Role In Chromosomal Replication Research Articles

The Toxoplasma Centrocone Houses Cell Cycle Regulatory Factors.

ABSTRACTOur knowledge of cell cycle regulatory mechanisms in apicomplexan parasites is very limited. In this study, we describe a novel Toxoplasma gondii factor that has a vital role in chromosome replication and the regulation of cytoplasmic and nuclear mitotic structures, and we named this factor ECR1 for essential for chromosome replication 1. ECR1 was discovered by complementation of a temperature-sensitive (ts) mutant that suffers lethal, uncontrolled chromosome replication at 40°C similar to a ts mutant carrying a defect in topoisomerase. ECR1 is a 52-kDa protein containing divergent RING and TRAF-Sina-like zinc binding domains that are dynamically expressed in the tachyzoite cell cycle. ECR1 first appears in the unique spindle compartment of the Apicomplexa (centrocone) of the nuclear envelope in early S phase and then in the nucleus in late S phase where it reaches maximum expression. Following nuclear division, but before daughter parasites separate from the mother parasite, ECR1 is downregulated and is absent in new daughter parasites. The proteomics of ECR1 identified interactions with the ubiquitin-mediated protein degradation machinery and the minichromosome maintenance complex, and the loss of ECR1 led to increased stability of a key member of this complex, MCM2. ECR1 also forms a stable complex with the cyclin-dependent kinase (CDK)-related kinase, T. gondii Crk5 (TgCrk5), which displays a similar cell cycle expression and localization during tachyzoite replication. Importantly, the localization of ECR1/TgCrk5 in the centrocone indicates that this Apicomplexa-specific spindle compartment houses important regulatory factors that control the parasite cell cycle.

Structure of a DNA Polymerase α-Primase Domain That Docks on the SV40 Helicase and Activates the Viral Primosome

DNA polymerase alpha-primase (pol-prim) plays a central role in DNA replication in higher eukaryotes, initiating synthesis on both leading and lagging strand single-stranded DNA templates. Pol-prim consists of a primase heterodimer that synthesizes RNA primers, a DNA polymerase that extends them, and a fourth subunit, p68 (also termed B-subunit), that is thought to regulate the complex. Although significant knowledge about single-subunit primases of prokaryotes has accumulated, the functions and regulation of pol-prim remain poorly understood. In the SV40 replication model, the p68 subunit is required for primosome activity and binds directly to the hexameric viral helicase T antigen, suggesting a functional link between T antigen-p68 interaction and primosome activity. To explore this link, we first mapped the interacting regions of the two proteins and discovered a previously unrecognized N-terminal globular domain of p68 (p68N) that physically interacts with the T antigen helicase domain. NMR spectroscopy was used to determine the solution structure of p68N and map its interface with the T antigen helicase domain. Structure-guided mutagenesis of p68 residues in the interface diminished T antigen-p68 interaction, confirming the interaction site. SV40 primosome activity of corresponding pol-prim mutants decreased in proportion to the reduction in p68N-T antigen affinity, confirming that p68-T antigen interaction is vital for primosome function. A model is presented for how this interaction regulates SV40 primosome activity, and the implications of our findings are discussed in regard to the molecular mechanisms of eukaryotic DNA replication initiation.

The yeast Pif1p DNA helicase preferentially unwinds RNA DNA substrates

Pif1p is the prototypical member of the PIF1 family of DNA helicases, a subfamily of SFI helicases conserved from yeast to humans. Baker's yeast Pif1p is involved in the maintenance of mitochondrial, ribosomal and telomeric DNA and may also have a general role in chromosomal replication by affecting Okazaki fragment maturation. Here we investigate the substrate preferences for Pif1p. The enzyme was preferentially active on RNA–DNA hybrids, as seen by faster unwinding rates on RNA–DNA hybrids compared to DNA–DNA hybrids. When using forked substrates, which have been shown previously to stimulate the enzyme, Pif1p demonstrated a preference for RNA–DNA hybrids. This preferential unwinding could not be correlated to preferential binding of Pif1p to the substrates that were the most readily unwound. Although the addition of the single-strand DNA-binding protein replication protein A (RPA) stimulated the helicase reaction on all substrates, it did not diminish the preference of Pif1p for RNA–DNA substrates. Thus, forked RNA–DNA substrates are the favored substrates for Pif1p in vitro. We discuss these findings in terms of the known biological roles of the enzyme.

DNA polymerase II as a fidelity factor in chromosomal DNA synthesis in Escherichia coli

Escherichia coli DNA polymerase III holoenzyme (HE) is the main replicase responsible for replication of the bacterial chromosome. E. coli contains four additional polymerases, and it is a relevant question whether these might also contribute to chromosomal replication and its fidelity. Here, we have investigated the role of DNA polymerase II (Pol II) (polB gene product). Mismatch repair-defective strains containing the polBex1 allele--encoding a polymerase-proficient but exonucleolytically defective Pol II--displayed a mutator activity for four different chromosomal lac mutational markers. The mutator effect was dependent on the chromosomal orientation of the lacZ gene. The results indicate that Pol II plays a role in chromosomal replication and that its role is not equal in leading- versus lagging-strand replication. In particular, the role of Pol II appeared larger in the lagging strand. When combined with dnaQ or dnaE mutator alleles, polBex1 showed strong, near multiplicative effects. The results fit a model in which Pol II acts as proofreader for HE-produced misinsertion errors. A second role of Pol II is to protect mismatched 3' termini against the mutagenic action of polymerase IV (dinB product). Overall, Pol II may be considered a main player in the polymerase trafficking at the replication fork.

Restart of DNA replication in Gram-positive bacteria: functional characterisation of the Bacillus subtilis PriA initiator.

The PriA protein was identified in Escherichia coli as a factor involved in the replication of extrachromosomal elements such as bacteriophage phiX174 and plasmid pBR322. Recent data show that PriA plays an important role in chromosomal replication, by promoting reassembly of the replication machinery during reinitiation of inactivated forks. A gene encoding a product 32% identical to the E.coli PriA protein has been identified in Bacillus subtilis. To characterise this protein, designated PriA(Bs), we constructed priA(Bs) mutants. These mutants are poorly viable, filamentous and sensitive to rich medium and UV irradiation. Replication of pAMbeta1-type plasmids, which is initiated through the formation of a D-loop structure, and the activity of the primosome assembly site ssiA of plasmid pAMbeta1 are strongly affected in the mutants. The purified PriA(Bs) protein binds preferentially to the active strand of ssiA, even in the presence of B.subtilis SSB protein (SSB(Bs)). PriA(Bs) also binds stably and specifically to an artificial D-loop structure in vitro. These data show that PriA(Bs) recognises two specific substrates, ssiA and D-loops, and suggest that it triggers primosome assembly on them. PriA(Bs) also displays a single-stranded DNA-dependent ATPase activity, which is reduced in the presence of SSB(Bs), unless the ssiA sequence is present on the ssDNA substrate. Finally, PriA(Bs) is shown to be an active helicase. Altogether, these results demonstrate a clear functional identity between PriA(Ec) and PriA(Bs). However, PriA(Bs) does not complement an E.coli priA null mutant strain. This host specificity may be due to the divergence between the proteins composing the E.coli and B.subtilis PriA-dependent primosomes.

Interaction of the beta sliding clamp with MutS, ligase, and DNA polymerase I.

The beta and proliferating cell nuclear antigen (PCNA) sliding clamps were first identified as components of their respective replicases, and thus were assigned a role in chromosome replication. Further studies have shown that the eukaryotic clamp, PCNA, interacts with several other proteins that are involved in excision repair, mismatch repair, cellular regulation, and DNA processing, indicating a much wider role than replication alone. Indeed, the Escherichia coli beta clamp is known to function with DNA polymerases II and V, indicating that beta also interacts with more than just the chromosomal replicase, DNA polymerase III. This report demonstrates three previously undetected protein-protein interactions with the beta clamp. Thus, beta interacts with MutS, DNA ligase, and DNA polymerase I. Given the diverse use of these proteins in repair and other DNA transactions, this expanded list of beta interactive proteins suggests that the prokaryotic beta ring participates in a wide variety of reactions beyond its role in chromosomal replication.

CspD, a novel DNA replication inhibitor induced during the stationary phase in Escherichia coli.

CspD is a stationary phase-induced, stress response protein in the CspA family of Escherichia coli. Here, we demonstrate that overproduction of CspD is lethal, with the cells displaying a morphology typical of cells with impaired DNA replication. CspD consists mainly of beta-strands, and the purified protein exists exclusively as a dimer and binds to single-stranded (ss)DNA and RNA in a dose-dependent manner without apparent sequence specificity. CsdD effectively inhibits both the initiation and the elongation steps of minichromosome replication in vitro. Electron microscopic studies revealed that CspD tightly packs ssDNA, resulting in structures distinctly different from those of SSB-coated DNA. We propose that CspD dimers, with two independent beta-sheets interacting with ssDNA, function as a novel inhibitor of DNA replication and play a regulatory role in chromosomal replication in nutrient-depleted cells.

Altered spectra of hypermutation in DNA repair-deficient mice.

Affinity maturation of the humoral immune response is based on the ability of immunoglobulin variable genes to undergo a process of rapid and extensive somatic mutation followed by antigenic selection for antibodies with higher affinity. While the behaviour of this somatic hypermutation phenomenon has been well characterized over the last 20 years, the molecular mechanism responsible for inserting mutations has remained shrouded. To better understand this mechanism, we studied the interplay between hypermutation and other DNA associated activities such as DNA repair. There was no effect on the frequency and pattern of hypermutation in mice deficient for nucleotide excision repair, base excision repair and ataxia-telangiectasia mutated gene repair of double strand breaks. However, variable genes from mice lacking some components of mismatch repair had an increased frequency of tandem mutations and had more mutations of G and C nucleotides. These results suggest that the DNA polymerase(s) involved in the hypermutation pathway produces a unique spectra of mutations, which is then altered by mismatch repair and antigenic selection. We, also describe the differential pattern of expression of some nuclear DNA polymerases in hypermutating versus non-hypermutating B lymphocytes. The rapidly dividing germinal centre B cells expressed DNA polymerases alpha, beta, delta, epsilon and zeta, whereas the resting non-germinal centre cells did not express polymerases alpha or epsilon at detectable levels, although they did express polymerases beta, delta and zeta. The lack of expression of polymerase epsilon in the non-germinal centre cells suggests that this enzyme has a critical role in chromosomal replication but does not participate in DNA repair in these cells.

Differential expression of DNA polymerase ϵ in resting and activated B lymphocytes is consistent with an in vivo role in replication and not repair

DNA polymerases may be differentially expressed by cells during periods of quiescence and proliferation. Murine B cells are an ideal population to study because their division time varies widely in vivo, and different subsets can be easily isolated. Consequently, we analyzed RNA from resting cells (B220 +peanut agglutinin −) and activated germinal center cells (B220 +peanut agglutinin +) from spleens by reverse transcriptase-PCR using primers for five nuclear polymerases and their associated subunits. Gel analyses of the amplified products showed that the rapidly-dividing germinal center B cells expressed DNA polymerases α, β, δ, ϵ, and ζ. The resting B cells did not express polymerases α or ϵ at detectable levels, although they did express polymerases β, δ, and ζ. Thus, polymerase ϵ, as well as α, appears to have a primary role in chromosomal replication of murine B lymphocytes. Further, the lack of expression of polymerase ϵ in resting cells indicates that this enzyme is not used in any DNA repair pathways by these cells. The expression of polymerase ζ by resting cells suggests that it has another role in DNA repair, perhaps recombination, in addition to its function of bypassing damage during chromosomal replication.

Mammalian DNA topoisomerase IIIalpha is essential in early embryogenesis.

Targeted disruption of the mouse TOP3alpha gene encoding DNA topoisomerase IIIalpha was carried out to study the physiological functions of the mammalian type IA DNA topoisomerase. Whereas heterozygous top3alpha+/- mutant mice were found to resemble phenotypically their TOP3alpha+/+ litermates, no viable top3alpha-/- homozygotes were found among over 100 progeny of top3alpha+/- intercrosses. Examination of embryos dissected from decidual swellings and in vitro culturing of blastocysts from top3alpha+/- intercrosses showed that implantation of top3alpha-/- embryos and the induction of decidualization could occur, but viability of these embryos was severely compromised at an early stage of development. The requirement of mouse DNA topoisomerase IIIalpha during early embryogenesis is discussed in terms of its plausible role in chromosome replication and its interaction with the RecQ/SGS1 family of DNA helicases, whose members include the Bloom's syndrome and the Werner's syndrome gene products.

Schizosaccharomyces pombe cdc20+ encodes DNA polymerase epsilon and is required for chromosomal replication but not for the S phase checkpoint.

In fission yeast both DNA polymerase alpha (pol alpha) and delta (pol delta) are required for DNA chromosomal replication. Here we demonstrate that Schizosaccharomyces pombe cdc20+ encodes the catalytic subunit of DNA polymerase epsilon (pol epsilon) and that this enzyme is also required for DNA replication. Following a shift to the restrictive temperature, cdc20 temperature-sensitive mutant cells block at the onset of DNA replication, suggesting that cdc20+ is required early in S phase very near to the initiation step. In the budding yeast Saccharomyces cerevisiae, it has been reported that in addition to its proposed role in chromosomal replication, DNA pol epsilon (encoded by POL2) also functions directly as an S phase checkpoint sensor [Navas, T. A., Zhou, Z. & Elledge, S. J. (1995) Cell 80, 29-39]. We have investigated whether cdc20+ is required for the checkpoint control operating in fission yeast, and our data indicate that pol epsilon does not have a role as a checkpoint sensor coordinating S phase with mitosis. In contrast, germinating spores disrupted for the gene encoding pol alpha rapidly enter mitosis in the absence of DNA synthesis, suggesting that in the absence of pol alpha, normal coordination between S phase and mitosis is lost. We propose that the checkpoint signal operating in S phase depends on assembly of the replication initiation complex, and that this signal is generated prior to the elongation stage of DNA synthesis.

Cellular role of DNA polymerase I

Escherichia coli possesses three well-established DNA polymerases, I, II, and III. DNA polymerase I (Pol I) is the main repair polymerase in E. coli and also has a minor but important role in chromosomal replication. A major advantage of Pol I as an experimental system is its simplicity; unlike other replication enzymes, it is active as a single subunit. To a large extent, mutagenesis appears to be the result of (dis)functions of the DNA replication machinery. It is the purpose of this review to provide an integrated view of this relationship with particular emphasis on the role of Pol I in mutagenic events.