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1.
Evidence that eosinophils catalyze the bromide-dependent decarboxylation of amino acids 总被引:1,自引:0,他引:1
Human eosinophils from subjects with or without myeloperoxidase (MPO) deficiency and guinea pig eosinophils are able to decarboxylate L- alanine in the presence of the cationic detergent cetyltrimethylammonium bromide (CTAB) but not in the presence of the nonionic detergent Triton X-100. Instead, both normal human neutrophils and guinea pig neutrophils decarboxylate L-alanine in the presence of either detergent. When the non-bromide-containing cationic detergent cetyltrimethylammonium hydroxide (CTAOH) is used instead of CTAB, the eosinophils from MPO-deficient subjects are unable to decarboxylate L- alanine. Decarboxylation occurs with the combination CTAOH-Br-, but not with the combinations CTAOH-I-, CTAOH-CI-, or CTAOH-F-. Bromide in the absence of CTAOH does not promote decarboxylation. Triton X-100 and deoxycholate are much less effective in promoting decarboxylation in the presence of bromide. L-Lysine and L-aspartic acid are decarboxylated to a considerably lower rate than L-alanine in the presence of CTAOH and Br-. It is concluded that the eosinophils can catalyze the bromide-dependent decarboxylation of the apolar amino acid L-alanine in the presence of a cationic detergent. 相似文献
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4.
Rqh1 blocks recombination between sister chromatids during double strand break repair, independent of its helicase activity
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Hope JC Mense SM Jalakas M Mitsumoto J Freyer GA 《Proceedings of the National Academy of Sciences of the United States of America》2006,103(15):5875-5880
Many questions remain about the process of DNA double strand break (DSB) repair by homologous recombination (HR), particularly concerning the exact function played by individual proteins and the details of specific steps in this process. Some recent studies have shown that RecQ DNA helicases have a function in HR. We studied the role of the RecQ helicase Rqh1 with HR proteins in the repair of a DSB created at a unique site within the Schizosaccharomyces pombe genome. We found that DSBs in rqh1(+) cells, are predominantly repaired by interchromosomal gene conversion, with HR between sister chromatids [sister-chromatid conversion (SCC)], occurring less frequently. In Deltarqh1 cells, repair by SCC is favored, and gene conversion rates slow significantly. When we limited the potential for SCC in Deltarqh1 cells by reducing the length of the G2 phase of the cell cycle, DSB repair continued to be predominated by SCC, whereas it was essentially eliminated in wild-type cells. These data indicate that Rqh1 acts to regulate DSB repair by blocking SCC. Interestingly, we found that this role for Rqh1 is independent of its helicase activity. In the course of these studies, we also found nonhomologous end joining to be largely faithful in S. pombe, contrary to current belief. These findings provide insight into the regulation of DSB repair by RecQ helicases. 相似文献
5.
Nina Y. Yao Roxana E. Georgescu Jeff Finkelstein Michael E. O'Donnell 《Proceedings of the National Academy of Sciences of the United States of America》2009,106(32):13236-13241
Single-molecule techniques are developed to examine mechanistic features of individual E. coli replisomes during synthesis of long DNA molecules. We find that single replisomes exhibit constant rates of fork movement, but the rates of different replisomes vary over a surprisingly wide range. Interestingly, lagging strand synthesis decreases the rate of the leading strand, suggesting that lagging strand operations exert a drag on replication fork progression. The opposite is true for processivity. The lagging strand significantly increases the processivity of the replisome, possibly reflecting the increased grip to DNA provided by 2 DNA polymerases anchored to sliding clamps on both the leading and lagging strands. 相似文献
6.
Increased efficiency of oligonucleotide-mediated gene repair through slowing replication fork progression 总被引:4,自引:0,他引:4
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Wu XS Xin L Yin WX Shang XY Lu L Watt RM Cheah KS Huang JD Liu DP Liang CC 《Proceedings of the National Academy of Sciences of the United States of America》2005,102(7):2508-2513
Targeted gene modification mediated by single-stranded oligonucleotides (SSOs) holds great potential for widespread use in a number of biological and biomedical fields, including functional genomics and gene therapy. By using this approach, specific genetic changes have been created in a number of prokaryotic and eukaryotic systems. In mammalian cells, the precise mechanism of SSO-mediated chromosome alteration remains to be established, and there have been problems in obtaining reproducible targeting efficiencies. It has previously been suggested that the chromatin structure, which changes throughout the cell cycle, may be a key factor underlying these variations in efficiency. This hypothesis prompted us to systematically investigate SSO-mediated gene repair at various phases of the cell cycle in a mammalian cell line. We found that the efficiency of SSO-mediated gene repair was elevated by approximately 10-fold in thymidine-treated S-phase cells. The increase in repair frequency correlated positively with the duration of SSO/thymidine coincubation with host cells after transfection. We supply evidence suggesting that these increased repair frequencies arise from a thymidine-induced slowdown of replication fork progression. Our studies provide fresh insight into the mechanism of SSO-mediated gene repair in mammalian cells and demonstrate how its efficiency may be reliably and substantially increased. 相似文献
7.
Nina Y. Yao Jeremy W. Schroeder Olga Yurieva Lyle A. Simmons Mike E. O’Donnell 《Proceedings of the National Academy of Sciences of the United States of America》2013,110(32):12942-12947
The concentration of ribonucleoside triphosphates (rNTPs) in cells is far greater than the concentration of deoxyribonucleoside triphosphates (dNTPs), and this pool imbalance presents a challenge for DNA polymerases (Pols) to select their proper substrate. This report examines the effect of nucleotide pool imbalance on the rate and fidelity of the Escherichia coli replisome. We find that rNTPs decrease replication fork rate by competing with dNTPs at the active site of the C-family Pol III replicase at a step that does not require correct base-pairing. The effect of rNTPs on Pol rate generalizes to B-family eukaryotic replicases, Pols δ and ε. Imbalance of the dNTP pool also slows the replisome and thus is not specific to rNTPs. We observe a measurable frequency of rNMP incorporation that predicts one rNTP incorporated every 2.3 kb during chromosome replication. Given the frequency of rNMP incorporation, the repair of rNMPs is likely rapid. RNase HII nicks DNA at single rNMP residues to initiate replacement with dNMP. Considering that rNMPs will mark the new strand, RNase HII may direct strand-specificity for mismatch repair (MMR). How the newly synthesized strand is recognized for MMR is uncertain in eukaryotes and most bacteria, which lack a methyl-directed nicking system. Here we demonstrate that Bacillus subtilis incorporates rNMPs in vivo, that RNase HII plays a role in their removal, and the RNase HII gene deletion enhances mutagenesis, suggesting a possible role of incorporated rNMPs in MMR.The structures of ribonucleoside triphosphates (rNTPs) and deoxyribonucleoside triphosphates (dNTPs) differ by a single atom, yet each must be distinguished by DNA and RNA polymerases (Pols). This is more challenging for DNA Pols because the intracellular concentration of rNTPs is 10–100-fold higher than that of dNTPs (1–3). DNA is more stabile than RNA, and rNMP residues in DNA could lead to spontaneous strand breaks. Hence, it is important to genomic integrity that DNA Pols exclude rNMPs from incorporation into the genome (1, 4).Structural studies reveal that DNA Pols distinguish ribo and deoxyribo sugars via a “steric gate,” in which a bulky residue or main chain atom sterically occludes binding of the ribo 2′OH (5, 6). However, the single-atom difference between rNTPs and dNTPs imposes an upper limit to sugar recognition, and thus DNA Pols incorporate rNMPs at a low frequency. For example, studies in yeast demonstrate that rNMPs are incorporated in vivo, and studies in vitro demonstrate a frequency of rNMP incorporation predicting that 10,000 rNMPs or more may be incorporated each replication cycle (2, 7, 8). Given their abundant incorporation, there are probably multiple pathways to remove rNMPs, as their persistence is associated with genomic instability in yeast (7, 9).The current study reconstitutes the Escherichia coli replisome and examines the cost of rNTP/dNTP nucleotide pool imbalance on the frequency of rNMP incorporation and the rate of fork progression. We find that a nucleotide pool imbalance slows the replisome two- to threefold by competing with dNTPs at the active site of the Pol III replicase at a step that does not require correct base-pairing. We determine the rNMP incorporation frequency and estimate that at intracellular concentrations of nucleotides, Pol III incorporates one rNMP every 2.3 kb, for a total of about 2,000 rNMPs per daughter chromosome. We also find that the replisome pauses 4–30-fold at a template rNMP, which could possibly promote genomic instability if rNMPs were not removed before the next round of replication.Despite detrimental aspects of the rNTP/dNTP pool imbalance, there exists a potential benefit of rNMP incorporation: It has been proposed that replicative Pols may incorporate rNMPs for particular tasks, one of which may mark the newly replicated DNA strand for mismatch repair (MMR) (2). Some bacteria (e.g., E. coli) direct MMR using a DNA methylase and MutH endonuclease to recognize and nick the newly replicated strand (1, 10, 11). However, eukaryotes and most bacteria (e.g., Bacillus subtilis) do not use methyl direction for MMR. Because rNMP incorporation is specific to the newly synthesized strand, the nick generated during its repair could be recruited to direct MMR. We investigated this hypothesis in B. subtilis, a Gram-positive bacterium that lacks a methyl-directed MMR system, and report an increase in mutagenesis after deletion of two RNase H genes. 相似文献
8.
Johnson DE Takahashi M Hamdan SM Lee SJ Richardson CC 《Proceedings of the National Academy of Sciences of the United States of America》2007,104(13):5312-5317
T7 gene 5 DNA polymerase (gp5) and its processivity factor, Escherichia coli thioredoxin, together with the T7 gene 4 DNA helicase, catalyze strand displacement synthesis on duplex DNA processively (>17,000 nucleotides per binding event). The processive DNA synthesis is resistant to the addition of a DNA trap. However, when the polymerase-thioredoxin complex actively synthesizing DNA is challenged with excess DNA polymerase-thioredoxin exchange occurs readily. The exchange can be monitored by the use of a genetically altered T7 DNA polymerase (gp5-Y526F) in which tyrosine-526 is replaced with phenylalanine. DNA synthesis catalyzed by gp5-Y526F is resistant to inhibition by chain-terminating dideoxynucleotides because gp5-Y526F is deficient in the incorporation of these analogs relative to the wild-type enzyme. The exchange also occurs during coordinated DNA synthesis in which leading- and lagging-strand synthesis occur at the same rate. On ssDNA templates with the T7 DNA polymerase alone, such exchange is not evident, suggesting that free polymerase is first recruited to the replisome by means of T7 gene 4 helicase. The ability to exchange DNA polymerases within the replisome without affecting processivity provides advantages for fidelity as well as the cycling of the polymerase from a completed Okazaki fragment to a new primer on the lagging strand. 相似文献
9.
Zhang L Kim KP Kleckner NE Storlazzi A 《Proceedings of the National Academy of Sciences of the United States of America》2011,108(50):20036-20041
Meiotic recombination initiates via programmed double-strand breaks (DSBs). We investigate whether, at a given initiation site, DSBs occur independently among the four available chromatids. For a single DSB "hot spot", the proportions of nuclei exhibiting zero, one, or two (or more) observable events were defined by tetrad analysis and compared with those predicted by different DSB distribution scenarios. Wild-type patterns are incompatible with independent distribution of DSBs among the four chromatids. In most or all nuclei, DSBs occur one-per-pair of chromatids, presumptively sisters. In many nuclei, only one DSB occurs per four chromatids, confirming the existence of trans inhibition where a DSB on one chromosome interactively inhibits DSB formation on the partner chromosome. Several mutants exhibit only a one-per-pair constraint, a phenotype we propose to imply loss of trans inhibition. Signal transduction kinases Mec1 (ATR) and Tel1 (ATM) exhibit this phenotype and thus could be mediators of this effect. Spreading trans inhibition can explain even spacing of total recombinational interactions and implies that establishment of interhomolog interactions and DSB formation are homeostatic processes. The two types of constraints on DSB formation provide two different safeguards against recombination failure during meiosis. 相似文献
10.
Evidence from in vitro replication that O6-methylguanine can adopt multiple conformations.
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M K Dosanjh E L Loechler B Singer 《Proceedings of the National Academy of Sciences of the United States of America》1993,90(9):3983-3987
The effect of O6-methylguanine (m6G) on replication, in a partially double-stranded defined 25-base oligonucleotide, has been studied under nonlimiting conditions of unmodified dNTPs and over an extended time period, using the Klenow fragment of Escherichia coli DNA polymerase I. The sequence surrounding m6G has flanking cytosines (C-m6G-C), and the initial steady-state kinetics have been reported. When the primer was annealed so that the first base to be replicated was m6G, replication was virtually complete in approximately 5 min, although the reaction appears biphasic. When annealed with a primer where thymine or cytosine is paired opposite template m6G, about half the molecules were replicated in the first 15 sec, and no significant further replication was seen over a 1-hr period. When m6G was dealkylated by DNA-O6-methylguanine-methyltransferase, replication was rapid with no blockage. These data suggest that there can be two (or more) conformations of m6G. In these studies the term syn refers to conformers interfering with base-pairing, whereas anti refers to those allowing such base-pairing. Previous physical studies by others indicate that syn- and anti-conformers of the methyl group relative to the N1 of guanine are possible. Here molecular modeling/computational studies are described, suggesting that syn- and anti-m6G can be of similar energy in DNA, and, therefore, these two conformers may explain the two types of species observed during in vitro replication. An alternative explanation could be the possibility that the different species may manifest differential interactions of m6G with Klenow fragment. These results may provide a rationale for why m6G lesions in vivo have been reported to be lethal as well as mutagenic. 相似文献
11.
Sarah L. Noton Vanessa M. Cowton Chadene R. Zack David R. McGivern Rachel Fearns 《Proceedings of the National Academy of Sciences of the United States of America》2010,107(22):10226-10231
RNA virus polymerases must initiate replicative RNA synthesis with extremely high accuracy to maintain their genome termini and to avoid generating defective genomes. For the single-stranded negative-sense RNA viruses, it is not known how this accuracy is achieved. To investigate this question, mutations were introduced into the 3′ terminal base of a respiratory syncytial virus (RSV) template, and the RNA products were examined to determine the impact of the mutation. To perform the assay, RNA replication was reconstituted using a modified minireplicon system in which replication was limited to a single step. Importantly, this system allowed analysis of RSV RNA generated intracellularly, but from a defined template that was not subject to selection by replication. Sequence analysis of RNA products generated from templates containing 1U-C and 1U-A substitutions showed that, in both cases, replication products were initiated with a nontemplated, WT A residue, rather than a templated G or U residue, indicating that the polymerase selects the terminal NTP independently of the template. Examination of a template in which the position 1 nucleotide was deleted supported these findings. This mutant directed efficient replication at ∼60% of WT levels, and its product was found to be initiated at the WT position (−1 relative to the template) with a WT A residue. These findings show that the RSV replicase selects ATP and initiates at the correct position, independently of the first nucleotide of the template, suggesting a mechanism by which highly accurate replication initiation is achieved. 相似文献
12.
Christelle Vasnier Arnaud de Muyt Liangran Zhang Sophie Tessé Nancy E. Kleckner Denise Zickler Eric Espagne 《Proceedings of the National Academy of Sciences of the United States of America》2014,111(38):E4015-E4023
Karyogamy, the process of nuclear fusion is required for two haploid gamete nuclei to form a zygote. Also, in haplobiontic organisms, karyogamy is required to produce the diploid nucleus/cell that then enters meiosis. We identify sun like protein 1 (Slp1), member of the mid–Sad1p, UNC-84–domain ubiquitous family, as essential for karyogamy in the filamentous fungus Sordaria macrospora, thus uncovering a new function for this protein family. Slp1 is required at the last step, nuclear fusion, not for earlier events including nuclear movements, recognition, and juxtaposition. Correspondingly, like other family members, Slp1 localizes to the endoplasmic reticulum and also to its extensions comprising the nuclear envelope. Remarkably, despite the absence of nuclear fusion in the slp1 null mutant, meiosis proceeds efficiently in the two haploid “twin” nuclei, by the same program and timing as in diploid nuclei with a single dramatic exception: the normal prophase program of recombination and synapsis between homologous chromosomes, including loading of recombination and synaptonemal complex proteins, occurs instead between sister chromatids. Moreover, the numbers of recombination-initiating double-strand breaks (DSBs) and ensuing recombinational interactions, including foci of the essential crossover factor Homo sapiens enhancer of invasion 10 (Hei10), occur at half the diploid level in each haploid nucleus, implying per-chromosome specification of DSB formation. Further, the distribution of Hei10 foci shows interference like in diploid meiosis. Centromere and spindle dynamics, however, still occur in the diploid mode during the two meiotic divisions. These observations imply that the prophase program senses absence of karyogamy and/or absence of a homolog partner and adjusts the interchromosomal interaction program accordingly.Karyogamy is the process by which two nuclei fuse to produce a single nucleus. This process is critical in diploid organisms (e.g., mammals and plants) when haploid egg and sperm nuclei fuse to produce a diploid nucleus and zygote. In organisms with a haploid vegetative cycle (e.g., fungi and most algae), karyogamy is required to produce the diploid nucleus/cell, which will then enter meiosis. In both situations, karyogamy involves two steps: nuclear movement leading to nuclear juxtaposition (congression) and final fusion of the nuclear membranes. Cooperation of the cytoskeleton components is required for nuclear movement and correct positioning of the nuclei (e.g., ref. 1).In budding yeast, premeiotic karyogamy requires several genes (named Kar1 to Kar9) with their mutant defects corresponding to two steps: nuclear congression, which involves cytoskeleton components, motor proteins and the spindle pole body (SPB, mammal centrosome equivalent) and fusion of the two haploid nuclear envelopes (NEs), which involves either the endoplasmic reticulum (ER) per se or protein translocation into the perinuclear space (reviewed in refs. 2–4 and references therein). Kar homologs are present in fission yeast and Candida albicans (5, 6) and two fission-yeast proteins required for proper microtubule (MT) integrity, Mal3 and Mto1, are also required for karyogamy (7). Blast search of the basidiomycete Cryptococcus neoformans genome identified four of the known KAR genes; interestingly only the kar7 mutant showed a defect in vegetative nuclear movement and hyphae mating but not in premeiotic karyogamy (8), likely reflecting the different evolution of ascomycetes and basidiomycetes.Whereas karyogamy studies of fungi have provided significant insight into the steps and molecules involved, less is known concerning the outcome of the remaining nonfused haploid nuclei. Such “twin” meiosis has been analyzed in fission yeast in three cases: in the presence of the mating-type allele h90 (9) or in absence of either the microtubule plus-end tracking protein Eb1/Mal3 (7, 10) or the type 1 membrane protein Tht1 (11), but without detailed analysis of the meiotic process per se.To further investigate these issues, we cloned and characterized the two Sad1p, UNC-84 (SUN)-domain genes of the fungus Sordaria macrospora. In most organisms investigated thus far, SUN-domain proteins are NE associated and are good candidates to play roles in karyogamy because they (i) transmit forces between the nucleus and cytoskeletal complexes; (ii) have conserved roles in meiotic chromosome movements, and (iii) in yeasts, are associated with the SPB (reviewed in ref. 12). One Sordaria gene is a member of the canonical SUN1/SUN2/MPS3/SAD1 family. Being essential for cell viability, it could not be analyzed for roles in the sexual cycle. The other gene is not essential for viability and is a member of the SLP1/OPT/SUCO/SUN3/4/5/SUNB mid–SUN-domain family (13–15). Like the other proteins of this family, Sordaria sun like protein 1 (Slp1) is localized in both the ER and NE. The precise role of those proteins remains unknown. Budding yeast Slp1 is implicated in folding of integral membrane proteins and is involved directly or indirectly in Sun1/Mps3 localization to the NE (15, 16). Dictyostellum discoideum SunB protein plays roles in both cell proliferation and differentiation (14). The mouse ortholog osteopotentia (Opt) protein is a key regulator of bone formation (17). The two splicing variants (CH1 and C1orf9) of the human SUCO gene were respectively found overexpressed in breast cancer cells or linked to the hereditary prostate cancer locus, HPC1 (18, 19).Interestingly, the Sordaria SLP1 gene is absolutely crucial for karyogamy but not for the overall meiotic program. In an slp1Δ null mutant, the two juxtaposed haploid nuclei progress synchronously and efficiently through the meiotic program, but the normal program of recombination-mediated interactions between homologous chromosomes (including synaptonemal complex formation and crossover interference) (20) now occurs regularly and efficiently between sister chromatids. These and other observations imply that the two haploid twin nuclei sense their change in status, either by detecting absence of karyogamy or by directly detecting absence of a homolog partner, and appropriately alter the program of effects to rescue a minimal level of gamete formation. 相似文献
13.
Irina Bruck Daniel L. Kaplan 《Proceedings of the National Academy of Sciences of the United States of America》2015,112(36):11223-11228
Dbf4-dependent kinase (DDK) phosphorylates minichromosome maintenance 2 (Mcm2) during S phase in yeast, and Sld3 recruits cell division cycle 45 (Cdc45) to minichromosome maintenance 2-7 (Mcm2-7). We show here DDK-phosphoryled Mcm2 preferentially interacts with Cdc45 in vivo, and that Sld3 stimulates DDK phosphorylation of Mcm2 by 11-fold. We identified a mutation of the replication initiation factor Sld3, Sld3-m16, that is specifically defective in stimulating DDK phosphorylation of Mcm2. Wild-type expression levels of sld3-m16 result in severe growth and DNA replication defects. Cells expressing sld3-m16 exhibit no detectable Mcm2 phosphorylation in vivo, reduced replication protein A-ChIP signal at an origin, and diminished Go, Ichi, Ni, and San association with Mcm2-7. Treslin, the human homolog of Sld3, stimulates human DDK phosphorylation of human Mcm2 by 15-fold. DDK phosphorylation of human Mcm2 decreases the affinity of Mcm5 for Mcm2, suggesting a potential mechanism for helicase ring opening. These data suggest a conserved mechanism for replication initiation: Sld3/Treslin coordinates Cdc45 recruitment to Mcm2-7 with DDK phosphorylation of Mcm2 during S phase.The replication fork helicase in eukaryotes is composed of Cdc45, the Mcm2-7 heterohexameric ATPase, and the tetrameric GINS (Go, Ichi, Ni, and San) complex (CMG assembly) (1). The replication fork helicase (CMG) assembles in S phase in a manner that is dependent upon the replication initiation factors Sld2, Sld3, and Dpb11 (2). Sld3 (Treslin/TICRR in humans), Sld2 (RecQL4/RecQ4 in humans), and Dpb11 (TopBP1 in humans) are required for the initiation of DNA replication, but these proteins do not travel with the replication fork (3). The S-phase-specific kinases, cyclin-dependent kinase (CDK) and the Dbf4-dependent kinase (DDK), are also required for CMG assembly and origin activation (4, 5). In late M and G1 phases, the Mcm2-7 complex loads to encircle dsDNA as a double hexamer (6, 7). During S phase, a single strand of DNA is extruded from the central channel of Mcm2-7, and this event is required because the CMG complex unwinds DNA by a steric exclusion mechanism (8).Central to the initiation of DNA replication is the coordination of entry into S phase with origin firing (4, 5). Levels of the S phase-specific kinases, S-CDK and DDK, rise during the onset of S phase, and these two kinases are central to coordinating S-phase entry with origin firing (4, 5). S-CDK phosphorylates Sld2 and Sld3, and these phosphorylation events are the essential functions of S-CDK (9, 10). S-CDK phosphorylation of Sld3 is conserved in human Treslin (11). S-CDK phosphorylation of Sld2 promotes the association of Sld2 with yeast Dpb11 (12), and also the association of Sld2 with T-rich ssDNA (13). S-CDK phosphorylation of Sld3 stimulates the association of Sld3 with Dpb11 (9, 10). The associations of Sld2 with Dpb11 and Sld3 with Dpb11 have been proposed to be important for the recruitment of GINS to origins, through the generation of a preloading complex (Pre-LC), composed of Sld2, GINS, Polε, and Dpb11 (14). S-CDK–catalyzed formation of an Sld3-Dpb11-Sld2 complex has also been proposed to be important to generate a ternary ssDNA-binding complex of high affinity, because Sld2, Sld3, and Dpb11 bind to T-rich ssDNA (13, 15, 16).The essential role of DDK in yeast cells is the phosphorylation of subunits of the Mcm2-7 complex (17). DDK phosphorylation of Mcm4 is important for cell growth, and this phosphorylation event alleviates an inhibitory function of the N terminus of Mcm4 (18). DDK phosphorylation of Mcm4 may also promote the interaction between Cdc45 and Mcm2-7 (18). DDK phosphorylation of Mcm6 may also be important for cell growth (19). Mcm2 is also a target for DDK (20), and DDK phosphorylation of Mcm2 is also required for DNA replication under normal growth conditions (21). Furthermore, expression of a mutant of mcm2 (mcm2-S164A,S170A) that is not phosphorylated by DDK exerts a dominant-negative severe growth defect in budding yeast that is bypassed by the mcm5-bob1 (mcm5-P83L) mutation (21). The biochemical mechanism of this genetic suppression has also been examined. DDK phosphorylation of Mcm2 reduces the affinity of budding yeast Mcm2 for Mcm5, and the mcm5-bob1 mutation also reduces this affinity (21). This reduced affinity may help open the “Mcm2-Mcm5 gate,” which may be important for the extrusion of ssDNA from the central channel of Mcm2-7 during S phase, a requirement for origin activation (22).Cdc45 binds weakly to Mcm2-7 in the absence of accessory factors (23). Sld3 binds tightly to Mcm2-7 and Cdc45, and thus Sld3 recruits Cdc45 to Mcm2-7 complexes (2, 23). This step may further require DDK and involve the nonessential initiation factor Sld7 (24). During origin activation, Sld3 is removed from Mcm2-7, presumably through the exposure of sequestering T-rich ssDNA (16). GINS can substitute for Sld3 as a factor that promotes the association of Cdc45 with Mcm2-7, thereby forming the stable Cdc45-Mcm2-7-GINS (CMG) replicative helicase complex (23, 25).The mechanism of GINS recruitment may involve the formation of the S-CDK–dependent preloading complex, wherein the pre-LC recruits GINS to Mcm2-7, analogous to how Sld3 recruits Cdc45 to Mcm2-7 (14). A second proposal posits that Sld3, Sld2, and Dpb11 compete with GINS for binding to Mcm2-7 before origin activation, blocking the premature interaction between GINS and Mcm2-7 before origin activation (15, 16, 26). However, when T-rich ssDNA is extruded from the central channel of Mcm2-7, an ssDNA binding surface for Sld3-Sld2-Dpb11 is generated (15, 16, 26). Sld3-Sld2-Dpb11 dissociates from Mcm2-7 once the origin is melted, because Sld3-Sld2-Dpb11 has a higher affinity for ssDNA then Mcm2-7 (15, 16, 26). The dissociation of Sld3-Sld2-Dpb11 from Mcm2-7 allows GINS to bind Mcm2-7 by a passive, sequestration mechanism (15, 16, 26). The two models are not incompatible with one another, and they may both be correct.There is an excess of Mcm2-7 double hexamer complexes loaded onto dsDNA in M phase and G1 relative to the number of Mcm2-7 double hexamer complexes that actually fire. Remarkably, the activated Mcm2-7 complexes share several features in common: DDK phosphorylation of Mcm2-7, initiation factor (Sld3, Sld2, Dpb11, Mcm10, and Sld7) binding to Mcm2-7, and Cdc45/GINS attachment to Mcm2-7. What coordinates these different activities at a particular Mcm2-7 double hexamer? In other words, what prevents Cdc45 from binding to one Mcm2-7 double hexamer while DDK phosphorylates a different Mcm2-7 double hexamer? We sought to address this fundamental question in this paper.We show that DDK-phosphorylated Mcm2 preferentially interacts with Cdc45 in vivo compared with Mcm2 (all Mcm2 in cell, phosphorylated and unphosphorylated), suggesting that Cdc45 recruitment to Mcm2-7 is correlated with DDK phosphorylation of Mcm2. We also show that Sld3 substantially stimulates DDK phosphorylation of Mcm2 in vitro. We identified a mutant of Sld3, sld3-m16, that is defective in the stimulation of DDK phosphorylation of Mcm2. When sld3-m16 is expressed in budding yeast cells, a dominant negative severe growth defect is observed that is bypassed by mcm5-bob1. Wild-type expression levels of sld3-m16 confer a growth and DNA replication defect, with decreased DDK phosphorylation of Mcm2. Furthermore, expression of sld3-m16 results in diminished replication protein A (RPA)-ChIP signal at an origin, and decreased GINS interaction with Mcm2-7. Furthermore, human Treslin substantially stimulates DDK phosphorylation of Mcm2 at serines 53 and 108. Finally, a mutant of human Mcm2 that mimics DDK-phosphorylated Mcm2 exhibits diminished interaction with human Mcm5, suggesting a mechanism for “gate” opening. We conclude that Sld3 coordinates helicase phosphorylation with helicase assembly. 相似文献
14.
DNA intermediates at the Escherichia coli replication fork: effect of dUTP. 总被引:4,自引:0,他引:4
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B M Olivera 《Proceedings of the National Academy of Sciences of the United States of America》1978,75(1):238-242
We have directly tested the hypothesis that elevated levels of dUTP cause the formation of small DNA fragments at the replication fork of Escherichia coli. Addition of increasing levels of dUTP to lysates on cellophane discs results in an increasing number of strand scissions in the newly replicated DNA. Lysates of strains defective in dUTPase produce many more scissions at the same level of dUTP. The size distribution of Okazaki pieces obtained in vivo can be reconstituted in vitro on cellophane discs if appropriate levels of dUTP are present. Although uracil excision leads to the apparent production of Okazaki pieces from both daughter strands, DNA synthesis is actually asymmetric under these conditions. De novo chain initiation events occur on only one strand. It is suggested that asymmetry of synthesis in vivo may be masked by uracil excision and other postreplication processing mechanisms. 相似文献
15.
Evidence that a single DNA ligase is involved in replication and recombination in yeast. 总被引:7,自引:0,他引:7
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F Fabre H Roman 《Proceedings of the National Academy of Sciences of the United States of America》1979,76(9):4586-4588
The possible existence in yeast of different nuclear DNA ligase enzymes led us to ask whether induced recombination (gene conversion) involves the same ligase as that involved in DNA replication. The conditional cdc9 mutant is known to be defective, under restrictive conditions, in the rejoining of Okazaki fragments. We show here that under the same conditions, x-ray-induced convertants within the cdc9 locus are produced with kinetics indicating that most, if not all, of the conversion events require the participation of the cdc9-controlled ligase. Thus, the same DNA ligase is involved in DNA replication and in induced gene conversion. 相似文献
16.
Segregation of DNA polynucleotide strands into sister chromatids and the use of endoreduplicated cells to track sister chromatid exchanges induced by crosslinks, alkylations, or x-ray damage. 总被引:2,自引:0,他引:2
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S Wolff V Afzal 《Proceedings of the National Academy of Sciences of the United States of America》1996,93(12):5765-5769
The method of Matsumoto and Ohta [Matsumoto, K. & Ohta, T. (1992) Chromosoma 102, 60-65; Matsumoto, K. & Ohta, T. (1995) Mutat. Res. 326, 93-98] to induce large numbers of endoreduplicated Chinese hamster ovary cells has now been coupled with the fluorescence-plus-Giemsa method of Perry and Wolff [Perry, P. & Wolff, S. (1974) Nature (London) 251, 156-158] to produce harlequin endoreduplicated chromosomes that after the third round of DNA replication are composed of a chromosome with a light chromatid and a dark chromatid in close apposition to its sister chromosome containing two light chromatids. Unless the pattern is disrupted by sister chromatid exchange (SCE), the dark chromatid is always in the center, so that the order of the chromatids is light-dark light-light. The advent of this method, which permits the observation of SCEs in endoreduplicated cells, makes it possible to determine with great ease in which cell cycle an SCE occurred. This now allows us to approach several vexing questions about the induction of SCEs (genetic damage and its repair) after exposure to various types of mutagenic carcinogens. The present experiments have allowed us to observe how many cell cycles various types of lesions that are induced in DNA by a crosslinking agent, an alkylating agent, or ionizing radiation, and that are responsible for the induction of SCEs, persist before being repaired and thus lose their ability to inflict genetic damage. Other experiments with various types of mutagenic carcinogens and various types of cell lines that have defects in different DNA repair processes, such as mismatch repair, excision repair, crosslink repair, and DNA-strand-break repair, can now be carried out to determine the role of these types of repair in removing specific types of lesions. 相似文献
17.
Analysis of topoisomerase function in bacterial replication fork movement: use of DNA microarrays 总被引:22,自引:0,他引:22
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Khodursky AB Peter BJ Schmid MB DeRisi J Botstein D Brown PO Cozzarelli NR 《Proceedings of the National Academy of Sciences of the United States of America》2000,97(17):9419-9424
We used DNA microarrays of the Escherichia coli genome to trace the progression of chromosomal replication forks in synchronized cells. We found that both DNA gyrase and topoisomerase IV (topo IV) promote replication fork progression. When both enzymes were inhibited, the replication fork stopped rapidly. The elongation rate with topo IV alone was 1/3 of normal. Genetic data confirmed and extended these results. Inactivation of gyrase alone caused a slow stop of replication. Topo IV activity was sufficient to prevent accumulation of (+) supercoils in plasmid DNA in vivo, suggesting that topo IV can promote replication by removing (+) supercoils in front of the chromosomal fork. 相似文献
18.
Zhang Y Gaikwad NW Olson K Zahid M Cavalieri EL Rogan EG 《Metabolism: clinical and experimental》2007,56(7):887-894
Accumulating evidence suggests that specific metabolites of estrogens, namely, catechol estrogen quinones, react with DNA to form adducts and generate apurinic sites, which can lead to the mutations that induce breast cancer. Oxidation of estradiol (E(2)) produces 2 catechol estrogens, 4-hydroxyestradiol (4-OHE(2)) and 2-OHE(2) among the major metabolites. These, in turn, are oxidized to the quinones, E(2)-3,4-quinone (E(2)-3,4-Q) and E(2)-2,3-Q, which can react with DNA. Oxidation of E(2) to 2-OHE(2) is mainly catalyzed by cytochrome P450 (CYP) 1A1, and CYP3A4, whereas oxidation of E(2) to 4-OHE(2) in extrahepatic tissues is mainly catalyzed by CYP1B1 as well as some CYP3As. The potential involvement of CYP isoforms in the further oxidation of catechols to semiquinones and quinones has, however, not been investigated in detail. In this project, to identify the potential function of various CYPs in oxidizing catechol estrogens to quinones, we used different recombinant human CYP isoforms, namely, CYP1A1, CYP1B1, and CYP3A4, with the scope of oxidizing the catechol estrogens 2-OHE(2) and 4-OHE(2) to their respective estrogen quinones, which then reacted with DNA. The depurinating adducts 2-OHE(2)-6-N3Ade, 4-OHE(2)-1-N3Ade, and 4-OHE(2)-1-N7Gua were observed in the respective reaction systems by ultraperformance liquid chromatography/tandem mass spectrometry. Furthermore, more than 100-fold higher levels of estrogen-glutathione (GSH) conjugates were detected in the reactions. Glutathione conjugates were observed, in much smaller amounts, when control microsomes were used. Depurinating adducts, as well as GSH conjugates, were obtained when E(2)-3,4-Q was incubated with CYP1B1 or control microsomes in a 30-minute reaction, further demonstrating that GSH is present in these recombinant enzyme preparations. These experiments demonstrated that CYP1A1, CYP1B1, and CYP3A4 are able to oxidize catechol estrogens to their respective quinones, which can further react with GSH, protein, and DNA, the last resulting in depurinating adducts that can lead to mutagenesis. 相似文献
19.
20.
Evidence that transgenes encoding components of the Wnt signaling pathway preferentially induce mammary cancers from progenitor cells 总被引:40,自引:0,他引:40
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Li Y Welm B Podsypanina K Huang S Chamorro M Zhang X Rowlands T Egeblad M Cowin P Werb Z Tan LK Rosen JM Varmus HE 《Proceedings of the National Academy of Sciences of the United States of America》2003,100(26):15853-15858
Breast cancer is a genetically and clinically heterogeneous disease, and the contributions of different target cells and different oncogenic mutations to this heterogeneity are not well understood. Here we report that mammary tumors induced by components of the Wnt signaling pathway contain heterogeneous cell types and express early developmental markers, in contrast to tumors induced by other signaling elements. Expression of the Wnt-1 protooncogene in mammary glands of transgenic mice expands a population of epithelial cells expressing progenitor cell markers, keratin 6 and Sca-1; subsequent tumors express these markers and contain luminal epithelial and myoepithelial tumor cells that share a secondary mutation, loss of Pten, implying that they arose from a common progenitor. Mammary tumors arising in transgenic mice expressing beta-catenin and c-Myc, downstream components of the canonical Wnt signaling pathway, also contain a significant proportion of myoepithelial cells and cells expressing keratin 6. Progenitor cell markers and myoepithelial cells, however, are lacking in mammary tumors from transgenic mice expressing Neu, H-Ras, or polyoma middle T antigen. These results suggest that mammary stem cells and/or progenitors to mammary luminal epithelial and myoepithelial cells may be the targets for oncogenesis by Wnt-1 signaling elements. Thus, the developmental heterogeneity of different breast cancers is in part a consequence of differential effects of oncogenes on distinct cell types in the breast. 相似文献