Kinetic models reveal the in vivo mechanisms of mutagenesis in microbes and man

This review summarizes the evidence indicating that mutagenic mechanisms in vivo are essentially the same in all living cells. Unique metabolic reactions to a particular environmental stress apparently target specific genes for increased rates of transcription and mutation, resulting in higher mutation rates for those genes most likely to solve the problem. Kinetic models which have demonstrated predictive value are described and are shown to simulate mutagenesis in vivo in Escherichia coli, the p53 tumor suppressor gene, and somatic hypermutation. In all three models, direct correlations are seen between mutation frequencies and transcription rates. G and C nucleosides in single-stranded DNA (ssDNA) are intrinsically mutable, and G and C silent mutations in p53 and in VH framework regions provide compelling evidence for intrinsic mechanisms of mutability, since mutation outcomes are neutral and are not selected. During transcription, the availability of unpaired bases in the ssDNA of secondary structures is rate-limiting for, and determines the frequency of mutations in vivo. In vitro analyses also verify the conclusion that intrinsically mutable bases are in fact located in ssDNA loops of predicted stem-loop structures (SLSs)

I. VH gene transcription creates stabilized secondary structures for coordinated mutagenesis during somatic hypermutation

During the adaptive immune response, antigen challenge triggers a million-fold increase in mutation rates in the variable-region antibody genes. The frequency of mutation is causally and directly linked to transcription, which provides ssDNA and drives supercoiling that stabilizes secondary structures containing unpaired, intrinsically mutable bases. Simulation analysis of transcription in VH5 reveals a dominant 65nt secondary structure in the non-transcribed strand containing six sites of mutable ssDNA that have also been identified independently in human B cell lines and in primary mouse B cells. This dominant structure inter-converts briefly with less stable structures and is formed repeatedly during transcription, due to periodic pauses and backtracking. In effect, this creates a stable yet dynamic mutability platform consisting of ever-changing patterns of unpaired bases that are simultaneously exposed and therefore able to coordinate mutagenesis. Such a complex of secondary structures may be the source of ssDNA for enzyme-based diversification, which ultimately results in high affinity antibodies

Adding Natural Frequency Data to a Decision Aid for Colorectal Cancer Screening: Results of a Randomized Trial

Guidelines recommend that decision aids provide natural frequency data regarding baseline risk, risk reduction, and chances of false positives and negatives. Such quantitative information may confuse patients, especially those with low numeracy. We conducted a randomized trial to compare effects of 2 colorectal cancer (CRC) screening decision aids—one with and one without natural frequency data

Evolution of coordinated mutagenesis and somatic hypermutation in VH5

The VH5 human antibody gene was analyzed using a computer program (mfg) which simulates transcription, to better understand transcription-driven mutagenesis events that occur during phase 1 of somatic hypermutation. Results show that the great majority of mutations in the non-transcribed strand occur within loops of two predicted high-stability stem-loop structures, termed SLSs 14.9 and 13.9. In fact, 89% of the 2505 mutations reported are within the encoded complementarity-determining region (CDR) and occur in loops of these high-stability structures. In vitro studies were also done and verified the existence of SLS 14.9. Following the formation of SLSs 14.9 and 13.9, a sustained period of transcriptional activity occurs within a window size of 60-70 nucleotides. During this period, the stability of these two SLSs does not change, and may provide the substrate for base exchanges and mutagenesis. The data suggest that many mutable bases are exposed simultaneously at pause sites, allowing for coordinated mutagenesis

Presenting Stool Testing as the Default Option for Colorectal Cancer Screening: Results of a Randomized Trial

Individuals eligible for colorectal cancer (CRC) screening can choose from multiple approved tests, including colonoscopy and stool testing. The existence of multiple options allows patients to choose a preferred strategy but also may lead to indecision and delay. Behavioral economics suggests presenting one option as a default choice, i.e. the one that patients should receive if they do not wish to decide. We conducted a randomized trial to measure the impact of describing stool testing as the default option for CRC screening in a decision aid (DA)

Impact of including quantitative information in a decision aid for colorectal cancer screening: A randomized controlled trial

Objective: Guidelines recommend that decision aids provide quantitative information about risks and benefits of available options. Impact of providing this information is unknown. Methods: Randomized trial comparing two decision aids about colorectal cancer (CRC) screening with colonoscopy or fecal immunochemical test (FIT). 688 primary care patients due for CRC screening viewed a decision aid that uses words only (Verbal arm) vs. one that provides quantitative information (Quantitative arm). Main outcomes included perceived CRC risk, intent to be screened, and test preference, measured before and after viewing decision aid, and screening uptake at six months. Analyses were performed with ANCOVA and logistic regression. Results: Compared to the Verbal arm, those in the Quantitative arm had a larger increase in intent to undergo FIT (p = 0.011) and were more likely to switch their preferred test from non-FIT to FIT (28% vs. 19%, p = .010). There were decreases in perceived risk in the Verbal Arm but not the Quantitative Arm (p = 0.004). There was no difference in screening uptake. Numeracy did not moderate any effects. Conclusions: Quantitative information had relatively minor impact and no clearly negative effects, such as reducing uptake. Practice implications: Quantitative information may be useful but not essential for patients viewing decision aids

Providing Quantitative Information and a Nudge to Undergo Stool Testing in a Colorectal Cancer Screening Decision Aid: A Randomized Clinical Trial

Background. Guidelines recommend that patient decision aids should provide quantitative information about probabilities of potential outcomes, but the impact of this information is unknown. Behavioral economics suggests that patients confused by quantitative information could benefit from a “nudge” towards one option. We conducted a pilot randomized trial to estimate the effect sizes of presenting quantitative information and a nudge. Methods. Primary care patients (n = 213) eligible for colorectal cancer screening viewed basic screening information and were randomized to view (a) quantitative information (quantitative module), (b) a nudge towards stool testing with the fecal immunochemical test (FIT) (nudge module), (c) neither a nor b, or (d) both a and b. Outcome measures were perceived colorectal cancer risk, screening intent, preferred test, and decision conflict, measured before and after viewing the decision aid, and screening behavior at 6 months. Results. Patients viewing the quantitative module were more likely to be screened than those who did not (P = 0.012). Patients viewing the nudge module had a greater increase in perceived colorectal cancer risk than those who did not (P = 0.041). Those viewing the quantitative module had a smaller increase in perceived risk than those who did not (P = 0.046), and the effect was moderated by numeracy. Among patients with high numeracy who did not view the nudge module, those who viewed the quantitative module had a greater increase in intent to undergo FIT (P = 0.028) than did those who did not. Limitations. The limitations of this study were the limited sample size and single healthcare system. Conclusions. Adding quantitative information to a decision aid increased uptake of colorectal cancer screening, while adding a nudge to undergo FIT did not increase uptake. Further research on quantitative information in decision aids is warranted

The roles of transcription and genotoxins underlying p53 mutagenesis in vivo

Transcription drives supercoiling which forms and stabilizes single-stranded (ss) DNA secondary structures with loops exposing G and C bases that are intrinsically mutable and vulnerable to non-enzymatic hydrolytic reactions. Since many studies in prokaryotes have shown direct correlations between the frequencies of transcription and mutation, we conducted in silico analyses using the computer program, mfg, which simulates transcription and predicts the location of known mutable bases in loops of high-stability secondary structures. Mfg analyses of the p53 tumor suppressor gene predicted the location of mutable bases and mutation frequencies correlated with the extent to which these mutable bases were exposed in secondary structures. In vitro analyses have now confirmed that the 12 most mutable bases in p53 are in fact located in predicted ssDNA loops of these structures. Data show that genotoxins have two independent effects on mutagenesis and the incidence of cancer: Firstly, they activate p53 transcription, which increases the number of exposed mutable bases and also increases mutation frequency. Secondly, genotoxins increase the frequency of G-to-T transversions resulting in a decrease in G-to-A and C mutations. This precise compensatory shift in the \u27fate\u27 of G mutations has no impact on mutation frequency. Moreover, it is consistent with our proposed mechanism of mutagenesis in which the frequency of G exposure in ssDNA via transcription is rate limiting for mutation frequency in vivo

Uncoupling reproduction from metabolism extends chronological lifespan in yeast

Studies of replicative and chronological lifespan in Saccharomyces cerevisiae have advanced understanding of longevity in all eukaryotes. Chronological lifespan in this species is defined as the agedependent viability of nondividing cells. To date this parameter has only been estimated under calorie restriction, mimicked by starvation. Because postmitotic cells in higher eukaryotes often do not starve, we developed a model yeast system to study cells as they age in the absence of calorie restriction. Yeast cells were encapsulated in a matrix consisting of calcium alginate to form ~3 mm beads that were packed into bioreactors and fed ad libitum. Under these conditions cells ceased to divide, became heat shock and zymolyase resistant, yet retained high fermentative capacity. Over the course of 17 d, immobilized yeast cells maintained 10%. Immobilized cells exhibited a stable pattern of gene expression that differed markedly from growing or starving planktonic cells, highly expressing genes in glycolysis, cell wall remodeling, and stress resistance, but decreasing transcription of genes in the tricarboxylic acid cycle, and genes that regulate the cell cycle, including master cyclins CDC28 and CLN1. Stress resistance transcription factor MSN4 and its upstream effector RIM15 are conspicuously up-regulated in the immobilized state, and an immobilized rim15 knockout strain fails to exhibit the long-lived, growth-Arrested phenotype, suggesting that altered regulation of the Rim15-mediated nutrient-sensing pathway plays an important role in extending yeast chronological lifespan under calorie-unrestricted conditions