Base Damage within Single-Strand DNA Underlies In Vivo Hypermutability Induced by a Ubiquitous Environmental Agent

Duke University, United States of America
PLoS Genetics (Impact Factor: 7.53). 12/2012; 8(12):e1003149. DOI: 10.1371/journal.pgen.1003149
Source: PubMed


Chromosomal DNA must be in single-strand form for important transactions such as replication, transcription, and recombination to occur. The single-strand DNA (ssDNA) is more prone to damage than double-strand DNA (dsDNA), due to greater exposure of chemically reactive moieties in the nitrogenous bases. Thus, there can be agents that damage regions of ssDNA in vivo while being inert toward dsDNA. To assess the potential hazard posed by such agents, we devised an ssDNA-specific mutagenesis reporter system in budding yeast. The reporter strains bear the cdc13-1 temperature-sensitive mutation, such that shifting to 37°C results in telomere uncapping and ensuing 5' to 3' enzymatic resection. This exposes the reporter region, containing three closely-spaced reporter genes, as a long 3' ssDNA overhang. We validated the ability of the system to detect mutagenic damage within ssDNA by expressing a modified human single-strand specific cytosine deaminase, APOBEC3G. APOBEC3G induced a high density of substitutions at cytosines in the ssDNA overhang strand, resulting in frequent, simultaneous inactivation of two reporter genes. We then examined the mutagenicity of sulfites, a class of reactive sulfur oxides to which humans are exposed frequently via respiration and food intake. Sulfites, at a concentration similar to that found in some foods, induced a high density of mutations, almost always as substitutions at cytosines in the ssDNA overhang strand, resulting in simultaneous inactivation of at least two reporter genes. Furthermore, sulfites formed a long-lived adducted 2'-deoxyuracil intermediate in DNA that was resistant to excision by uracil-DNA N-glycosylase. This intermediate was bypassed by error-prone translesion DNA synthesis, frequently involving Pol ζ, during repair synthesis. Our results suggest that sulfite-induced lesions in DNA can be particularly deleterious, since cells might not possess the means to repair or bypass such lesions accurately.

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Available from: Kin Chan, Apr 28, 2014
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    • "In addition, genomewide sequencing of human cancers suggests that non-random, clustered mutations may be concentrated in long ssDNA regions, some of which might form R-loop structures (Roberts et al., 2012). Indeed, during transcription, possibly due to the relative chemical susceptibility to damage of ssDNA, there is an increase in the mutation rate associated with the activity of editing enzymes like activation-induced cytosine deaminase (AID) or apolipoprotein B mRNA-editing catalytic polypeptide proteins (APOBEC) (Alexandrov et al., 2013; Beale et al., 2004; Chan et al., 2012). Recently, the latter have been suggested to play a role in the mutational processes that affect breast cancer genomes (Burns et al., 2013; Nik-Zainal et al., 2012, 2014). "
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    ABSTRACT: The mechanisms contributing to transcription-associated genomic instability are both complex and incompletely understood. Although R-loops are normal transcriptional intermediates, they are also associated with genomic instability. Here, we show that BRCA1 is recruited to R-loops that form normally over a subset of transcription termination regions. There it mediates the recruitment of a specific, physiological binding partner, senataxin (SETX). Disruption of this complex led to R-loop-driven DNA damage at those loci as reflected by adjacent γ-H2AX accumulation and ssDNA breaks within the untranscribed strand of relevant R-loop structures. Genome-wide analysis revealed widespread BRCA1 binding enrichment at R-loop-rich termination regions (TRs) of actively transcribed genes. Strikingly, within some of these genes in BRCA1 null breast tumors, there are specific insertion/deletion mutations located close to R-loop-mediated BRCA1 binding sites within TRs. Thus, BRCA1/SETX complexes support a DNA repair mechanism that addresses R-loop-based DNA damage at transcriptional pause sites. Copyright © 2015 Elsevier Inc. All rights reserved.
    Molecular Cell 02/2015; 57(4):636-647. DOI:10.1016/j.molcel.2015.01.011 · 14.02 Impact Factor
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    • "Before kataegis was described, genome sequencing studies had revealed that many cancers have somatic mutations dominated by C-to-T transitions (Sjoblom et al., 2006; Greenman et al., 2007; Jones et al., 2010; Berger et al., 2011; Kumar et al., 2011; Parsons et al., 2011; Stransky et al., 2011; Taylor et al., 2013) and that overexpression of APOBEC1 was associated with cancer development (Yamanaka et al., 1995) when overexpression of APOBEC3A induced genomic damage and mutations (Stenglein et al., 2010; Landry et al., 2011; Suspene et al., 2011). The implication of APOBEC deaminases in kataegis was validated by several groups in yeast models (Taylor et al., 2004; Chan et al., 2012; Roberts et al., 2012) and in human cells (Burns et al., 2013), where overexpression of APOBEC3B was correlated with an elevated level of mutations in breast tumors and cell lines. Knockdown experiments showed that endogenous APOBEC3B was responsible for increased mutation frequencies and C-to-T transitions when APOBEC3B overexpression induced DNA damage and C-to-T mutations in human cells. "
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    ABSTRACT: Homologous recombination (HR) is an evolutionarily conserved process that plays a pivotal role in the equilibrium between genetic stability and diversity. HR is commonly considered to be error-free, but several studies have shown that HR can be error-prone. Here, we discuss the actual accuracy of HR. First, we present the product of genetic exchanges (gene conversion, GC, and crossing over, CO) and the mechanisms of HR during double strand break repair and replication restart. We discuss the intrinsic capacities of HR to generate genome rearrangements by GC or CO, either during DSB repair or replication restart. During this process, abortive HR intermediates generate genetic instability and cell toxicity. In addition to genome rearrangements, HR also primes error-prone DNA synthesis and favors mutagenesis on single stranded DNA, a key DNA intermediate during the HR process. The fact that cells have developed several mechanisms protecting against HR excess emphasize its potential risks. Consistent with this duality, several pro-oncogenic situations have been consistently associated with either decreased or increased HR levels. Nevertheless, this versatility also has advantages that we outline here. We conclude that HR is a double-edged sword, which on one hand controls the equilibrium between genome stability and diversity but, on the other hand, can jeopardize the maintenance of genomic integrity. Therefore, whether non-homologous end joining (which, in contrast with HR, is not intrinsically mutagenic) or HR is the more mutagenic process is a question that should be re-evaluated. Both processes can be "Dr. Jekyll" in maintaining genome stability/variability and "Mr. Hyde" in jeopardizing genome integrity.
    Frontiers in Genetics 06/2014; 5:175. DOI:10.3389/fgene.2014.00175
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    • "Expression of APOBEC cystidine deaminases in yeast generates mutations across the genome, a portion of which are found in clusters (17). Cystidine deaminases have been shown to generate such clustered mutations (18,19). In the present study, we examined whether APOBEC3B may also act as a molecule for tumor progression and metastasis in NSCLCs. "
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    ABSTRACT: Recent study results have demonstrated that a subclass of apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like (APOBEC) cytidine deaminase may induce mutation clusters in various types of cancer. From the Cancer Genome Altas, an APOBEC mutation pattern was identified in bladder, cervical, breast, head and neck and lung cancers. In the present study, APOBEC3B mRNA expression was investigated using quantitative reverse transcription-polymerase chain reaction (RT-qPCR) assay using LightCycler in surgically treated non-small-cell lung cancer (NSCLC) cases. Additionally, 88 surgically removed Japanese NSCLC cases were analyzed for mRNA level. The results showed that APOBEC3B/β-actin mRNA levels were significantly higher in lung cancer (1,598.481±6,465.781) when compared to adjacent normal lung tissues (2,116.639±8,337.331, P=0.5453). The tumor/normal (T/N) ratio of APOBEC3B/β-actin mRNA levels was not different within the gender, age, smoking status and pathological stages. The T/N ratio of APOBEC3B/β-actin mRNA levels was not significantly different in epidermal growth factor receptor (EGFR) or Kras mutation-positive cases as compared to the wild-type cases.
    05/2014; 2(3):392-395. DOI:10.3892/br.2014.256
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