Resumption Of DNA Replication Research Articles

Src family kinases maintain the balance between replication stress and the replication checkpoint.

Progression of DNA replication is tightly controlled by replication checkpoints to ensure the accurate and rapid duplication of genetic information. Upon replication stress, the replication checkpoint slows global DNA replication by inhibiting the late-firing origins and by slowing replication fork progression. Activation of the replication checkpoint has been studied in depth; however, little is known about the termination of the replication checkpoint. Here, we show that Src family kinases promote the recovery from replication checkpoints. shRNA knockdown of a Src family kinase, Lyn, and acute chemical inhibition of Src kinases prevented inactivation of Chk1 after removal of replication stress. Consistently, Src inhibition slowed resumption of DNA replication, after the removal of replication blocks. The effect of Src inhibition was not observed in the presence of an ATM/ATR inhibitor caffeine. These data indicate that Src kinases promote the resumption of DNA replication by suppressing ATR-dependent replication checkpoints. Surprisingly, the resumption of replication was delayed by caffeine. In addition, Src inhibition delayed recovery from replication fork collapse. We propose that Src kinases maintain the balance between replication stress and the activity of the replication checkpoint.

Ino80 Chromatin Remodeling Complex Promotes Recovery of Stalled Replication Forks

Chromatin remodeling complexes facilitate the access of enzymes that mediate transcription, replication or repair of DNA by modulating nucleosome position and/or composition. Ino80 is the DNA-dependent Snf2-like ATPase subunit of a complex whose nucleosome remodeling activity requires actin-related proteins, Arp4, Arp5 and Arp8, as well as two RuvB-like DNA helicase subunits. Budding yeast mutants deficient for Ino80 function are not only hypersensitive to reagents that induce DNA double-strand breaks, but also to those that impair replication fork progression. To understand why ino80 mutants are sensitive to agents that perturb DNA replication, we used chromatin immunoprecipitation to map the binding sites of the INO80 chromatin-remodeling complex on four budding yeast chromosomes. We found that Ino80 and Arp5 binding sites coincide with origins of DNA replication and tRNA genes. In addition, Ino80 was bound at 67% of the promoters of genes that are sensitive to ino80 mutation. When replication forks were arrested near origins in the presence of hydroxyurea (HU), the amount of INO80 complex at stalled forks and at unfired origins increased selectively. Importantly, the resumption of DNA replication after release from a HU block was impaired in ino80 mutants. These cells accumulated double-strand breaks as they attempted to restart replication. Consistently, ino80-deficient cells, although proficient for checkpoint activation, delay recovery from the checkpoint response. The INO80 chromatin remodeling complex is enriched at stalled replication forks, where it promotes the resumption of replication upon recovery from fork arrest.

Bloom's Syndrome Helicase and Mus81 are Required to Induce Transient Double-strand DNA Breaks in Response to DNA Replication Stress

Perturbed DNA replication either activates a cell cycle checkpoint, which halts DNA replication, or decreases the rate of DNA synthesis without activating a checkpoint. Here we report that at low doses, replication inhibitors did not activate a cell cycle checkpoint, but they did activate a process that required functional Bloom's syndrome-associated (BLM) helicase, Mus81 nuclease and ataxia telangiectasia mutated and Rad3-related (ATR) kinase to induce transient double-stranded DNA breaks. The induction of transient DNA breaks was accompanied by dissociation of proliferating cell nuclear antigen (PCNA) and DNA polymerase α from replication forks. In cells with functional BLM, Mus81 and ATR, the transient breaks were promptly repaired and DNA continued to replicate at a slow pace in the presence of replication inhibitors. In cells that lacked BLM, Mus81, or ATR, transient breaks did not form, DNA replication did not resume, and exposure to low doses of replication inhibitors was toxic. These observations suggest that BLM helicase, ATR kinase, and Mus81 nuclease are required to convert perturbed replication forks to DNA breaks when cells encounter conditions that decelerate DNA replication, thereby leading to the rapid repair of those breaks and resumption of DNA replication without incurring DNA damage and without activating a cell cycle checkpoint.

Sap1 Promotes the Association of the Replication Fork Protection Complex With Chromatin and Is Involved in the Replication Checkpoint in Schizosaccharomyces pombe

Sap1 is involved in replication fork pausing at rDNA repeats and functions during mating-type switching in Schizosaccharomyces pombe. These two roles are dependent on the ability of Sap1 to bind specific DNA sequences at the rDNA and mating-type loci, respectively. In S. pombe, Swi1 and Swi3 form the replication fork protection complex (FPC) and play important roles in the activation of the replication checkpoint and the stabilization of stalled replication forks. Here we describe the roles of Sap1 in the replication checkpoint. We show that Sap1 is involved in the activation of the replication checkpoint kinase Cds1 and that sap1 mutant cells accumulate spontaneous DNA damage during the S- and G2-phases, which is indicative of fork damage. We also show that sap1 mutants have a defect in the resumption of DNA replication after fork arrest. Sap1 is localized at the replication origin ori2004 and this localization is required for the association of the FPC with chromatin. We propose that Sap1 is required to recruit the FPC to chromatin, thereby contributing to the activation of the replication checkpoint and the stabilization of replication forks.

A novel intermediate in initiation complex assembly for fission yeast DNA replication.

Assembly of initiation factors on individual replication origins at onset of S phase is crucial for regulation of replication timing and repression of initiation by S-phase checkpoint control. We dissected the process of preinitiation complex formation using a point mutation in fission yeast nda4-108/mcm5 that shows tight genetic interactions with sna41(+)/cdc45(+). The mutation does not affect loading of MCM complex onto origins, but impairs Cdc45-loading, presumably because of a defect in interaction of MCM with Cdc45. In the mcm5 mutant, however, Sld3, which is required for Cdc45-loading, proficiently associates with origins. Origin-association of Sld3 without Cdc45 is also observed in the sna41/cdc45 mutant. These results suggest that Sld3-loading is independent of Cdc45-loading, which is different from those observed in budding yeast. Interestingly, returning the arrested mcm5 cells to the permissive temperature results in immediate loading of Cdc45 to the origin and resumption of DNA replication. These results suggest that the complex containing MCM and Sld3 is an intermediate for initiation of DNA replication in fission yeast.

Replication slippage involves DNA polymerase pausing and dissociation.

Genome rearrangements can take place by a process known as replication slippage or copy-choice recombination. The slippage occurs between repeated sequences in both prokaryotes and eukaryotes, and is invoked to explain microsatellite instability, which is related to several human diseases. We analysed the molecular mechanism of slippage between short direct repeats, using in vitro replication of a single-stranded DNA template that mimics the lagging strand synthesis. We show that slippage involves DNA polymerase pausing, which must take place within the direct repeat, and that the pausing polymerase dissociates from the DNA. We also present evidence that, upon polymerase dissociation, only the terminal portion of the newly synthesized strand separates from the template and anneals to another direct repeat. Resumption of DNA replication then completes the slippage process.

At the birth of molecular radiation biology.

Rational thinking builds on feelings, too. This article starts with a tribute to Richard Setlow, an eminent scientist; it retraces as well some studies in molecular genetics that helped to understand basic questions of radiation biology. In the mid-1950s, the induction of a dormant virus (prophage) by irradiation of its host was an intriguing phenomenon. Soon, it was found that prophage induction results from the inactivation of the prophage repressor. Similarly, a score of induced cellular SOS functions were found to be induced when the LexA repressor is inactivated. Repressor inactivation involves the formation of a newly formed distinctive structure: a RecA-polymer wrapped around single-stranded DNA left by the arrest of replication at damaged sites. By touching this RecA nucleofilament, the LexA repressor is inactivated, triggering the sequential expression of SOS functions. The RecA nucleofilament acts as a chaperone, allowing recombinational repair to occur after nucleotide excision repair is over. The UmuD'C complex, synthesized slowly and parsimoniously, peaks at the end of recombinational repair, ready to be positioned at the tip of a RecA nucleofilament, placing the UmuD'C complex right at a lesion. At this location, UmuD'C prevents recombinational repair, and now acts as an error-prone paucimerase that fills the discontinuity opposite the damaged DNA. Finally, the elimination of lesions from the path of DNA polymerase, allows the resumption of DNA replication, and the SOS repair cycle switches to a normal cell cycle.

Stationary phase induction of dnaN and recF, two genes of Escherichia coli involved in DNA replication and repair.

The beta subunit of DNA polymerase III holoenzyme, the Escherichia coli chromosomal replicase, is a sliding DNA clamp responsible for tethering the polymerase to DNA and endowing it with high processivity. The gene encoding beta, dnaN, maps between dnaA and recF, which are involved in initiation of DNA replication at oriC and resumption of DNA replication at disrupted replication forks, respectively. In exponentially growing cells, dnaN and recF are expressed predominantly from the dnaA promoters. However, we have found that stationary phase induction of the dnaN promoters drastically changes the expression pattern of the dnaA operon genes. As a striking consequence, synthesis of the beta subunit and RecF protein increases when cell metabolism is slowing down. Such an induction is dependent on the stationary phase sigma factor, RpoS, although the accumulation of this factor alone is not sufficient to activate the dnaN promoters. These promoters are located in DNA regions without static bending, and the -35 hexamer element is essential for their RpoS-dependent induction. Our results suggest that stationary phase-dependent mechanisms have evolved in order to coordinate expression of dnaN and recF independently of the dnaA regulatory region. These mechanisms might be part of a developmental programme aimed at maintaining DNA integrity under stress conditions.

A new mutation in Escherichia coli K12, isfA, which is responsible for inhibition of SOS functions.

A new mutation in Escherichia coli K12, isfA, is described, which causes inhibition of SOS functions. The mutation, discovered in a delta polA+ mutant, is responsible for inhibition of several phenomena related to the SOS response in polA+ strains: UV- and methyl methanesulfonate-induced mutagenesis, resumption of DNA replication in UV-irradiated cells, cell filamentation, prophage induction and increase in UV sensitivity. The isfA mutation also significantly reduces UV-induced expression of beta-galactosidase from recA::lacZ and umuC'::lacZ fusions. The results suggest that the isfA gene product may affect RecA* coprotease activity and may be involved in the regulation of the termination of the SOS response after completion of DNA repair. The isfA mutation was localized at 85 min on the E. coli chromosome, and preliminary experiments suggest that it may be dominant to the wild-type allele.

Replication of damaged DNA and the molecular mechanism of ultraviolet light mutagenesis.

On UV irradiation of Escherichia coli cells, DNA replication is transiently arrested to allow removal of DNA damage by DNA repair mechanisms. This is followed by a resumption of DNA replication, a major recovery function whose mechanism is poorly understood. During the post-UV irradiation period the SOS stress response is induced, giving rise to a multiplicity of phenomena, including UV mutagenesis. The prevailing model is that UV mutagenesis occurs by the filling in of single-stranded DNA gaps present opposite UV lesions in the irradiated chromosome. These gaps can be formed by the activity of DNA replication or repair on the damaged DNA. The gap filling involves polymerization through UV lesions (also termed bypass synthesis or error-prone repair) by DNA polymerase III. The primary source of mutations is the incorporation of incorrect nucleotides opposite lesions. UV mutagenesis is a genetically regulated process, and it requires the SOS-inducible proteins RecA, UmuD, and UmuC. It may represent a minor repair pathway or a genetic program to accelerate evolution of cells under environmental stress conditions.

Relationship of histone acetylation to DNA topology and transcription.

An autonomously replicating plasmid constructed from bovine papiloma virus (BPV) and pBR322 was stably maintained as a nuclear episome in a mouse cell culture. Addition to a cell culture of sodium butyrate (5 mM) induced an increase in plasmid DNA supercoiling of 3-5 turns, an increase in acetylation of cellular histones, and a decrease in plasmid transcription by 2- to 4-fold. After withdrawal of butyrate, DNA supercoiling began to fluctuate in a wave-like manner with an amplitude of up to 3 turns and a period of 3-4 h. These waves gradually faded by 24 h. The transcription of the plasmid and acetylation of cellular histones also oscillated with the same period. The wave-like alterations were not correlated with the cell cycle, for there was no resumption of DNA replication after butyrate withdrawal for at least 24 h. In vitro chemical acetylation of histones with acetyl adenylate also led to an increase in the superhelical density of plasmid DNA. The parallel changes in transcription, histone acetylation, and DNA supercoiling in vivo may indicate a functional innerconnection. Also, the observed in vivo variation in the level of DNA supercoiling directly indicates the possibility of its natural regulation in eukaryotic cells.

Mitochondrial gene expression during Xenopus laevis development: a molecular study.

Mitochondrial gene expression has been analysed during embryonic development of Xenopus laevis; the relative amounts of 12S and 16S ribosomal RNAs and of most mitochondrial messenger RNAs were determined by slot-blot and Northern-blot hybridization experiments with specific mitochondrial DNA probes. The rRNA content per embryo remained constant during early development, confirming earlier results of Chase and Dawid (1972, Dev. Biol., 27, 504-518.); on the contrary, all mRNAs decreased abruptly after fertilization within a few hours (by a factor of 5-10), remained at a very low level up to the late neurula stages and increased again during organogenesis. Since the mitochondrial DNA content does not vary during this period molecular analyses as well as biological observations suggest that the mitochondrial genome is completely inactivated at the beginning of embryonic development. The amounts of rRNAs and mRNAs evolve therefore as a function of time only according to their half-lives. Mitochondrial RNA accumulation resumes subsequently with a high rate when the general transcription in the embryo is starting again, and this occurs before the resumption of DNA replication in the organelle. It appears that the Xenopus embryonic development represents a quite clear example of regulation of the mitochondrial expression at the level of transcription.

Different radiosensitivities of Nicotiana plumbaginifolia leaves and regenerating protoplasts

The isolation of leaf protoplasts provides a system in which a homogeneous population of cells undergoes specific stimuli and goes through characteristic phases of dedifferentiation in a limited time. The whole process was calibrated with respect to the nuclear phases using Nicotiana plumbaginifolia and it was found to be remarkably synchronous. Taking advantage of this favourable situation, certain defined stages before and after protoplast release were checked for their radiosensitivity using γ-rays. Irradiated protoplasts from untreated leaves were definitely more resistant than leaves irradiated at identical dose levels. Their radiosensitivity increased again during the first day of culture up to a maximum immediately before the resumption of DNA replication. The sensitivity of the late S nuclear phase then returned to the initial level. The influence of cell progression on radiosensivity during culture was clearly established. Although no definite explanation can be given for the higher resistance of the protoplast compared to the initial leaf cell, some speculations involving cellular dehydration, interactions with components of the medium, or chromatin reorganisation are suggested.

Properties of a temperature-sensitive mutant of Staphylococcus aureus defective in DNA replication and cell division and replication of plasmids in the mutant.

The properties of a temperature-sensitive mutant (ts39) of Staphylococcus aureus NCTC 8235 are described. After transfer to the restrictive temperature (42 degrees C), absorbance increased 10-to 20-fold but DNA content did not increase beyond 150 to 200% and cell division continued at a greatly reduced rate. On transfer back to the permissive temperature, both cell division and DNA synthesis resumed if the transfer occurred after less than 120 min at 42 degrees C. Resumption of DNA replication was blocked by chloramphenicol (100 microgram ml-1). The results are discussed with reference to possible defects in DNA replication. Replication of the plasmids pI258 and pT10501 and the chromosome were affected to a similar extent in ts39. Growth at 42 degrees C resulted in the appearance of an increased amount of pI258 DNA in a form that sedimented slowly in a sucrose gradient.

UV-inducible repair II: Its role in various defective mutants of Escherichia coli K-12

Involvement of UV-inducible protein(s) in repair of various E. coli K-12 cell strains has been investigated using a procedure of double UV irradiation and postincubation with chloramphenicol. From the course of dose survival curves the following conclusions concerning significance of a UV-inducible protein have been drawn: 1. It is a very important for wild type cells; in these cells its early occurrence is necessary to prevent killing. 2. It is involved in repair of excision-deficient cells; however, its action early after UV is less urgent. 3. It is not involved at all in repair of lex mutant cells; 4. It exhibits some effect on survival of recA as well as recB mutant cells. We conclude that the protein is involved in excision repair as well as in resumption of DNA replication.

Initiation of DNA replication in Escherichia coli. III. Genetic analysis of the dna mutant exhibiting rifampicin-sensitive resumption of replication.

Temperature-sensitive mutants defective in the initiation of DNA replication are exposed to a non-permissive temperature to complete already initiated replication, and are transferred back to a permissive temperature. DNA synthesis can resume in the presence of rifampicin or rifampicin plus chloramphenicol in strain PC2 (dnaC2), but not in strain N167 (dna-167). In the presence of chloramphenicol alone, however, DNA synthesis can resume in both strains (Hirage and Saito, 1973, 1974). The double mutants carrying the dna-167 and dnaC2 mutations show the rifampicin-sensitive resumption of DNA replication as the dna-167 mutant. The rifampicin-sensitive character (designated as Rrr-) is closely linked with the temperature sensitivity of the dna-167 mutant in P1 transduction. The gene order is dna-167-tna-phoS-uncA-ilv. The Rrr- character does not correlate with the inactivation of the altered product of the mutated dna-167 gene at various temperatures in the double mutant carrying dna-167 and dnaC2. Although dnaC2 strains show the Rrr+ phenotype, the dnaC2 strains received the ilv-dnaA region of the Ts+ revertants obtained from a dna-167 strain show the Rrr- phenotype. These results suggest that the dna-167 mutant has two mutations which are closely linked to each other, controlling the Rrr- phenotype and the temperature sensitivity, respectively.

The differential response of resistant and sensitive strains of Escherichia coli to the cytotoxic effects of 5-aziridino-2,4-dinitrobenzamide (CB 1954)

The antineoplastic agent 5-aziridino-2,4-dinitrobenzamide (CB 1954) has an exceptionally high therapeutic index against the Walker rat carcinoma 256 but no effect on other experimental tumours. Escherichia coli B/r is much more resistant to cytotoxic alkylating agents than E. coli B s−1, and these strains were therefore used to determine the basis of the differential cytotoxicity of CB 1954. There is a semilogarithmic dose-survival relationship in both strains, B/r being some 26 times more resitant than B s−1. Studies using 3H-labelled CB 1954 showed that although the drug binds to the same extent to DNA in both strains, at the D 37 for B/r there are 560 alkylations per average genome compared with only 16 alkylations per genome in B s−1. These figures agree closely with those obtained with the bifunctional sulphur mustard but are 2–3 orders of magnitude lower than those found with monofunctional agents. The extent of reaction with RNA and protein in both strains is approximately the same, RNA binding more than DNA, and protein less, on a molar basis. Consideration of the relative target sizes of functional RNA and protein molecules in relation to binding at the D 37 excludes them as possible targets for inactivation. DNA synthesis and cell division in B/r resume at the control rate after a short, dose-depending lag following drug treatment, whereas in B s−1 DNA synthesis and cell division are permanently blocked. E. coli B/r has a greater capacity to excise CB 1954 residues from its DNA than B s−1 following treatment of both strains at the D 37 for B/r. The evidence suggests that, in these bacteria, CB 1954 exerts its cytotoxic effect by alkylation of DNA and subsequent hindrance of DNA replication and cell division. The resistant strain probably owes this resistance to its greater ability to remove that proportion of the alkylating residues sufficient to allow resumption of DNA replication and cell division.