Uncoupling of unwinding from DNA synthesis implies regulation of MCM helicase by Tof1/Mrc1/Csm3 checkpoint complex

The replicative DNA helicases can unwind DNA in the absence of polymerase activity in vitro. In contrast, replicative unwinding is coupled with DNA synthesis in vivo. The temperature-sensitive yeast polymerase alpha/primase mutants cdc17-1, pri2-1 and pri1-m4, which fail to execute the early step of DNA replication, have been used to investigate the interaction between replicative unwinding and DNA synthesis in vivo. We report that some of the plasmid molecules in these mutant strains became extensively negatively supercoiled when DNA synthesis is prevented. In contrast, additional negative supercoiling was not detected during formation of DNA initiation complex or hydroxyurea replication fork arrest. Together, these results indicate that the extensive negative supercoiling of DNA is a result of replicative unwinding, which is not followed by DNA synthesis. The limited number of unwound plasmid molecules and synthetic lethality of polymerase alpha or primase with checkpoint mutants suggest a checkpoint regulation of the replicative unwinding. In concordance with this suggestion, we found that the Tof1/Csm3/Mrc1 checkpoint complex interacts directly with the MCM helicase during both replication fork progression and when the replication fork is stalled.     

Properties of Oligonucleotide with Phenyl-Substituted Carbocyclic Nucleoside Analogs for the Formation of Duplex and Triplex DNA

(1S,3S,4R)-1-Phenyl-1-thymidyl-3-hydroxy-4-hydroxymethylcyclopentane (10) and their analogs were synthesized, incorporated into the oligodeoxynucleotides, and their properties were evaluated for the formation of duplex and triplex DNA. The known chiral cyclopentanone derivative was converted into the corresponding ketimine sulfonamide derivative, which was subjected to a stereoselective PhLi addition. The formed sulfonamide was hydrolyzed to afford the primary amino group, on which the thymine moiety was built. The benzyl protecting groups were removed to form the nucleoside analog having a phenyl group and the thymine unit at the 1′ position of a carbocyclic skeleton (10). In the estimation of the oligodeoxynucleotides incorporating 10 for duplex and triplex formation, the carbocyclic nucleoside analog 10 did not show the stabilizing effect for duplex formation; on the other hand, it stabilized the triplex. Therefore, the skeleton of the phenyl-substituted carbocyclic nucleoside analog 10 may be a platform for the formation of stable triplex DNA.     

Regulation of DNA replication by homopurine/homopyrimidine sequences

The simple repeating homopurine/homopyrimidine sequences dispersed throughout many eukaryotic genomes are known to form triple helical structures comprising three-stranded and single-stranded DNA. Several lines of evidence suggest that these structures influence DNA replication in cells. Homopurine/homopyrimidine sequences cloned into simian virus 40 (SV40) or SV40 origin-containing plasmids caused a reduced rate of DNA synthesis due to the pausing of replication forks. More prominent arrests were observed in in vitro experiments using single-stranded and double-stranded DNA with triplex-forming sequences. Nucleotides unable to form triplexes when present in the template DNA or when incorporated into the nascent strand prevented termination. Similarly, mutations destroying the triplex potential did not cause arrest while compensatory mutations restoring triplex potential restored it. These and other observations from a number of laboratories indicating that homopurine/homopyrimidine sequences act as arrest signals in vitro and as pause sites in vivo during replication fork movement suggest that these naturally occurring sequences play a regulatory role in DNA replication and gene amplification.     

Tomato protoplast DNA transformation: physical linkage and recombination of exogenous DNA sequences

Tomato protoplasts have been transformed with plasmid DNA's, containing a chimeric kanamycin resistance gene and putative tomato origins of replication. A calcium phosphate-DNA mediated transformation procedure was employed in combination with either polyethylene glycol or polyvinyl alcohol. There were no indications that the tomato DNA inserts conferred autonomous replication on the plasmids. Instead, Southern blot hybridization analysis of seven kanamycin resistant calli revealed the presence of at least one kanamycin resistance locus per transformant integrated in the tomato nuclear DNA. Generally one to three truncated plasmid copies were found integrated into the tomato nuclear DNA, often physically linked to each other. For one transformant we have been able to use the bacterial ampicillin resistance marker of the vector plasmid pUC9 to rescue a recombinant plasmid from the tomato genome. Analysis of the foreign sequences included in the rescued plasmid showed that integration had occurred in a non-repetitive DNA region. Calf-thymus DNA, used as a carrier in transformation procedure, was found to be covalently linked to plasmid DNA sequences in the genomic DNA of one transformant. A model is presented describing the fate of exogenously added DNA during the transformation of a plant cell. The results are discussed in reference to the possibility of isolating DNA sequences responsible for autonomous replication in tomato.     

Rad52 recruitment is DNA replication independent and regulated by Cdc28 and the Mec1 kinase

Recruitment of the homologous recombination machinery to sites of double‐strand breaks is a cell cycle‐regulated event requiring entry into S phase and CDK1 activity. Here, we demonstrate that the central recombination protein, Rad52, forms foci independent of DNA replication, and its recruitment requires B‐type cyclin/CDK1 activity. Induction of the intra‐S‐phase checkpoint by hydroxyurea (HU) inhibits Rad52 focus formation in response to ionizing radiation. This inhibition is dependent upon Mec1/Tel1 kinase activity, as HU‐treated cells form Rad52 foci in the presence of the PI3 kinase inhibitor caffeine. These Rad52 foci colocalize with foci formed by the replication clamp PCNA. These results indicate that Mec1 activity inhibits the recruitment of Rad52 to both sites of DNA damage and stalled replication forks during the intra‐S‐phase checkpoint. We propose that B‐type cyclins promote the recruitment of Rad52 to sites of DNA damage, whereas Mec1 inhibits spurious recombination at stalled replication forks.     

Mechanisms of Dealing with DNA Damage-Induced Replication Problems

During every S phase, cells need to duplicate their genomes so that both daughter cells inherit complete copies of genetic information. Given the large size of mammalian genomes and the required precision of DNA replication, genome duplication requires highly fine-tuned corrective and quality control processes. A major threat to the accuracy and efficiency of DNA synthesis is the presence of DNA lesions, caused by both endogenous and exogenous damaging agents. Replicative DNA polymerases, which carry out the bulk of DNA synthesis, evolved to do their job extremely precisely and efficiently. However, they are unable to use damaged DNA as a template and, consequently, are stopped at most DNA lesions. Failure to restart such stalled replication forks can result in major chromosomal aberrations and lead to cell dysfunction or death. Therefore, a well-coordinated response to replication perturbation is essential for cell survival and fitness. Here we review how this response involves activating checkpoint signaling and the use of specialized pathways promoting replication restart. Checkpoint signaling adjusts cell cycle progression to the emergency situation and thus gives cells more time to deal with the damage. Replication restart is mediated by two pathways. Homologous recombination uses homologous DNA sequence to repair or bypass the lesion and is therefore mainly error free. Error-prone translesion synthesis employs specialized, low fidelity polymerases to bypass the damage.     

Role of recombination occurring during DNA replication

Unequal crossing over between direct DNA repeats of sister chromosomes occurs during DNA replication in Escherichia coli. Such exchanges yield tandem duplications and thereby increase the expression of the genes involved. Nonhomologous cohesion of sister chromosomes and unequal crossing over were assumed to take place when the replication fork stops. When the replication forks moves continuously, homologous exchanges between sister chromosomes ensure their postreplication repair.     

Differences in the DNA replication of unicellular eukaryotes and metazoans: known unknowns

Although the basic mechanisms of DNA synthesis are conserved across species, there are differences between simple and complex organisms. In contrast to lower eukaryotes, replication origins in complex eukaryotes lack DNA sequence specificity, can be activated in response to stressful conditions and require poorly conserved factors for replication firing. The response to replication fork damage is monitored by conserved proteins, such as the TIPIN–TIM–CLASPIN complex. The absence of this complex induces severe effects on yeast replication, whereas in higher eukaryotes it is only crucial when the availability of replication origins is limiting. Finally, the dependence of DNA replication on homologous recombination proteins such as RAD51 and the MRE11–RAD50–NBS1 complex is also different; they are dispensable for yeast S‐phase but essential for accurate DNA replication in metazoans under unchallenged conditions. The reasons for these differences are not yet understood. Here, we focus on some of these known unknowns of DNA replication.     

Replication protein A complex in Thermococcus kodakarensis interacts with DNA polymerases and helps their effective strand synthesis

Replication protein A (RPA) is an essential component of DNA metabolic processes. RPA binds to single-stranded DNA (ssDNA) and interacts with multiple DNA-binding proteins. In this study, we showed that two DNA polymerases, PolB and PolD, from the hyperthermophilic archaeon Thermococcus kodakarensis interact directly with RPA in vitro. RPA was expected to play a role in resolving the secondary structure, which may stop the DNA synthesis reaction, in the template ssDNA. Our in vitro DNA synthesis assay showed that the pausing was resolved by RPA for both PolB and PolD. These results supported the fact that RPA interacts with DNA polymerases as a member of the replisome and is involved in the normal progression of DNA replication forks.     

Characterization and quantification of triple helix formation in chromosomal DNA

DNA-binding molecules that recognize specific sequences offer a high potential for the understanding of chromatin structure and associated biological processes in addition to their therapeutic potential, e.g. as positioning agents for validated anticancer drugs. A prerequisite for the development of DNA-binding molecules is the availability of appropriate methods to assess their binding properties quantitatively at the desired target sequence in the human genome. We have further developed a capture assay to assess triplex-forming oligonucleotide (TFO) binding efficiency quantitatively. This assay is based on bifunctional, psoralen and biotin-conjugated, TFOs and real-time PCR analysis. We have applied this novel quantification method to address two issues that are relevant for DNA-binding molecules. First, we have compared directly the extent of TFO-binding in three experimental settings with increasing similarity to the situation in vivo, i.e. naked genomic DNA, isolated cell nuclei, or whole cells. This comparison allows us to characterize factors that influence genomic triplex formation, e.g. chromosomal DNA organization or intracellular milieu. In isolated nuclei, the binding was threefold lower compared to naked DNA, consistent with a decreased target accessibility int he nucleosomal environment. Binding was detected in whole cells, indicating that the TFO enters the nucleus and binds to its target in intact cells in vivo, but the efficiency was decreased (tenfold) compared to nuclei. Secondly, we applied the method to characterize the binding properties of two different TFOs targeting the same sequence. We found that an antiparallel-binding GT-containing TFO bound more efficiently, but with less target sequence selectivity compared to a parallel-binding CU-containing TFO. Collectively, a sensitive method to characterize genomic triplex formation was described. This may be useful for the determination of factors driving TFO binding efficiency and, thus, may improve the usefulness of triplex-mediated gene targeting for studies of chromatin structure as well as for therapeutic antigene strategies.     

Sgs1 function in the repair of DNA replication intermediates is separable from its role in homologous recombinational repair

Mutations in human homologues of the bacterial RecQ helicase cause diseases leading to cancer predisposition and/or shortened lifespan (Werner, Bloom, and Rothmund–Thomson syndromes). The budding yeast Saccharomyces cerevisiae has one RecQ helicase, Sgs1, which functions with Top3 and Rmi1 in DNA repair. Here, we report separation‐of‐function alleles of SGS1 that suppress the slow growth of top3Δ and rmi1Δ cells similar to an SGS1 deletion, but are resistant to DNA damage similar to wild‐type SGS1. In one allele, the second acidic region is deleted, and in the other, only a single aspartic acid residue 664 is deleted. sgs1‐D664Δ, unlike sgs1Δ, neither disrupts DNA recombination nor has synthetic growth defects when combined with DNA repair mutants. However, during S phase, it accumulates replication‐associated X‐shaped structures at damaged replication forks. Furthermore, fluorescent microscopy reveals that the sgs1‐D664Δ allele exhibits increased spontaneous RPA foci, suggesting that the persistent X‐structures may contain single‐stranded DNA. Taken together, these results suggest that the Sgs1 function in repair of DNA replication intermediates can be uncoupled from its role in homologous recombinational repair.     

Comparison of DNA Strand Break Induction in CHO Cells Measured by Alkaline Elution and by Fluorometric Analysis of DNA Unwinding (FADU)

DNA damage in X-irradiated CHO cells was measured by alkaline filter elution and compared to fluorometric analysis of DNA unwinding (FADU). The FADU method proved to be as sensitive as the alkaline filter elution technique in detecting X-ray induced DNA breaks. Strand break induction was also measured after treatment with four radical generating chemicals (hydrogen peroxide, bleomycin, mitomycin C and methyl viologen) using the FADU technique.     

Comparison of DNA Strand Break Induction in CHO Cells Measured by Alkaline Elution and by Fluorometric Analysis of DNA Unwinding (FADU)

DNA damage in X-irradiated CHO cells was measured by alkaline filter elution and compared to fluorometric analysis of DNA unwinding (FADU). The FADU method proved to be as sensitive as the alkaline filter elution technique in detecting X-ray induced DNA breaks. Strand break induction was also measured after treatment with four radical generating chemicals (hydrogen peroxide, bleomycin, mitomycin C and methyl viologen) using the FADU technique.     

Homologous recombination in DNA repair and DNA damage tolerance

Homologous recombination （HR） comprises a series of interrelated pathways that function in the repair of DNA double-stranded breaks （DSBs） and interstrand crosslinks （ICLs）. In addition, recombination provides critical support for DNA replication in the recovery of stalled or broken replication forks, contributing to tolerance of DNA damage. A central core of proteins, most critically the RecA homolog Rad51, catalyzes the key reactions that typify HR： homology search and DNA strand invasion. The diverse functions of recombination are reflected in the need for context-specific factors that perform supplemental functions in conjunction with the core proteins. The inability to properly repair complex DNA damage and resolve DNA replication stress leads to genomic instability and contributes to cancer etiology. Mutations in the BRCA2 recombination gene cause predisposition to breast and ovarian cancer as well as Fanconi anemia, a cancer predisposition syndrome characterized by a defect in the repair of DNA interstrand crosslinks. The cellular functions of recombination are also germane to DNA-based treatment modalities of cancer, which target replicating cells by the direct or indirect induction of DNA lesions that are substrates for recombination pathways. This review focuses on mechanistic aspects of HR relating to DSB and ICL repair as well as replication fork support.     

Nucleoprotein complexes released from lymphoma nuclei that contain the abl oncogene and RNA and DNA polymerase and RNA primase activities.

We report on the discovery and isolation of DNA- and RNA-containing macromolecular nuclear complexes whose purified major DNA possessed electrophoretic mobilities of approximately 90 and approximately 25 kbp. The deoxyribonucleoprotein-ribonucleoprotein complexes contain RNA and DNA polymerase and primase activities and were isolated from nuclei of murine RAW117 large-cell lymphoma cells by restriction digestion with Msp-I, gentle extraction with solutions containing MgCl2, but without chelating agents, and low ionic strength gel electrophoresis. Two-dimensional (isoelectric focusing/M(r)) gel electrophoresis and silver staining of the proteins of the complexes after treatment with DNase I indicated the presence of approximately 30 protein components. In vitro DNA and RNA polymerase/primase assays showed that the DNP/RNP complexes had very high enzyme specific activities. Using the DNP/RNP complexes a discrete DNA polymerase alpha product of approximately 85 kbp was synthesized that was not synthesized in the presence of the DNA polymerase alpha inhibitor aphidicolin. RNA polymerase assays in the presence of excess alpha-amanitin indicated that the complexes possessed significant RNA polymerase I activity. Preparing the complexes at various times after the release of cells from a double thymidine block showed the complexes as well as the complex-associated enzyme activities to be cell-cycle dependent. The DNA and RNA polymerase-related activities were highest in late S phase, 7 and 9 h, respectively, after release from the double thymidine block. The complexes synthesized a specific in vitro DNA polymerase product using endogenous substrate and nucleotide precursors.(ABSTRACT TRUNCATED AT 250 WORDS)     

BRCA2 is needed for both repair and cell cycle arrest in mammalian cells exposed to S23906, an anticancer monofunctional DNA binder

Repair of DNA-targeted anticancer agents is an active area of investigation of both fundamental and clinical interest. However, most studies have focused on a small number of compounds limiting our understanding of both DNA repair and the DNA damage response. {"type":"entrez-protein","attrs":{"text":"S23906","term_id":"96914","term_text":"pir||S23906"}}S23906 is an acronycine derivative that shows strong activity toward solid tumors in experimental models. {"type":"entrez-protein","attrs":{"text":"S23906","term_id":"96914","term_text":"pir||S23906"}}S23906 forms bulky monofunctional DNA adducts in the minor groove which leads to destabilization of the double-stranded helix. We now report that {"type":"entrez-protein","attrs":{"text":"S23906","term_id":"96914","term_text":"pir||S23906"}}S23906 induces formation of DNA double strand breaks that are processed through homologous recombination (HR) but not Non-Homologous End-Joining (NHEJ) repair. Interestingly, {"type":"entrez-protein","attrs":{"text":"S23906","term_id":"96914","term_text":"pir||S23906"}}S23906 exposure was accompanied by a higher sensitivity of BRCA2-deficient cells compared to other HR deficient cell lines and by an S-phase accumulation in wild-type (wt), but not in BRCA2-deficient cells. Recently, we have shown that {"type":"entrez-protein","attrs":{"text":"S23906","term_id":"96914","term_text":"pir||S23906"}}S23906-induced S phase arrest was mediated by the checkpoint kinase Chk1. However, its activated phosphorylated form is equally induced by {"type":"entrez-protein","attrs":{"text":"S23906","term_id":"96914","term_text":"pir||S23906"}}S23906 in wt and BRCA2-deficient cells, likely indicating a role for BRCA2 downstream of Chk1. Accordingly, override of the S phase arrest by either 7-hydroxystaurosporine (UCN-01) or AZD7762 potentiates the cytotoxic activity of {"type":"entrez-protein","attrs":{"text":"S23906","term_id":"96914","term_text":"pir||S23906"}}S23906 in wt, but not in BRCA2-deficient cells. Together, our findings suggest that the pronounced sensitivity of BRCA2-deficient cells to {"type":"entrez-protein","attrs":{"text":"S23906","term_id":"96914","term_text":"pir||S23906"}}S23906 is due to both a defective S-phase arrest and the absence of HR repair. Tumors with deficiencies for proteins involved in HR, and BRCA2 in particular, may thus show increased sensitivity to {"type":"entrez-protein","attrs":{"text":"S23906","term_id":"96914","term_text":"pir||S23906"}}S23906, thereby providing a rationale for patient selection in clinical trials.