DNA slip-outs cause RNA polymerase II arrest in vitro: potential implications for genetic instability

The abnormal number of repeats found in triplet repeat diseases arises from ‘repeat instability’, in which the repetitive section of DNA is subject to a change in copy number. Recent studies implicate transcription in a mechanism for repeat instability proposed to involve RNA polymerase II (RNAPII) arrest caused by a CTG slip-out, triggering transcription-coupled repair (TCR), futile cycles of which may lead to repeat expansion or contraction. In the present study, we use defined DNA constructs to directly test whether the structures formed by CAG and CTG repeat slip-outs can cause transcription arrest in vitro. We found that a slip-out of (CAG)20 or (CTG)20 repeats on either strand causes RNAPII arrest in HeLa cell nuclear extracts. Perfect hairpins and loops on either strand also cause RNAPII arrest. These findings are consistent with a transcription-induced repeat instability model in which transcription arrest in mammalian cells may initiate a ‘gratuitous’ TCR event leading to a change in repeat copy number. An understanding of the underlying mechanism of repeat instability could lead to intervention to slow down expansion and delay the onset of many neurodegenerative diseases in which triplet repeat expansion is implicated

8-Oxo-7,8-dihydroguanine in DNA does not constitute a barrier to transcription, but is converted into transcription-blocking damage by OGG1

The common DNA base modification 8-oxo-7,8-dihydroguanine (8-oxo-G) affects the efficiency and fidelity of transcription. We constructed plasmid substrates carrying single 8-oxo-G residues, specifically positioned in the transcribed or the non-transcribed DNA strands, to investigate their effects on the expression of an EGFP reporter gene and to explore the role of base excision repair in the mechanism of transcription inhibition. We report that 8-oxo-G does not directly block transcription in cells, since a single 8-oxo-G in the transcribed DNA strand did not reduce the EGFP expression levels in repair-deficient (OGG1-null) mouse embryonic fibroblast cell lines. Rather, inhibition of transcription by 8-oxo-G fully depends on 8-oxoguanine DNA glycosylase (OGG1) and, at the same time, does not require the localization of the lesion in the transcribed DNA strand. We propose that the interruption of transcription is induced by base excision repair intermediates and, therefore, could be a common consequence of various DNA base modifications. Concordantly, the non-blocking DNA modification uracil was also found to inhibit transcription, but in an OGG1-independent manner

A novel method for the genome-wide high resolution analysis of DNA damage

DNA damage occurs via endogenous and exogenous genotoxic agents and compromises a genome’s integrity. Knowing where damage occurs within a genome is crucial to understanding the repair mechanisms which protect this integrity. This paper describes a new development based on microarray technology which uses ultraviolet light induced DNA damage as a paradigm to determine the position and frequency of DNA damage and its subsequent repair throughout the entire yeast genome

Thin-film optoacoustic transducers for the subcellular Brillouin oscillation imaging of individual biological cells

Mechanical characterisation and imaging of biological tissue has piqued interest in the applicability to cell and tissue biology. One method, based on detection of Brillouin oscillations, has already lead to demonstrations on biological cells using ultrasound in the GHz range. In this paper we present a technique to extend this picosecond laser ultrasound technique from point measurements and line scans into high resolution acoustic imaging. Our technique uses a three layered metal-dielectric-metal film under the cell as a transducer for the generation of ultrasound. The design of this transducer and measuring system is optimised to address a limiting SNR factor related to the cell fragility; its sensitivity to laser light. Our approach shields the cell from laser radiation while having acoustic generation, optical detection and aiding heat dissipation. For that, Brillouin detection is performed in transmission rather than reflection. The conditions necessary to perform successfully this kind of detection are discussed and experimental results on phantom, fixed and living cells are presented

Controlling the motor activity of a transcription-repair coupling factor: autoinhibition and the role of RNA polymerase

Motor proteins that couple ATP hydrolysis to movement along nucleic acids play a variety of essential roles in DNA metabolism. Often these enzymes function as components of macromolecular complexes, and DNA translocation by the motor protein drives movement of other components of the complex. In order to understand how the activity of motor proteins is regulated within multi-protein complexes we have studied the bacterial transcription-repair coupling factor, Mfd, which is a helicase superfamily 2 member that binds to RNA polymerase (RNAP) and removes stalled transcription complexes from DNA. Using an oligonucleotide displacement assay that monitors protein movement on double-stranded DNA we show that Mfd has little motor activity in isolation, but exhibits efficient oligonucleotide displacement activity when bound to a stalled transcription complex. Deletion of the C-terminal domain of Mfd increases the ATPase activity of the protein and allows efficient oligo-displacement in the absence of RNAP. Our results suggest that an autoinhibitory domain ensures the motor activity of Mfd is only functional within the correct macromolecular context: recruitment of Mfd to a stalled transcription complex relieves the autoinhibition and unmasks the motor activity

Exploiting CpG Hypermutability to Identify Phenotypically Significant Variation Within Human Protein-Coding Genes

The CpG dinucleotide is disproportionately represented in human genetic variation due to the hypermutability of 5-methyl-cytosine (5mC). We exploit this hypermutability and a novel codon substitution model to identify candidate functionally important exonic nucleotides. Population genetic theory suggests that codon positions with high cross-species CpG frequency will derive from stronger purifying selection. Using the phylogeny-based maximum likelihood inference framework, we applied codon substitution models with context-dependent parameters to measure the mutagenic and selective processes affecting CpG dinucleotides within exonic sequence. The suitability of these models was validated on >2,000 protein coding genes from a naturally occurring biological control, four yeast species that do not methylate their DNA. As expected, our analyses of yeast revealed no evidence for an elevated CpG transition rate or for substitution suppression affecting CpG-containing codons. Our analyses of >12,000 protein-coding genes from four primate lineages confirm the systemic influence of 5mC hypermutability on the divergence of these genes. After adjusting for confounding influences of mutation and the properties of the encoded amino acids, we confirmed that CpG-containing codons are under greater purifying selection in primates. Genes with significant evidence of enhanced suppression of nonsynonymous CpG changes were also shown to be significantly enriched in Online Mendelian Inheritance in Man. We developed a method for ranking candidate phenotypically influential CpG positions in human genes. Application of this method indicates that of the ∼1 million exonic CpG dinucleotides within humans, ∼20% are strong candidates for both hypermutability and disease association

Formation and genotoxicity of a guanine–cytosine intrastrand cross-link lesion in vivo

Reactive oxygen species (ROS) can be induced by both endogenous and exogenous processes, and they can damage biological molecules including nucleic acids. Exposure of isolated DNA to X/γ-rays and Fenton reagents was shown to lead to the formation of intrastrand cross-link lesions where the neighboring nucleobases in the same DNA strand are covalently bonded. By employing HPLC coupled with tandem mass spectrometry (LC-MS/MS) with the isotope dilution method, we assessed quantitatively the formation of a guanine–cytosine (G[8-5]C) intrastrand cross-link lesion in HeLa-S3 cells upon exposure to γ-rays. The yield of the G[8-5]C cross-link was 0.037 lesions per 109 nucleosides per Gy, which was ∼300 times lower than that of 5-formyl-2′-deoxyuridine (0.011 lesions per 106 nucleosides per Gy) under identical exposure conditions. We further constructed a single-stranded M13 genome harboring a site-specifically incorporated G[8-5]C lesion and developed a novel mass spectrometry-based method for interrogating the products emanating from the replication of the genome in Escherichia coli cells. The results demonstrated that G[8-5]C blocked considerably DNA replication as represented by a 20% bypass efficiency, and the lesion was significantly mutagenic in vivo, which included a 8.7% G→T and a 1.2% G→C transversion mutations. DNA replication in E. coli hosts deficient in SOS-induced polymerases revealed that polymerase V was responsible for the error-prone translesion synthesis in vivo

Dissociation of CAK from Core TFIIH Reveals a Functional Link between XP-G/CS and the TFIIH Disassembly State

Transcription factor II H (TFIIH) is comprised of core TFIIH and Cdk-activating kinase (CAK) complexes. Here, we investigated the molecular and cellular manifestation of the TFIIH compositional changes by XPG truncation mutations. We showed that both core TFIIH and CAK are rapidly recruited to damage sites in repair-proficient cells. Chromatin immunoprecipitation against TFIIH and CAK components revealed a physical engagement of CAK in nucleotide excision repair (NER). While XPD recruitment to DNA damage was normal, CAK was not recruited in severe XP-G and XP-G/CS cells, indicating that the associations of CAK and XPD to core TFIIH are differentially affected. A CAK inhibition approach showed that CAK activity is not required for assembling pre-incision machinery in vivo or for removing genomic photolesions. Instead, CAK is involved in Ser5-phosphorylation and UV-induced degradation of RNA polymerase II. The CAK inhibition impaired transcription from undamaged and UV-damaged reporter, and partially decreased transcription of p53-dependent genes. The overall results demonstrated that a) XP-G/CS mutations affect the disassembly state of TFIIH resulting in the dissociation of CAK, but not XPD from core TFIIH, and b) CAK activity is not essential for global genomic repair but involved in general transcription and damage-induced RNA polymerase II degradation