139,039 research outputs found
Characteristics of oligonucleotide frequencies across genomes: Conservation versus variation, strand symmetry, and evolutionary implications
No full textOne of the objectives of evolutionary genomics is to reveal the genetic information contained in the primordial genome (called the primary genetic information in this paper, with the primordial genome defined here as the most primitive nucleic acid genome for earth’s life) by searching for primitive traits or relics remained in modern genomes. As the shorter a sequence is, the less probable it would be modified during genome evolution. For that reason, some characteristics of very short nucleotide sequences would have considerable chances to persist during billions of years of evolution. Consequently, conservation of certain genomic features of mononucleotides, dinucleotides, and higher-order oligonucleotides across various genomes may exist; some, if not all, of these features would be relics of the primary genetic information. Based on this assumption, we analyzed the pattern of frequencies of mononucleotides, dinucleotides, and higher-order oligonucleotides of the whole-genome sequences from 458 species (including archaea, bacteria, and eukaryotes). Also, we studied the phenomenon of strand symmetry in these genomes. The results show that the conservation of frequencies of some dinucleotides and higher-order oligonucleotides across genomes does exist, and that strand symmetry is a ubiquitous and explicit phenomenon that may contribute to frequency conservation. We propose a new hypothesis for the origin of strand symmetry and frequency conservation as well as for the constitution of early genomes. We conclude that the phenomena of strand symmetry and the pattern of frequency conservation would be original features of the primary genetic information
Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis
No full textIn all organisms, the protein machinery responsible for the replication of DNA, the replisome, is faced with a directionality problem. The antiparallel nature of duplex DNA permits the leading-strand polymerase to advance in a continuous fashion, but forces the lagging-strand polymerase to synthesize in the opposite direction. By extending RNA primers, the lagging-strand polymerase restarts at short intervals and produces Okazaki fragments. At least in prokaryotic systems, this directionality problem is solved by the formation of a loop in the lagging strand of the replication fork to reorient the lagging-strand DNA polymerase so that it advances in parallel with the leading-strand polymerase. The replication loop grows and shrinks during each cycle of Okazaki fragment synthesis. Here we use single-molecule techniques to visualize, in real time, the formation and release of replication loops by individual replisomes of bacteriophage T7 supporting coordinated DNA replication. Analysis of the distributions of loop sizes and lag times between loops reveals that initiation of primer synthesis and the completion of an Okazaki fragment each serve as a trigger for loop release. The presence of two triggers may represent a fail-safe mechanism ensuring the timely reset of the replisome after the synthesis of every Okazaki fragment.
Application of Pulsed Field Gel Electrophoresis to Determine γ-ray-induced Double-strand Breaks in Yeast Chromosomal Molecules
No full textThe frequency of DNA double-strand breaks (dsb) was determined in yeast cells exposed to γ-rays under anoxic conditions. Genomic DNA of treated cells was separated by pulsed field gel electrophoresis, and two different approaches for the evaluation of the gels were employed: (1) The DNA mass distribution profile obtained by electrophoresis was compared to computed profiles, and the number of DSB per unit length was then derived in terms of a fitting procedure; (2) hybridization of selected chromosomes was performed, and a comparison of the hybridization signals in treated and untreated samples was then used to derive the frequency of dsb
hSSB1 interacts directly with the MRN complex stimulating its recruitment to DNA double-strand breaks and its endo-nuclease activity
No full texthSSB1 is a recently discovered single-stranded DNA binding protein that is essential for efficient repair of DNA double-strand breaks (DSBs) by the homologous recombination pathway. hSSB1 is required for the efficient recruitment of the MRN complex to sites of DSBs and for the efficient initiation of ATM dependent signalling. Here we explore the interplay between hSSB1 and MRN. We demonstrate that hSSB1 binds directly to NBS1, a component of the MRN complex, in a DNA damage independent manner. Consistent with the direct interaction, we observe that hSSB1 greatly stimulates the endo-nuclease activity of the MRN complex, a process that requires the C-terminal tail of hSSB1. Interestingly, analysis of two point mutations in NBS1, associated with Nijmegen breakage syndrome, revealed weaker binding to hSSB1, suggesting a possible disease mechanism.Peer reviewe
Targeting the DNA double strand break repair machinery in prostate cancer
No full textRegardless of the achievable remissions with first line hormone therapy in patients with prostate cancer (CaP), the disease escapes the hormone dependent stage to a more aggressive status where chemotherapy is the only effective treatment and no treatment is curative. This makes it very important to identify new targets that can improve the outcome of treatment. ATM and DNA-PK are the two kinases responsible for signalling and repairing double strand breaks (DSB). Thus, both kinases are pertinent targets in CaP treatment to enhance the activity of the numerous DNA DSB inducing agents used in CaP treatment such as ionizing radiation (IR). Colony formation assay was used to assess the sensitivity of hormone dependent, p53 wt (LNCaP) and hormone independent p53 mutant (PC3) CaP cell lines to the cytotoxic effect of IR and Doxorubicin in the presence or absence of Ku55933 and NU7441 which are small molecule inhibitors of ATM and DNA-PK, respectively. Flow cytometry based methods were used to assess the effect of the two inhibitors on cell cycle, apoptosis and H2AX foci formation. Neutral comet assay was used to assess the induction of DNA DSBs. Ku55933 or NU7441 alone increased the sensitivity of CaP cell lines to the DNA damaging agents, however combining both inhibitors together resulted in further enhancement of sensitivity. The cell cycle profile of both cell lines was altered with increased cell death, DNA DSBs and H2AX foci formation. This study justifies further evaluation of the ATM and DNA-PK inhibitors for clinical application in CaP patients. Additionally, the augmented effect resulting from combining both inhibitors may have a significant implication for the treatment of CaP patients who have a defect in one of the two DSB repair pathway
Expression of catalytic mutants of the mtDNA helicase Twinkle and polymerase POLG causes distinct replication stalling phenotypes.
No full textThe mechanism of mitochondrial DNA replication is a subject of intense debate. One model proposes a strand-asynchronous replication in which both strands of the circular genome are replicated semi-independently while the other model proposes both a bidirectional coupled leading- and lagging-strand synthesis mode and a unidirectional mode in which the lagging-strand is initially laid-down as RNA by an unknown mechanism (RITOLS mode). Both the strand-asynchronous and RITOLS model have in common a delayed synthesis of the DNA-lagging strand. Mitochondrial DNA is replicated by a limited set of proteins including DNA polymerase gamma (POLG) and the helicase Twinkle. Here, we report the effects of expression of various catalytically deficient mutants of POLG1 and Twinkle in human cell culture. Both groups of mutants reduced mitochondrial DNA copy number by severe replication stalling. However, the analysis showed that while induction of POLG1 mutants still displayed delayed lagging-strand synthesis, Twinkle-induced stalling resulted in maturated, essentially fully double-stranded DNA intermediates. In the latter case, limited inhibition of POLG with dideoxycytidine restored the delay between leading- and lagging-strand synthesis. The observed cause-effect relationship suggests that Twinkle-induced stalling increases lagging-strand initiation events and/or maturation mimicking conventional strand-coupled replication
Sex differences in Cognitive Abilities Test scores: a UK national picture
No full textBackground and aims. There is uncertainty about the extent or even existence of sex differences in the mean and variability of reasoning test scores ( Jensen, 1998; Lynn, 1994, ; Mackintosh, 1996). This paper analyses the Cognitive Abilities Test (CAT) scores of a large and representative sample of UK pupils to determine the extent of any sex differences.
Sample. A nationally representative UK sample of over 320,000 school pupils aged 11-12 years was assessed on the CAT (third edition) between September 2001 and August 2003. The CAT includes separate nationally standardized tests for verbal, quantitative, and non-verbal reasoning. The size and recency of the sample is unprecedented in research on this issue.
Methods. The sheer size of the sample ensures that any sex difference will achieve statistical significance. Therefore, effect sizes (d) and variance ratios (VR) are employed to evaluate the magnitude of sex differences in mean scores and in score variability, respectively.
Results. The mean verbal reasoning score for girls was 2.2 standard score points higher than the mean for boys, but only 0.3 standard points in favour of girls for non-verbal reasoning (NVR), and 0.7 points in favour of boys for quantitative reasoning (QR). However, for all three tests there were substantial sex differences in the standard deviation of scores, with greater variance among boys. Boys were over represented relative to girls at both the top and the bottom extremes for all tests, with the exception of the top 10% in verbal reasoning.
Conclusions. Given the small differences in means, explanations for sex differences in wider domains such examination attainment at age 16 need to look beyond conceptions of `ability'. Boys tend to be both the lowest and the highest performers in terms of their reasoning abilities, which warns against the danger of stereotyping boys as low achievers
NF-κB regulates DNA double-strand break repair in conjunction with BRCA1-CtIP complexes
No full textNF-κB is involved in immune responses, inflammation, oncogenesis, cell proliferation and apoptosis. Even though NF-κB can be activated by DNA damage via Ataxia telangiectasia-mutated (ATM) signalling, little was known about an involvement in DNA repair. In this work, we dissected distinct DNA double-strand break (DSB) repair mechanisms revealing a stimulatory role of NF-κB in homologous recombination (HR). This effect was independent of chromatin context, cell cycle distribution or cross-talk with p53. It was not mediated by the transcriptional NF-κB targets Bcl2, BAX or Ku70, known for their dual roles in apoptosis and DSB repair. A contribution by Bcl-xL was abrogated when caspases were inhibited. Notably, HR induction by NF-κB required the targets ATM and BRCA2. Additionally, we provide evidence that NF-κB interacts with CtIP-BRCA1 complexes and promotes BRCA1 stabilization, and thereby contributes to HR induction. Immunofluorescence analysis revealed accelerated formation of replication protein A (RPA) and Rad51 foci upon NF-κB activation indicating HR stimulation through DSB resection by the interacting CtIP-BRCA1 complex and Rad51 filament formation. Taken together, these results define multiple NF-κB-dependent mechanisms regulating HR induction, and thereby providing a novel intriguing explanation for both NF-κB-mediated resistance to chemo- and radiotherapies as well as for the sensitization by pharmaceutical intervention of NF-κB activatio
Use of a unique reporter cassette to examine 5´ to 3´ resection at a site specific DNA double-strand break during meiosis
No full textMeiotic recombination in Saccharomyces cerevisiae is initiated by the formation of DNA double strand breaks (DSBs), which are created by Spoil protein. Recombination preferentially occurs between homologous chromosomes, in order to establish interhomologue connections. These connections serve as a platform for genetic recombination and to promote accurate homologue disjunction at the first meiotic division (MI). Specific mechanisms are in place to ensure that meiotic DSB repair is directed towards interchromosomal repair, and genes thought to be involved in these mechanisms were examined in a DSB assay, where interchromosomal repair was precluded. Genes involved in the formation and processing of Spol1-DSBs were also examined. In meiosis, the regulation of resectioning is critical to repair outcome, and this assay was designed to measure two different lengths of resection tract. In a mek1 mutant, there was an increase in the generation of longer resection tracts, suggesting that Mek1 protein may exert its influence over repair template choice by negatively regulating DSB resectioning. An sae2 mutant was found to generate fewer shorter resection tracts, and was delayed for DSB repair. This suggested that Sae2 protein may have an early role in resectioning, by influencing repair template choice. Mutants of the MRX complex were all compromised for DSB repair, while an exol mutant failed to generate long resection tracts only. Finally, from work on a dmcl mutant, the prospect of protein sequestration at sites of excess single stranded DNA was proposed
Site-specific DNA double-strand breaks greatly increase stable transformation efficiency in Trypanosoma brucei.
No full textGenetic manipulation in African trypanosomes typically relies upon electroporation with chromosomal integration of DNA constructs by homologous recombination. Relatively little is known about chromosomal recombination and repair in these organisms however and low transformation efficiency and position effects can limit forward genetic approaches. In yeast and mammalian cells, site-specific DNA double-strand breaks (DSBs) stimulate targeted integration through homologous recombination-based repair where the exogenous DNA serves as the template. We have explored the effect of DSBs on targeted integration in bloodstream-form Trypanosoma brucei, focusing on the ribosomal RNA-spacer target commonly used to integrate recombinant constructs. DSB-repair within the ribosomal RNA tandem gene-repeats is likely dominated by single-strand annealing allowing approximately 80% of cells to survive the break. In the presence of exogenous DNA, transformation efficiency is increased approximately 250-fold by DSB-induction. In the example presented, more than 1% of cells that survive the procedure were transformed generating 80,000 transformants from a typical experiment
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