Mutational Strand Bias Research Articles

Asymmetron: a toolkit for the identification of strand asymmetry patterns in biological sequences.

DNA strand asymmetries can have a major effect on several biological functions, including replication, transcription and transcription factor binding. As such, DNA strand asymmetries and mutational strand bias can provide information about biological function. However, a versatile tool to explore this does not exist. Here, we present Asymmetron, a user-friendly computational tool that performs statistical analysis and visualizations for the evaluation of strand asymmetries. Asymmetron takes as input DNA features provided with strand annotation and outputs strand asymmetries for consecutive occurrences of a single DNA feature or between pairs of features. We illustrate the use of Asymmetron by identifying transcriptional and replicative strand asymmetries of germline structural variant breakpoints. We also show that the orientation of the binding sites of 45% of human transcription factors analyzed have a significant DNA strand bias in transcribed regions, that is also corroborated in ChIP-seq analyses, and is likely associated with transcription. In summary, we provide a novel tool to assess DNA strand asymmetries and show how it can be used to derive new insights across a variety of biological disciplines.

Genomic Lesions Unrepaired at Mitosis Affect Clonal Evolution in Cancer

Mutagen-induced DNA lesions were not all resolved prior to mitosis, yielding mutational strand bias.

Mutagenic potential of hypoxanthine in live human cells

Hypoxanthine (Hx) is a major DNA lesion generated by deamination of adenine during chronic inflammatory conditions, which is an underlying cause of various diseases including cancer of colon, liver, pancreas, bladder and stomach. There is evidence that deamination of DNA bases induces mutations, but no study has directly linked Hx accumulation to mutagenesis and strand-specific mutations yet in human cells.Using a site-specific mutagenesis approach, we report the first direct evidence of mutation potential and pattern of Hx in live human cells. We investigated Hx-induced mutations in human nonmalignant HEK293 and cancer HCT116 cell lines and found that Hx is mutagenic in both HEK293 and HCT116 cell lines. There is a strand bias for Hx-mediated mutations in both the cell lines; the Hx in lagging strand is more mutagenic than in leading strand. There is also some difference in cell types regarding the strand bias for mutation types; HEK293 cells showed largely deletion (>80%) mutations in both leading and lagging strand and the rest were insertions and A:T→G:C transition mutations in leading and lagging strands, respectively, whereas in HCT116 cells we observed 60% A:T→G:C transition mutations in the leading strand and 100% deletions in the lagging strand. Overall, Hx is a highly mutagenic lesion capable of generating A:T→G:C transitions and large deletions with a significant variation in leading and lagging strands in human cells. In recent meta-analysis study A→G (T→C) mutations were found to be a prominent signature in a variety of cancers, including a majority types that are induced by inflammation. The deletions are known to be a major cause of copy-number variations or CNVs, which is a major underlying cause of many human diseases including mental illness, developmental disorders and cancer. Thus, Hx, a major DNA lesion induced by different deamination mechanisms, has potential to initiate inflammation-driven carcinogenesis in addition to various human pathophysiological consequences.

Simultaneous DNA and RNA Mapping of Somatic Mitochondrial Mutations across Diverse Human Cancers.

Somatic mutations in the nuclear genome are required for tumor formation, but the functional consequences of somatic mitochondrial DNA (mtDNA) mutations are less understood. Here we identify somatic mtDNA mutations across 527 tumors and 14 cancer types, using an approach that takes advantage of evidence from both genomic and transcriptomic sequencing. We find that there is selective pressure against deleterious coding mutations, supporting that functional mitochondria are required in tumor cells, and also observe a strong mutational strand bias, compatible with endogenous replication-coupled errors as the major source of mutations. Interestingly, while allelic ratios in general were consistent in RNA compared to DNA, some mutations in tRNAs displayed strong allelic imbalances caused by accumulation of unprocessed tRNA precursors. The effect was explained by altered secondary structure, demonstrating that correct tRNA folding is a major determinant for processing of polycistronic mitochondrial transcripts. Additionally, the data suggest that tRNA clusters are preferably processed in the 3′ to 5′ direction. Our study gives insights into mtDNA function in cancer and answers questions regarding mitochondrial tRNA biogenesis that are difficult to address in controlled experimental systems.

Mechanisms of mutagenesis in vivo due to imbalanced dNTP pools

The mechanisms by which imbalanced dNTPs induce mutations have been well characterized within a test tube, but not in vivo. We have examined mechanisms by which dNTP imbalances induce genome instability in strains of Saccharomyces cerevisiae with different amino acid substitutions in Rnr1, the large subunit of ribonucleotide reductase. These strains have different dNTP imbalances that correlate with elevated CAN1 mutation rates, with both substitution and insertion–deletion rates increasing by 10- to 300-fold. The locations of the mutations in a strain with elevated dTTP and dCTP are completely different from those in a strain with elevated dATP and dGTP. Thus, imbalanced dNTPs reduce genome stability in a manner that is highly dependent on the nature and degree of the imbalance. Mutagenesis is enhanced despite the availability of proofreading and mismatch repair. The mutations can be explained by imbalanced dNTP-induced increases in misinsertion, strand misalignment and mismatch extension at the expense of proofreading. This implies that the relative dNTP concentrations measured in extracts are truly available to a replication fork in vivo. An interesting mutational strand bias is observed in one rnr1 strain, suggesting that the S-phase checkpoint selectively prevents replication errors during leading strand replication.

Transcription-Induced Mutational Strand Bias and Its Effect on Substitution Rates in Human Genes

If substitution rates are not the same on the two complementary DNA strands, a substitution is considered strand asymmetric. Such substitutional strand asymmetries are determined here for the three most frequent types of substitution on the human genome (C --> T, A --> G, and G --> T). Substitution rate differences between both strands are estimated for 4,590 human genes by aligning all repeats occurring within the introns with their ancestral consensus sequences. For 1,630 of these genes, both coding strand and noncoding strand rates could be compared with rates in gene-flanking regions. All three rates considered are found to be on average higher on the coding strand and lower on the transcribed strand in comparison to their values in the gene-flanking regions. This finding points to the simultaneous action of rate-increasing effects on the coding strand--such as increased adenine and cytosine deamination--and transcription-coupled repair as a rate-reducing effect on the transcribed strand. The common behavior of the three rates leads to strong correlations of the rate asymmetries: Whenever one rate is strand biased, the other two rates are likely to show the same bias. Furthermore, we determine all three rate asymmetries as a function of time: the A --> G and G --> T rate asymmetries are both found to be constant in time, whereas the C --> T rate asymmetry shows a pronounced time dependence, an observation that explains the difference between our results and those of an earlier work by Green et al. (2003. Transcription-associated mutational asymmetry in mammalian evolution. Nat Genet. 33:514-517.). Finally, we show that in addition to transcription also the replication process biases the substitution rates in genes.

First AID (Activation-induced Cytidine Deaminase) Is Needed to Produce High Affinity Isotype-switched Antibodies

An answer to the question "why don't you die when I sneeze?" resides in the ability of higher organisms to mount an aggressive defense in response to an invasion by foreign substances called antigens. In part this defense mechanism is accomplished through the synthesis of high affinity antibody (Ab) 2 proteins that effectively defuse the attack. Initially, lymphoid B cells rearrange immunoglobulin (Ig) genes using V(D)J recombination to generate a diverse repertoire of low affinity Abs. Subsequently these Ig genes undergo somatic hypermutation (SHM) and class switch recombination (CSR) to generate high affinity Ab molecules of different isotypes. Activation-induced cytidine deaminase (AID) is required to initiate both CSR and SHM. The current understanding of immunological diversification can be attributed to the contributions made by cellular and molecular immunologists with biochemical enzymology recently entering the arena. This minireview discusses advances in the understanding ofAb diversification from a biochemical perspective emphasizing the enzymatic properties of AID in initiating SHM and CSR and exploring the role of downstream mutation fixation events mediated by mismatch repair (MMR), basic excision repair (BER), and error-prone DNA polymerases (EP pols).

Yeast Origins Establish a Strand Bias for Replicational Mutagenesis

To determine whether replicational mutagenesis in the yeast genome is influenced by the positions of active origins, a reporter gene was placed in two orientations at multiple locations within a 39,000 bp region of chromosome III possessing two strong origins. The frequency of mutations resulting from misincorporation of adenine opposite 8-hydroxyguanine in one strand and 6-hydroxylaminopurine opposite cytosine in the other strand differed by 3- to 10-fold, depending on the gene orientation and its distance from the origins. The observed patterns indicate that active origins establish a strand bias for mutations that is maintained over thousands of base pairs and results from lower nucleotide selectivity and/or less efficient proofreading or mismatch repair during leading strand DNA replication.

UV-induced photolesions, their repair and mutations

UV-induced cyclobutane pyrimidine dimers (CPD) are selectively removed from the transcribed strand of transcriptionally active genes in V79 Chinese hamster cells. This strand specificity of repair corresponds well with the observation that UV-induced mutations in the HPRT gene are primarily generated by DNA photolesions in the non-transcribed strand. This strand bias for mutations is, however, much more pronounced at 2 J/m 2 than at the higher dose of 12 J/m 2. An alternative explanation for strand specificity of mutations would be that most of the mutations are caused by pyrimidone 6-4 pyrimidine photoproducts (6-4 PP). Indeed experiments with a V79-derived cell line capable of repairing 6-4 PP but not CPD have revealed direct evidence for 6-4 PP as the mutagenic lesion in UV-irradiated hamster cells. This implies that 6-4 PP are also preferentially repaired in the transcribed strand. We have investigated the repair of DNA photolesions in the HPRT gene by measuring the distribution of bromodeoxyuridine-labeled repair patches in the transcribed and non-transcribed strands of genes employing a newly developed immunoextraction procedure. Three cell lines with different capacities to remove CPD and 6-4 PP from the HPRT gene and from the genome overall were used. We found no evidence for preferential repair of 6-4 PP in the transcribed strand of the HPRT gene in cells exposed to 10 J/m 2. These data are in favor of a lack of strand-specific repair of 6-4 PP underlying the much less pronounced strand bias for induced mutations at high UV dose. However, the conclusive test would be the demonstration of preferential repair of 6-4 PP in the transcribed strand of transcriptionally active genes in cells exposed to 2 J/m 2.

Analysis of Mutations Occurring at the Human hprt Locus

We have recently established a computerized database containing information on mutants at the human hypoxanthine guanine phosphoribosyl transferase ( hprt) locus. The database contains sequence information on over 1000 mutants. We now present an analysis of the information in the database. 542 single base substitution mutants in the hprt coding region exist, and we have examined (1) the number of mutations and the number of mutable sites in each exon, (2) transcribed versus non-transcribed strand bias for mutations, (3) the frequency of the 5′ and 3′ nearest neighbors to a mutated base, and (4) the distribution of amino acid substitutions. The distribution of both DNA mutations and amino acid mutations was not uniform, several clusterings of mutations were observed and we propose several possible mechanisms to account for the hotspots. We also examined mRNA splicing mutants, mutants with small deletions, and frameshift mutants.

DNA strand-specific mutations induced by (+/-)-3 alpha,4 beta-dihydroxy- 1 alpha,2 alpha-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene in the dihydrofolate reductase gene.

We previously showed that the preferred mutation induced by (+/-)-3 alpha,4 beta-dihydroxy-1 alpha,2 alpha-epoxy- 1,2,3,4-tetrahydrobenzo[c]phenanthrene (BcPHDE) in the dihydrofolate reductase gene in Chinese hamster ovary cells was a purine to thymine transversion on the nontranscribed strand at the sequence 5'-RRR-3' (R is a purine and the mutated base is underlined). To determine whether the observed mutational strand specificity was due to bias in the phenotypic selection, we designed a nonsense-codon reversion assay in which a triple purine target was present on both strands and all R----T transversion mutations yielded amino acid substitutions that were compatible with dihydrofolate reductase enzyme activity. From the size of the targets, a 2:1 ratio of mutations at the purines on the nontranscribed strand was expected if the DNA strands were mutationally equivalent. We isolated a total of 66 BcPHDE-induced revertants of two mutants that carry point mutations at either the 5' or the 3' end of the gene. All reversions at the 5' end arose by substitution on the nontranscribed strand; those at the 3' end showed a strand bias that favored this strand by 7:1. For both mutants, R----T transversions accounted for 88% of all the induced base changes. Thus, in this system, mutational strand bias is independent of the selection for phenotype. The results are consistent with the model of preferential repair of the transcribed strand as proposed by others. The involvement of RNA polymerase in the selective repair recruitment is discussed.