Monofunctional alkylating agent-induced S-phase-dependent DNA damage

Alkylating agents are S-phase-dependent clastogenic agents: Chromosome aberrations are not observed unless the treated cells have first undergone a replicative DNA synthesis. While DNA gaps resulting from misreplication of the alkylated template are believed to underlie aberration formation, the specific alkylated DNA lesions that produce these DNA gaps are not known. To quantitate the DNA strand break induction that results from replication of an alkylated DNA template and attempt to identify those alkylated lesions which underlie DNA strand breakage. [14C]thymidine-labeled Chinese hamster ovary (CHO) cells were treated with either N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or methyl methanesulfonate (MMS) in G1 and then allowed to progress through S phase in the presence of [3H]thymidine. When analyzed at the subsequent mitosis, DNA strand breaks were found in the nonalkylated ([3H]thymidine-labeled) DNA strand. This did not appear to be the consequence of any recombinational or endonuclease-mediated event and was more likely due to DNA gaps produced by incomplete replication off the alkylated template. A portion of these breaks probably result from a failure to replicate past 3-methyladenine. Differences between MNNG and MMS in the frequency of S-phase-dependent breaks they produce relative to the overall alkylation damage suggest that the O6-methylguanine lesion might also be involved in S-phase-dependent DNA strand breakage.     

Hemicatenanes form upon inhibition of DNA replication

Plasmid DNA incubated in interphase Xenopus egg extracts is normally assembled into chromatin and then into synthetic nuclei which undergo one round of regulated replication. During a study of restriction endonuclease cut plasmid replication intermediates (RIs) by the Brewer–Fangman 2D gel electrophoresis technique, we have observed the formation of a strong spike of X-shaped DNA molecules in extracts that otherwise yield very little or no RIs. Formation of these joint molecules is also efficiently induced in replication-competent extracts upon inhibition of replication fork progression by aphidicolin. Although their electrophoretic properties are quite similar to those of Holliday junctions, 2D gels of doubly cut plasmids show that these junctions can link two plasmid molecules at any pair of DNA sequences, with no regard for sequence homology at the branch points. Neutral–neutral–alkaline 3D gels show that the junctions only contain single strands of parental size and no recombinant strands. A hemicatenane, in which one strand of a duplex is wound around one strand of another duplex, is the most likely structure to account for these observations. The mechanism of formation of these novel joint DNA molecules and their biological implications are discussed.     

STRANDEDNESS OF VICIA FABA CHROMOSOMES AS REVEALED BY ENZYME DIGESTION STUDIES

Chromosomes and nuclei isolated from neutral formalin-fixed Vicia faba lateral roots were treated with trypsin, pepsin, RNase, or DNase. Only trypsin affected the morphology of the chromosomes and nuclei. The appearance of the chromosomes after trypsin digestion indicated that each chromatid contained four strands that could be seen with an ordinary light microscope. The experiments are interpreted as indicating that mitotic chromosomes of Vicia faba are multistranded and that the linear continuity of the chromosome is dependent on protein.     

A complex RNA motif defined by three discontinuous 5-nucleotide-long strands is essential for Flavivirus RNA replication

Tertiary or higher-order RNA motifs that regulate replication of positive-strand RNA viruses are as yet poorly understood. Using Japanese encephalitis virus (JEV), we now show that a key element in JEV RNA replication is a complex RNA motif that includes a string of three discontinuous complementary sequences (TDCS). The TDCS consists of three 5-nt-long strands, the left (L) strand upstream of the translation initiator AUG adjacent to the 5′-end of the genome, and the middle (M) and right (R) strands corresponding to the base of the Flavivirus-conserved 3′ stem–loop structure near the 3′-end of the RNA. The three strands are arranged in an antiparallel configuration, with two sets of base-pairing interactions creating L-M and M-R duplexes. Disrupting either or both of these duplex regions of TDCS completely abolished RNA replication, whereas reconstructing both duplex regions, albeit with mutated sequences, fully restored RNA replication. Modeling of replication-competent genomes recovered from a large pool of pseudorevertants originating from six replication-incompetent TDCS mutants suggests that both duplex base-pairing potentials of TDCS are required for RNA replication. In all cases, acquisition of novel sequences within the 3′M-R duplex facilitated a long-range RNA–RNA interaction of its 3′M strand with either the authentic 5′L strand or its alternative (invariably located upstream of the 5′ initiator), thereby restoring replicability. We also found that a TDCS homolog is conserved in other flaviviruses. These data suggest that two duplex base-pairings defined by the TDCS play an essential regulatory role in a key step(s) of Flavivirus RNA replication.     

Origin-Dependent Inverted-Repeat Amplification: Tests of a Model for Inverted DNA Amplification

DNA replication errors are a major driver of evolution—from single nucleotide polymorphisms to large-scale copy number variations (CNVs). Here we test a specific replication-based model to explain the generation of interstitial, inverted triplications. While no genetic information is lost, the novel inversion junctions and increased copy number of the included sequences create the potential for adaptive phenotypes. The model—Origin-Dependent Inverted-Repeat Amplification (ODIRA)—proposes that a replication error at pre-existing short, interrupted, inverted repeats in genomic sequences generates an extrachromosomal, inverted dimeric, autonomously replicating intermediate; subsequent genomic integration of the dimer yields this class of CNV without loss of distal chromosomal sequences. We used a combination of in vitro and in vivo approaches to test the feasibility of the proposed replication error and its downstream consequences on chromosome structure in the yeast Saccharomyces cerevisiae. We show that the proposed replication error—the ligation of leading and lagging nascent strands to create “closed” forks—can occur in vitro at short, interrupted inverted repeats. The removal of molecules with two closed forks results in a hairpin-capped linear duplex that we show replicates in vivo to create an inverted, dimeric plasmid that subsequently integrates into the genome by homologous recombination, creating an inverted triplication. While other models have been proposed to explain inverted triplications and their derivatives, our model can also explain the generation of human, de novo, inverted amplicons that have a 2:1 mixture of sequences from both homologues of a single parent—a feature readily explained by a plasmid intermediate that arises from one homologue and integrates into the other homologue prior to meiosis. Our tests of key features of ODIRA lend support to this mechanism and suggest further avenues of enquiry to unravel the origins of interstitial, inverted CNVs pivotal in human health and evolution.     

Asymmetric Strand Segregation: Epigenetic Costs of Genetic Fidelity?

Asymmetric strand segregation has been proposed as a mechanism to minimize effective mutation rates in epithelial tissues. Under asymmetric strand segregation, the double-stranded molecule that contains the oldest DNA strand is preferentially targeted to the somatic stem cell after each round of DNA replication. This oldest DNA strand is expected to have fewer errors than younger strands because some of the errors that arise on daughter strands during their synthesis fail to be repaired. Empirical findings suggest the possibility of asymmetric strand segregation in a subset of mammalian cell lineages, indicating that it may indeed function to increase genetic fidelity. However, the implications of asymmetric strand segregation for the fidelity of epigenetic information remain unexplored. Here, I explore the impact of strand-segregation dynamics on epigenetic fidelity using a mathematical-modelling approach that draws on the known molecular mechanisms of DNA methylation and existing rate estimates from empirical methylation data. I find that, for a wide range of starting methylation densities, asymmetric—but not symmetric—strand segregation leads to systematic increases in methylation levels if parent strands are subject to de novo methylation events. I found that epigenetic fidelity can be compromised when enhanced genetic fidelity is achieved through asymmetric strand segregation. Strand segregation dynamics could thus explain the increased DNA methylation densities that are observed in structured cellular populations during aging and in disease.     

Low-Molecular-Weight DNA Replication Intermediates in Escherichia coli: Mechanism of Formation and Strand Specificity

Chromosomal DNA replication intermediates, revealed in ligase-deficient conditions in vivo, are of low molecular weight (LMW) independently of the organism, suggesting discontinuous replication of both the leading and the lagging DNA strands. Yet, in vitro experiments with purified enzymes replicating sigma-structured substrates show continuous synthesis of the leading DNA strand in complete absence of ligase, supporting the textbook model of semi-discontinuous DNA replication. The discrepancy between the in vivo and in vitro results is rationalized by proposing that various excision repair events nick continuously synthesized leading strands after synthesis, producing the observed LMW intermediates. Here, we show that, in an Escherichia coli ligase-deficient strain with all known excision repair pathways inactivated, new DNA is still synthesized discontinuously. Furthermore, hybridization to strand-specific targets demonstrates that the LMW replication intermediates come from both the lagging and the leading strands. These results support the model of discontinuous leading strand synthesis in E. coli.     

Deficiency of RecA-dependent RecFOR and RecBCD pathways causes increased instability of the (GAA*TTC)n sequence when GAA is the lagging strand template

The most common mutation in Friedreich ataxia is an expanded (GAA·TTC)_n sequence, which is highly unstable in human somatic cells and in the germline. The mechanisms responsible for this genetic instability are poorly understood. We previously showed that cloned (GAA·TTC)_n sequences replicated in Escherichia coli are more unstable when GAA is the lagging strand template, suggesting erroneous lagging strand synthesis as the likely mechanism for the genetic instability. Here we show that the increase in genetic instability when GAA serves as the lagging strand template is seen in RecA-deficient but not RecA-proficient strains. We also found the same orientation-dependent increase in instability in a RecA⁺ temperature-sensitive E. coli SSB mutant strain (ssb-1). Since stalling of replication is known to occur within the (GAA·TTC)_n sequence when GAA is the lagging strand template, we hypothesized that genetic stability of the (GAA·TTC)_n sequence may require efficient RecA-dependent recombinational restart of stalled replication forks. Consistent with this hypothesis, we noted significantly increased instability when GAA was the lagging strand template in strains that were deficient in components of the RecFOR and RecBCD pathways. Our data implicate defective processing of stalled replication forks as a mechanism for genetic instability of the (GAA·TTC)_n sequence.     

Mutation fixation in MNNG-treated Haemophilus influenzae as determined by transformation

A transformation assay has been used to follow the fixation of mutations to novobiocin resistance induced by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) in Haemophilus influenzae. Very few mutations are produced by recently treated DNA, but many are produced by the DNA from cells that have been incubated for a time after exposure to MNNG. The time course of this mutation fixation is shown to coincide reasonably well with the time course of semiconservative DNA synthesis, as judged by uptake studies and by isopycnic centrifugation of density-labeled cells. Incubation with bromodeoxyuridine (BrdUrd) during the fixation period decreases the number of mutations that are fixed, showing in another way the importance of DNA synthesis for fixation.Mutations fixed in the presence of BrdUrd are not more sensitive to 313-nm radiation than those fixed in its absence, suggesting that these residual mutations are fixed in the absence of extensive DNA replication. Mutations newly fixed in the absence of BrdUrd are much more sensitive to 313-nm radiation than are the same mutations some cell generations later. This shows that the newly fixed mutations are in a state that is different from their final form, either because they are in regions of DNA with special configurations of the strands or because they are in a region of DNA that is a hybrid between an old, alkylated strand and a new strand with some bases different from normal. The data suggest that it is unlikely that anything like all the mutations that are fixed in H. influenzae arise by direct action of MNNG on the replication fork. Many of the results can be explained in terms of fixation during semiconservative replication of premutational lesions, some of which are initially located some distance from the replication fork. The final yield would then depend on the relative rates of removal of the lesions by repair and of fixation by replication.     

Mutagenesis of acridine orange in mitotic cleavage nuclei of the silkworm, Bombyx mori

The present study was done to determine whether acridine orange (AO) is mutagenic for the mitotic cleavage nuclei in the silkworm. The mutation frequency was estimated by the specific locus method using egg-color genes. AO was injected into the body cavity of marked female pupae (homozygous for pe and re genes) in active vitellogenesis (prophase I oocytes). The moths emerging from the treated pupae were mated to wild type male moths. AO increased the frequency of mosaic type mutations, indicating that AO has a positive mutagenic action on the paternal chromosomes in the mitotic cleavage nuclei in the silkworm.     

DNA structure and the Werner protein modulate human DNA polymerase delta-dependent replication dynamics within the common fragile site FRA16D

Common fragile sites (CFS) are chromosomal regions that exhibit instability during DNA replication stress. Although the mechanism of CFS expression has not been fully elucidated, one known feature is a severely delayed S-phase. We used an in vitro primer extension assay to examine the progression of DNA synthesis through various sequences within FRA16D by the replicative human DNA polymerases δ and α, and with human cell-free extracts. We found that specific cis-acting sequence elements perturb DNA elongation, causing inconsistent DNA synthesis rates between regions on the same strand and complementary strands. Pol δ was significantly inhibited in regions containing hairpins and microsatellites, [AT/TA]₂₄ and [A/T]_19–28, compared with a control region with minimal secondary structure. Pol δ processivity was enhanced by full length Werner Syndrome protein (WRN) and by WRN fragments containing either the helicase domain or DNA-binding C-terminal domain. In cell-free extracts, stalling was eliminated at smaller hairpins, but persisted in larger hairpins and microsatellites. Our data support a model whereby CFS expression during cellular stress is due to a combination of factors—density of specific DNA secondary-structures within a genomic region and asymmetric rates of strand synthesis.     

Mismatch Repair Balances Leading and Lagging Strand DNA Replication Fidelity

The two DNA strands of the nuclear genome are replicated asymmetrically using three DNA polymerases, α, δ, and ε. Current evidence suggests that DNA polymerase ε (Pol ε) is the primary leading strand replicase, whereas Pols α and δ primarily perform lagging strand replication. The fact that these polymerases differ in fidelity and error specificity is interesting in light of the fact that the stability of the nuclear genome depends in part on the ability of mismatch repair (MMR) to correct different mismatches generated in different contexts during replication. Here we provide the first comparison, to our knowledge, of the efficiency of MMR of leading and lagging strand replication errors. We first use the strand-biased ribonucleotide incorporation propensity of a Pol ε mutator variant to confirm that Pol ε is the primary leading strand replicase in Saccharomyces cerevisiae. We then use polymerase-specific error signatures to show that MMR efficiency in vivo strongly depends on the polymerase, the mismatch composition, and the location of the mismatch. An extreme case of variation by location is a T-T mismatch that is refractory to MMR. This mismatch is flanked by an AT-rich triplet repeat sequence that, when interrupted, restores MMR to >95% efficiency. Thus this natural DNA sequence suppresses MMR, placing a nearby base pair at high risk of mutation due to leading strand replication infidelity. We find that, overall, MMR most efficiently corrects the most potentially deleterious errors (indels) and then the most common substitution mismatches. In combination with earlier studies, the results suggest that significant differences exist in the generation and repair of Pol α, δ, and ε replication errors, but in a generally complementary manner that results in high-fidelity replication of both DNA strands of the yeast nuclear genome.     

DNA Strand Breaks in Mitotic Germ Cells of Caenorhabditis elegans Evaluated by Comet Assay

DNA damage responses are important for the maintenance of genome stability and the survival of organisms. Such responses are activated in the presence of DNA damage and lead to cell cycle arrest, apoptosis, and DNA repair. In Caenorhabditis elegans, double-strand breaks induced by DNA damaging agents have been detected indirectly by antibodies against DSB recognizing proteins. In this study we used a comet assay to detect DNA strand breaks and to measure the elimination of DNA strand breaks in mitotic germline nuclei of C. elegans. We found that C. elegans brc-1 mutants were more sensitive to ionizing radiation and camptothecin than the N2 wild-type strain and repaired DNA strand breaks less efficiently than N2. This study is the first demonstration of direct measurement of DNA strand breaks in mitotic germline nuclei of C. elegans. This newly developed assay can be applied to detect DNA strand breaks in different C. elegans mutants that are sensitive to DNA damaging agents.     

Caenorhabditis elegans HIM-18/SLX-4 Interacts with SLX-1 and XPF-1 and Maintains Genomic Integrity in the Germline by Processing Recombination Intermediates

Homologous recombination (HR) is essential for the repair of blocked or collapsed replication forks and for the production of crossovers between homologs that promote accurate meiotic chromosome segregation. Here, we identify HIM-18, an ortholog of MUS312/Slx4, as a critical player required in vivo for processing late HR intermediates in Caenorhabditis elegans. DNA damage sensitivity and an accumulation of HR intermediates (RAD-51 foci) during premeiotic entry suggest that HIM-18 is required for HR–mediated repair at stalled replication forks. A reduction in crossover recombination frequencies—accompanied by an increase in HR intermediates during meiosis, germ cell apoptosis, unstable bivalent attachments, and subsequent chromosome nondisjunction—support a role for HIM-18 in converting HR intermediates into crossover products. Such a role is suggested by physical interaction of HIM-18 with the nucleases SLX-1 and XPF-1 and by the synthetic lethality of him-18 with him-6, the C. elegans BLM homolog. We propose that HIM-18 facilitates processing of HR intermediates resulting from replication fork collapse and programmed meiotic DSBs in the C. elegans germline.     

Replication of bacteriophage M13. 8. Differential effects of rifampicin and nalidixic acid on the synthesis of the two strands of M13 duplex DNA

Replication of bacteriophage M13 replicative forms is inhibited by rifampicin, an antibiotic that specifically inhibits the Escherichia coli RNA polymerase, and by nalidixic acid, an inhibitor of phage and bacterial DNA replication. Synthesis of the M13 complementary strand during RF³ replication was at least tenfold more sensitive to inhibition by rifampicin and by nalidixic acid than was that of the viral strand. Since M13 complementary strand synthesis is relatively insensitive to chloramphenicol, an inhibitor of protein synthesis, its inhibition by rifampicin suggests that complementary strands are initiated during RF replication by an RNA priming mechanism similar to that involved in parental RF formation. The nalidixic acid-sensitivity of complementary strand synthesis during RF replication clearly distinguishes this process from the nalidixic acid-resistant formation of the parental complementary strand in the conversion of the infecting single strand to RF.Production of progeny viral strands is indirectly affected by rifampiein in two ways. It prevents the conversion of supercoiled RF (RFI) to the open form (RFII), an essential step both in RF replication and in single-strand synthesis. In addition, rifampiein interferes with the expression of gene 5, an M13 gene function required for the accumulation of progeny viral strands.     

A Multi-Step Process of Viral Adaptation to a Mutagenic Nucleoside Analogue by Modulation of Transition Types Leads to Extinction-Escape

Resistance of viruses to mutagenic agents is an important problem for the development of lethal mutagenesis as an antiviral strategy. Previous studies with RNA viruses have documented that resistance to the mutagenic nucleoside analogue ribavirin (1-β-D-ribofuranosyl-1-H-1,2,4-triazole-3-carboxamide) is mediated by amino acid substitutions in the viral polymerase that either increase the general template copying fidelity of the enzyme or decrease the incorporation of ribavirin into RNA. Here we describe experiments that show that replication of the important picornavirus pathogen foot-and-mouth disease virus (FMDV) in the presence of increasing concentrations of ribavirin results in the sequential incorporation of three amino acid substitutions (M296I, P44S and P169S) in the viral polymerase (3D). The main biological effect of these substitutions is to attenuate the consequences of the mutagenic activity of ribavirin —by avoiding the biased repertoire of transition mutations produced by this purine analogue—and to maintain the replicative fitness of the virus which is able to escape extinction by ribavirin. This is achieved through alteration of the pairing behavior of ribavirin-triphosphate (RTP), as evidenced by in vitro polymerization assays with purified mutant 3Ds. Comparison of the three-dimensional structure of wild type and mutant polymerases suggests that the amino acid substitutions alter the position of the template RNA in the entry channel of the enzyme, thereby affecting nucleotide recognition. The results provide evidence of a new mechanism of resistance to a mutagenic nucleoside analogue which allows the virus to maintain a balance among mutation types introduced into progeny genomes during replication under strong mutagenic pressure.     

Structure and replication of Phaseolus polytene chromosomes

The normal morphology of the polytene chromosomes of the embryo suspensor of Phaseolus coccineus is that of a tightly condensed cord with heavily Feulgen staining centromeric heterochromatic regions (α-heterochromatin) and other accessory heterochromatic regions (β-heterochromatin). The replication pattern of the chromosomes has been determined by autoradiographic analysis of material pulsed with ³H-thymidine for various lengths of time. The DNA replication cycle reqires 4–6 hours for completion. During replication chromosome structure becomes diffuse and the β-heterochromatic regions are indistinguishable from the euchromatic regions. The euchromatin is the first to replicate, and replication begins simultaneously at numerous sites in the euchromatin. The β-heterochromatin replicates next, and finally the centromeric heterochromatin. Replication is essentially complete in each of these parts of the chromosome before DNA synthesis begins in the next. The chromosomes are composed of numerous longitudinally running Feulgen positive strands, the equivalent portions of which replicate simultaneously. This indicates that there must be close control of the replication cycle in sister strands.     

DNA polymerase IV mediates efficient and quick recovery of replication forks stalled at N2-dG adducts

Escherichia coli DNA polymerase IV (Pol IV, also known as DinB) is a Y-family DNA polymerase capable of catalyzing translesion DNA synthesis (TLS) on certain DNA lesions, and accumulating data suggest that Pol IV may play an important role in copying various kinds of spontaneous DNA damage including N²-dG adducts and alkylated bases. Pol IV has a unique ability to coexist with Pol III on the same β clamp and to positively dissociate Pol III from β clamp in a concentration-dependent manner. Reconstituting the entire process of TLS in vitro using E. coli replication machinery and Pol IV, we observed that a replication fork stalled at (−)-trans-anti-benzo[a]pyrene-N²-dG lesion on the leading strand was efficiently and quickly recovered via two sequential switches from Pol III to Pol IV and back to Pol III. Our results suggest that TLS by Pol IV smoothes the way for the replication fork with minimal interruption.