ends serve as origins and termini for replication of adenovirus type 2 DNA.     

In vitro termination of adenovirus DNA synthesis by a soluble replication complex.

The double-stranded DNA from a soluble DNA replication complex that was labeled with deoxyribonucleoside triphosphates and completed in vitro was digested with EcoRI, Sma I, and Hpa I restriction endonucleases. All regions of the adenovirus type 2 genome were labeled in vitro, but restriction fragments derived from the ends of the DNA molecules were relatively more highly labeled than those derived from internal regions. The in vitro endogenous DNA polymerase reaction also exhibited strand-specific labeling near the molecular ends, in that restriciton fragments from the left end were labeled predominantly in the r strand and fragments from the right end were labeled predominantly in the l strand.     

Synthesis of single-stranded plasmid pT181 DNA in vitro. Initiation and termination of DNA replication

The origin of replication of plasmid pT181 is nicked by the plasmid-encoded RepC protein. The free 3'-hydroxyl end at the nick is presumably used as primer for leading strand DNA synthesis. In vitro replication of pT181 was found to generate single-stranded DNA in addition to the supercoiled, double-stranded DNA. The single-stranded DNA was circular and corresponded to the pT181 leading strand. Recombinant plasmids were constructed that contain two pT181 origins of replication in either direct or inverted orientation. In vitro replication of the plasmid carrying two origins in direct orientation was shown to generate circular, single-stranded DNA that corresponded to initiation of replication at one origin sequence and termination at the other origin. These results demonstrate that the origin of pT181 leading strand DNA replication also serves as the site for termination of replication. Interestingly, the presence of two origins in inverted orientation resulted in initiation of replication at one origin and stalling of the replisome at the other origin. These results suggest that RepC can reinitiate replication at the second origin by nicking partially replicated, relaxed DNA. These data are consistent with the replication of pT181 by a rolling circle mechanism and indicate that single-stranded DNA is an intermediate in pT181 replication.     

Appearance of a late stage during mammalian DNA replication when cells resume formation of 10 kb DNA replication intermediates

After the joining of human large DNA replication intermediates and before the appearance of mature chromatin DNA, there exists a distinct stage--'the post-elongation stage'. This stage reappears during recovery of DNA synthesis simultaneously with the reappearance of a large DNA replication intermediate, 10 kb DNA.