Intrinsic Mutation Rate Research Articles

Abstract B043: Quantifying selection intensity and epistatic interactions among gene variants within angiosarcoma

Abstract Evolutionary processes underlie cancer development. Mutations occur and accumulate in somatic tissues, and some mutations result in increases in cell division or survival and fix in cell populations more often than expected by chance. Computational techniques that account for differences in mutation rate across the genome and between tissues have helped pinpoint which genes are contributing to tumor growth within a wide array of cancer types. However, rare cancers are often understudied due to lack of funding, availability of data, and applicable tools that are capable of incorporating data from different sources. Recent initiatives, such as the Angiosarcoma Project, have increased data availability for the study of angiosarcoma, a rare cancer with approximately 300 new cases a year. In this study, we incorporate whole-genome, whole-exome, and targeted sequencing data from different sources—accounting for study-specific differences in genome coverage—into an evolutionary analysis of angiosarcoma tumorigenesis. Prior to our study, potential genes with oncogenic activity for angiosarcoma have been primarily evaluated based on genetic variant prevalence among tumor samples, but substitution prevalence is dependent on intrinsic mutation rate and thus relative prevalence of variants may not correspond with relative tumorigenic potential. We calculate the intensity of selection, a measure of the magnitude of increase of cell division and survival, of individual variants driving angiosarcoma, and complement existing studies that have previously determined KDR and TP53 mutations to be a driver of angiosarcoma, and we also implicate novel putative drivers that have high calculated selection intensity, including BRAF V600E, IGDCC4 S1020A, and SETD2 Q1152E. Additionally, we evaluated epistatic interactions among genes and found that selection for TP53 is increased after mutation of other genes such as MUC16, FAT1, and NOTCH2. This study demonstrates the feasibility of applying computational models informed by evolutionary approaches to a rare disease as well as identifies gene variants that likely have a large impact on angiosarcoma tumorigenesis. Citation Format: Caralynn E. Hampson, Vincent L. Cannataro. Quantifying selection intensity and epistatic interactions among gene variants within angiosarcoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr B043.

High-resolution mapping reveals the mechanism and contribution of genome insertions and deletions to RNA virus evolution

RNA viruses rapidly adapt to selective conditions due to the high intrinsic mutation rates of their RNA-dependent RNA polymerases (RdRps). Insertions and deletions (indels) in viral genomes are major contributors to both deleterious mutational load and evolutionary novelty, but remain understudied. To characterize the mechanistic details of their formation and evolutionary dynamics during infection, we developed a hybrid experimental-bioinformatic approach. This approach, called MultiMatch, extracts insertions and deletions from ultradeep sequencing experiments, including those occurring at extremely low frequencies, allowing us to map their genomic distribution and quantify the rates at which they occur. Mapping indel mutations in adapting poliovirus and dengue virus populations, we determine the rates of indel generation and identify mechanistic and functional constraints shaping indel diversity. Using poliovirus RdRp variants of distinct fidelity and genome recombination rates, we demonstrate tradeoffs between fidelity and Indel generation. Additionally, we show that maintaining translation frame and viral RNA structures constrain the Indel landscape and that, due to these significant fitness effects, Indels exert a significant deleterious load on adapting viral populations. Conversely, we uncover positively selected Indels that modulate RNA structure, generate protein variants, and produce defective interfering genomes in viral populations. Together, our analyses establish the kinetic and mechanistic tradeoffs between misincorporation, recombination, and Indel rates and reveal functional principles defining the central role of Indels in virus evolution, emergence, and the regulation of viral infection.

Death and Progress: How Evolvability is Influenced by Intrinsic Mortality.

Many factors influence the evolvability of populations, and this article illustrates how intrinsic mortality (death induced through internal factors) in an evolving population contributes favorably to evolvability on a fixed deceptive fitness landscape. We test for evolvability using the hierarchical if-and-only-if (h-iff) function as a deceptive fitness landscape together with a steady state genetic algorithm (SSGA) with a variable mutation rate and indiscriminate intrinsic mortality rate. The mutation rate and the intrinsic mortality rate display a relationship for finding the global maximum. This relationship was also found when implementing the same deceptive fitness landscape in a spatial model consisting of an evolving population. We also compared the performance of the optimal mutation and mortality rate with a state-of-the-art evolutionary algorithm called age-fitness Pareto optimization (AFPO) and show how the two approaches traverse the h-iff landscape differently. Our results indicate that the intrinsic mortality rate and mutation rate induce random genetic drift that allows a population to efficiently traverse a deceptive fitness landscape. This article gives an overview of how intrinsic mortality influences the evolvability of a population. It thereby supports the premise that programmed death of individuals could have a beneficial effect on the evolvability of the entire population.

Prophage-Driven Genomic Structural Changes Promote Bartonella Vertical Evolution.

Bartonella is a genetically diverse group of vector-borne bacteria. Over 40 species have been characterized to date, mainly from mammalian reservoirs and arthropod vectors. Rodent reservoirs harbor one of the largest Bartonella diversity described to date, and novel species and genetic variants are continuously identified from these hosts. Yet, it is still unknown if this significant genetic diversity stems from adaptation to different niches or from intrinsic high mutation rates. Here, we explored the vertical occurrence of spontaneous genomic alterations in 18 lines derived from two rodent-associated Bartonella elizabethae-like strains, evolved in nonselective agar plates under conditions mimicking their vector- and mammalian-associated temperatures, and the transmission cycles between them (i.e., 26 °C, 37 °C, and alterations between the two), using mutation accumulation experiments. After ∼1,000 generations, evolved genomes revealed few point mutations (average of one-point mutation per line), evidencing conserved single-nucleotide mutation rates. Interestingly, three large structural genomic changes (two large deletions and an inversion) were identified over all lines, associated with prophages and surface adhesin genes. Particularly, a prophage, deleted during constant propagation at 37 °C, was associated with an increased autonomous replication at 26 °C (the flea-associated temperature). Complementary molecular analyses of wild strains, isolated from desert rodents and their fleas, further supported the occurrence of structural genomic variations and prophage-associated deletions in nature. Our findings suggest that structural genomic changes represent an effective intrinsic mechanism to generate diversity in slow-growing bacteria and emphasize the role of prophages as promoters of diversity in nature.

Reduced intrinsic DNA curvature leads to increased mutation rate

BackgroundMutation rates vary across the genome. Many trans factors that influence mutation rates have been identified, as have specific sequence motifs at the 1–7-bp scale, but cis elements remain poorly characterized. The lack of understanding regarding why different sequences have different mutation rates hampers our ability to identify positive selection in evolution and to identify driver mutations in tumorigenesis.ResultsHere, we use a combination of synthetic genes and sequences of thousands of isolated yeast colonies to show that intrinsic DNA curvature is a major cis determinant of mutation rate. Mutation rate negatively correlates with DNA curvature within genes, and a 10% decrease in curvature results in a 70% increase in mutation rate. Consistently, both yeast and humans accumulate mutations in regions with small curvature. We further show that this effect is due to differences in the intrinsic mutation rate, likely due to differences in mutagen sensitivity and not due to differences in the local activity of DNA repair.ConclusionsOur study establishes a framework for understanding the cis properties of DNA sequence in modulating the local mutation rate and identifies a novel causal source of non-uniform mutation rates across the genome.

Cellular Assays for Studying the Fe-S Cluster Containing Base Excision Repair Glycosylase MUTYH and Homologs.

Many DNA repair enzymes, including the human adenine glycosylase MUTYH, require iron-sulfur (Fe-S) cluster cofactors for DNA damage recognition and subsequent repair. MUTYH prokaryotic and eukaryotic homologs are a family of adenine (A) glycosylases that cleave A when mispaired with the oxidatively damaged guanine lesion, 8-oxo-7,8-dihydroguanine (OG). Faulty OG:A repair has been linked to the inheritance of missense mutations in the MUTYH gene. These inherited mutations can result in the onset of a familial colorectal cancer disorder known as MUTYH-associated polyposis (MAP). While in vitro studies can be exceptional at unraveling how MutY interacts with its OG:A substrate, cell-based assays are needed to provide a cellular context to these studies. In addition, strategic comparison of in vitro and in vivo studies can provide exquisite insight into the search, selection, excision process, and the coordination with protein partners, required to mediate full repair of the lesion. A commonly used assay is the rifampicin resistance assay that provides an indirect evaluation of the intrinsic mutation rate in Escherichia coli (E. coli or Ec), read out as antibiotic-resistant cell growth. Our laboratory has also developed a bacterial plasmid-based assay that allows for direct evaluation of repair of a defined OG:A mispair. This assay provides a means to assess the impact of catalytic defects in affinity and excision on overall repair. Finally, a mammalian GFP-based reporter assay has been developed that more accurately models features of mammalian cells. Taken together, these assays provide a cellular context to the repair activity of MUTYH and its homologs that illuminates the role these enzymes play in preventing mutations and disease.

Abstract P4-04-01: PDL-1 expression in primary breast cancers with germline mutations in BRCA 1 and 2

Abstract Background: The PD-1/PDL-1 pathway is a major inhibitory regulator of the immune response to tumors. Tumors responding to anti-PD-1 therapy appear to be characterized by a high "load" of somatic mutations, e.g.carcinogen-induced cancers (melanoma, lung) and tumors with a high intrinsic mutation rate (colorectal cancers with defects in DNA mismatch repair).High mutational load in these tumors may cause the expression of neoantigens which may induce an immune response unless the inhibitory PD-1/PDL-1 pathway is upregulated.Tumors in carriers of germline mutations in BRCA1/2 lack effective DNA repair and are genomically unstable, with a high mutational load and the possible expression of neoantigens. Breast cancers arising in carriers of germline mutations in BRCA1 and BRCA2 may rely on the PD-1/PDL-1 pathway to avoid immune destruction. Methods: Samples from thirty (30) treatment-naïve, primary breast cancers from 30 women with known germline mutations in BRCA 1 or BRCA2 were identified by records review in the Cedars-Sinai Department of Pathology, and selected for analysis of PD-1 and PDL-1 expression. Samples were analyzed for PD1 and PDL-1 expression utilizing immunohistochemical staining. Sections from FFPE tissue blocks were analysed with anti-PD-L1 clone 22C3(Merck); anti-PD-L1 clone SP142; or anti-PD-1 clone NAT105. IHC Scoring analyzed both tumor cell and non-tumor cell (inflammatory) infiltrate. A 0-5 scoring system (0= neg, 1 = rare, 2 = low, 3 = mod, 4 = high, 5 = very high) was applied. Tumors with IHC scores of 2 or greater were considered "positive" for expression of PD1 or PDL-1. Clinical Data: Age range 39-90 years, median age 61. Twenty(20) tumors were from BRCA 1 mutations carriers, and 10 were BRCA2. Sixteen (16) were basal type breast cancer (13 BRCA1, 3 BRCA2), 14 were estrogen receptor positive(ER+)(7 BRCA1, 7BRCA2), with 7 Luminal A and 7 Luminal B. There were no HER2 -amplified tumors in the cohort.Results: PD1 expression was observed in 11/30 (37%) of the cohort, and PDL-1 expression was detected in 21/30 (70%) of the cohort. PDL-1 expression was primarily seen in non-tumor, infiltrating immune cells. PDL-1 expression was seen in 15/20 (75%) of tumors from BRCA1 mutation carriers, and 6/10 (60%) of tumors from BRCA2 carriers. PDL-1 expression was present in 13/16 (81%) basal tumors and 8/14 (57%) ER+ tumors. Within the cohort of PDL-1 expressing cancers, 7/21 (33%) were scored as "high" or "very high", 5 basal breast cancers and 2 ER+ cancers. Conclusion: The current study has identified a high rate of PDL-1 expression in untreated primary breast cancers with germline BRCA1 and BRCA2 mutations, regardless of intrinsic subtype. Although the highest rate of expression was seen in basal breast cancers (81%), the majority of ER+ breast cancers, both Luminal A and Luminal B, also expressed PDL-1. Anti-PD-1 therapy has yielded a response rate of 19% in metastatic triple negative breast cancer, unselected for BRCA mutation.The current study suggests that tumors in carriers of germline mutations in BRCA1 or BRCA2 , regardless of intrinsic subtype, may rely on immune checkpoint inhibition for their growth and survival, and that therapy directed against the PD-1/PDL-1 pathway may be of benefit in this cohort of patients. Citation Format: Audeh MW, Dadmanesh F, Yearley J. PDL-1 expression in primary breast cancers with germline mutations in BRCA 1 and 2. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-04-01.

Contributions of intrinsic mutation rate and selfish selection to levels of de novo HRAS mutations in the paternal germline

The RAS proto-oncogene Harvey rat sarcoma viral oncogene homolog (HRAS) encodes a small GTPase that transduces signals from cell surface receptors to intracellular effectors to control cellular behavior. Although somatic HRAS mutations have been described in many cancers, germline mutations cause Costello syndrome (CS), a congenital disorder associated with predisposition to malignancy. Based on the epidemiology of CS and the occurrence of HRAS mutations in spermatocytic seminoma, we proposed that activating HRAS mutations become enriched in sperm through a process akin to tumorigenesis, termed selfish spermatogonial selection. To test this hypothesis, we quantified the levels, in blood and sperm samples, of HRAS mutations at the p.G12 codon and compared the results to changes at the p.A11 codon, at which activating mutations do not occur. The data strongly support the role of selection in determining HRAS mutation levels in sperm, and hence the occurrence of CS, but we also found differences from the mutation pattern in tumorigenesis. First, the relative prevalence of mutations in sperm correlates weakly with their in vitro activating properties and occurrence in cancers. Second, specific tandem base substitutions (predominantly GC>TT/AA) occur in sperm but not in cancers; genomewide analysis showed that this same mutation is also overrepresented in constitutional pathogenic and polymorphic variants, suggesting a heightened vulnerability to these mutations in the germline. We developed a statistical model to show how both intrinsic mutation rate and selfish selection contribute to the mutational burden borne by the paternal germline.

Modelling the evolution of genetic instability during tumour progression.

The role of genetic instability in driving carcinogenesis remains controversial. Genetic instability should accelerate carcinogenesis by increasing the rate of advantageous driver mutations; however, genetic instability can also potentially retard tumour growth by increasing the rate of deleterious mutation. As such, it is unclear whether genetically unstable clones would tend to be more selectively advantageous than their genetically stable counterparts within a growing tumour. Here, we show the circumstances where genetic instability evolves during tumour progression towards cancer. We employ a Wright–Fisher type model that describes the evolution of tumour subclones. Clones can acquire both advantageous and deleterious mutations, and mutator mutations that increase a cell's intrinsic mutation rate. Within the model, cancers evolve with a mutator phenotype when driver mutations bestow only moderate increases in fitness: very strong or weak selection for driver mutations suppresses the evolution of a mutator phenotype. Genetic instability occurs secondarily to selectively advantageous driver mutations. Deleterious mutations have relatively little effect on the evolution of genetic instability unless selection for additional driver mutations is very weak or if deleterious mutations are very common. Our model provides a framework for studying the evolution of genetic instability in tumour progression. Our analysis highlights the central role of selection in shaping patterns of mutation in carcinogenesis.

Highlight: Family Ties--New Evidence Simplifies Human Evolutionary Tree

Rest easy, it seems our ancestors were not the interspecies Casanovas once supposed. After humans went their separate way from chimpanzees, we made a clean break of it, each group mating within its species, a new analysis suggested by geneticist Tadashi and coworkers. This is a thorough rewrite of the evolutionary narrative told by David Reich, Nick Patterson, and colleagues at the Broad Institute in Cambridge, MA. In 2006, the popular press rang with near-salacious headlines such as “Late swinging in the evolutionary tree?” from The Philadelphia Inquirer and “Grandpa! Leave that chimp alone! Who knows what it might lead to?” from The Times of London. That year in Nature, Reich and Patterson presented evidence that, following an initial genetic split 6 million years ago, humans and chimpanzees interbred for a time as a hybridized species. The analysis was not universally well received. “We’d like to have a more Victorian view of our genome,” David Page of the Whitehead Institute in Cambridge told the New York Times. ”This reminds us that we are really animals.” However, beyond the giggle-inducing implications, a number of phylogenists did not like it for more practical reasons. The explanation was complicated and based on thin evidence. “In it very small numbers were given large importance,” said Dan Graur, who studies molecular evolution at the University of Houston. “This very complicated model has been making its way into literature and textbooks. But the problem is that people have ignored the fact of recombination.” During meiosis, matching chromosomes can swap whole sections of material, leaving Junior with new-sprung genetic diversity, but phylogenists with a newly complicated picture. However, in a new study published in Genome Biology and Evolution (Hara et al. 2012), researchers from Japan’s National Institute of Advanced Science and Technology in Tokyo and the Graduate University for Advanced Studies in Kanagawa managed to sidestep this problem entirely. “What was done in this case this was quite amazing,” said Graur, who was not involved in the work. “The limits are not contaminated by half-maternal, half-paternal material.” By comparing shorter segments that are unlikely to have recombined since humans shared a common ancestor with the great apes, they were able to trace evolutionary history without seeing any hint that different linages had once converged into one. “Everyone who is doing primate evolution will be affected by this,” Graur said. When asked how much their work negates previous work in the field, Imanishi was modest: “Our estimates of human-ape divergence times and the ancestral population sizes are not much different from those of previous works,” he wrote. “However, by using the largest dataset of genome sequences and applying a much better model to the data, we succeeded in presenting the most accurate estimates ever.” Their work, so far, has been warmly received by colleagues. “It seems that many evolutionary biologists, including Dr. Naruya Saitou of National Institute of Genetics of Japan […] did not like the hypothesis by Patterson et al. and wanted to deny it. We could demonstrate what they intuitively thought.” In addition, the researchers are intrigued by several other aspects of their study. They were also surprised by the finding that each chromosome seems to have an intrinsic mutation rate. “We concluded that variation in tau [speciation time] across the chromosomes would be due to the difference in mutation rate across the chromosomes,” writes Yuichiro Hara. “But we could not find the cause of the variation in mutation rate. We are interested in the mechanisms of genome evolution in closely related lineages (Hara and Imanishi, 2011, BMC Evol. Biol.), too, and thus clarification of this is our next goal.” And beyond that the team is also interested in seeing their novel analytical method applied to other species. Graur is similarly eager to see this method become more widespread, “These days complicated models are used as a first resort rather than a last resort. It’s nice to see such a straightforward and ingenious approach to solve a problem that was quite complicated.” He is especially interested in seeing the relationship between humans, Neanderthals, and Denisovians explored. Some claim that modern Papua New Guineans carry fragments of the Denisovian genome and paleolithic hominids believed to exist based on a tiny amount of DNA extracted from a finger bone found several years ago in a Siberian cave. Graur did have one criticism. “If I was writing up this work, I would have said that everything that was said before about the speciation was junk. The authors are very polite. They could have been more forceful.”

Frequent pseudogenization and loss of the plastid‐encoded sulfate‐transport genecysA throughout the evolution of liverworts

The presence or absence of a functional copy of a plastid gene may reflect relaxed selection, and may be phylogenetically significant, reflecting shared ancestry. In some liverworts, the plastid gene cysA is a pseudogene (inferred to be nonfunctional). We surveyed 63 liverworts from all major clades to determine whether the loss of cysA is phylogenetically significant, whether intact copies of cysA are under selective constraints, and whether rates of nucleotide substitution differ in other plastid genes from taxa with and without a functional copy of cysA. Primers annealing to flanking and internal regions were used to amplify and sequence cysA from 61 liverworts. Two additional cysA sequences were downloaded from NCBI. The ancestral states of cysA were reconstructed on a phylogenetic hypothesis inferred from seven markers. Rates of nucleotide substitution were estimated for three plastid loci to reflect the intrinsic mutation rate in the plastid genome. The ratio of nonsynonymous to synonymous substitutions was estimated for intact copies of cysA to infer selective constraints. Throughout liverworts, cysA has been lost up to 29 times, yet intact copies of cysA are evolving under selective constraints. Gene loss is more frequent in groups with an increased substitution rate in the plastid genome. The number of inferred losses of cysA in liverworts exceeds any other reported plastid gene. Despite frequent losses, cysA is evolving under purifying selection in liverworts that retain the gene. It appears that cysA is lost more frequently in lineages characterized by a higher rate of nucleotide substitutions in the plastid.

Targeting the Human DEAD-Box Polypeptide 3 (DDX3) RNA Helicase as a Novel Strategy to Inhibit Viral Replication

Compounds currently used for the treatment of HIV-1 Infections are targeted to viral proteins. However, the high intrinsic mutation and replication rates of HIV-1 often lead to the emergence of drug resistant strains and consequent therapeutic failure. On this basis, cellular cofactors represent attractive new targets for HIV-1 chemotherapy, since targeting a cellular factor that is required for viral replication should help to overcome the problem of viral resistance. We and others have recently reported the identification of compounds suppressing HIV-1 replication by targeting the cellular DEAD-box helicase DDX3. These results provide a proof-of-principle for the feasibility of blocking HIV-1 infection by rendering the host cell environment less favorable for the virus. The rationale for such an approach and its implications in potentially overcoming the problem of drug resistance related to drugs targeting viral proteins will be discussed in the context of the known cellular functions of the DEAD-box helicase DDX3.

Directed adenovirus evolution using engineered mutator viral polymerases

Adenoviruses (Ads) are the most frequently used viruses for oncolytic and gene therapy purposes. Most Ad-based vectors have been generated through rational design. Although this led to significant vector improvements, it is often hampered by an insufficient understanding of Ad’s intricate functions and interactions. Here, to evade this issue, we adopted a novel, mutator Ad polymerase-based, ‘accelerated-evolution’ approach that can serve as general method to generate or optimize adenoviral vectors. First, we site specifically substituted Ad polymerase residues located in either the nucleotide binding pocket or the exonuclease domain. This yielded several polymerase mutants that, while fully supportive of viral replication, increased Ad’s intrinsic mutation rate. Mutator activities of these mutants were revealed by performing deep sequencing on pools of replicated viruses. The strongest identified mutators carried replacements of residues implicated in ssDNA binding at the exonuclease active site. Next, we exploited these mutators to generate the genetic diversity required for directed Ad evolution. Using this new forward genetics approach, we isolated viral mutants with improved cytolytic activity. These mutants revealed a common mutation in a splice acceptor site preceding the gene for the adenovirus death protein (ADP). Accordingly, the isolated viruses showed high and untimely expression of ADP, correlating with a severe deregulation of E3 transcript splicing.

Understanding human genetic variation in the era of high‐throughput sequencing

The EMBO/EMBL symposium 'Human Variation: Cause and Consequence' highlighted advances in understanding the molecular basis of human genetic variation and its myriad implications for biology, human origins and disease. As high-throughput sequencing allows us to define genetic variation and its functional consequences at genome-wide resolution for a large number of people, important questions need to be asked about how to use new technologies to maximize the translational relevance of genetic research for society and the individual patient.

Inhibitors of human immunodeficiency virus-1 replication targeting the human DEAD-box polypeptide 3 (DDX3) RNA helicase

Compounds currently used for the treatment of HIV-1 infections are targeted to viral proteins. However, the high intrinsic mutation and replication rates of HIV-1 led to the emergence of drug resistant strains with a consequent therapeutic failure. On this basis, cellular cofactors represent attractive new targets for HIV-1 chemotherapy, since targeting a cellular factor that is required for viral replication should help to overcome the problem of viral resistance. We aimed to develop through rational design a series of non-nucleosidic inhibitors of the HIV-1 cellular cofactor DDX3 and show that they can be used to block viral proliferation. Methods The X-ray crystallographic structure of human helicase DDX3 in complex with AMP has been used to generate a structure-based pharmacophoric model to be inserted in a computational protocol for the identification of small inhibitors of the ATPase activity of DDX3. Next, the pharmacophore was used as the three-dimensional query of a virtual screening approach to filter databases of commercially available compounds in order to identify chemical scaffolds with putative affinity toward the DDX3 ATP binding site. Positive hits were tested in antienzymatic and antiviral assays. An iterative process of synthesis, testing and optimization was used to derive lead compounds. Results One positive hit was identified in the first virtual screening with an IC50 against the enzymatic activity of DDX3 of 5 μM. The mechanism of action was found uncompetitive with respect to ATP and dependent on the multimeric state of the enzyme and several derivatives have been synthesized. Among those, some compounds showed nanomolar potencies against the enzymatic activity of DDX3 and micromolar potencies against HIV-1 replication in PBMCs, with no detectable toxicities.

Lethal Mutagenesis of Bacteria

Lethal mutagenesis, the killing of a microbial pathogen with a chemical mutagen, is a potential broad-spectrum antiviral treatment. It operates by raising the genomic mutation rate to the point that the deleterious load causes the population to decline. Its use has been limited to RNA viruses because of their high intrinsic mutation rates. Microbes with DNA genomes, which include many viruses and bacteria, have not been considered for this type of treatment because their low intrinsic mutation rates seem difficult to elevate enough to cause extinction. Surprisingly, models of lethal mutagenesis indicate that bacteria may be candidates for lethal mutagenesis. In contrast to viruses, bacteria reproduce by binary fission, and this property ensures their extinction if subjected to a mutation rate >0.69 deleterious mutations per generation. The extinction threshold is further lowered when bacteria die from environmental causes, such as washout or host clearance. In practice, mutagenesis can require many generations before extinction is achieved, allowing the bacterial population to grow to large absolute numbers before the load of deleterious mutations causes the decline. Therefore, if effective treatment requires rapid population decline, mutation rates >>0.69 may be necessary to achieve treatment success. Implications for the treatment of bacteria with mutagens, for the evolution of mutator strains in bacterial populations, and also for the evolution of mutation rate in cancer are discussed.

C-Myc: Linking Transformation and Genomic Instability

CMYC has long been known to be among the most frequently de-regulated oncogenes in human cancer. Only recently, however has a clear understanding begun to emerge of how it promotes transformation. Through its role as a transcription factor, c-Myc alters the expression of hundreds of target genes, many of which are themselves oncogenes or tumor suppressors. The deregulation of c-Myc is both necessary and sufficient for the "acute" type of rapid in vitro transformation that occurs in certain established rodent cell lines. Transformation of primary rodent cells in vitro is also rapid but requires the contribution of at least one additional cooperating oncogene such as Ras. In contrast, the "chronic" form of in vivo transformation by c-Myc is a rare event that requires the acquisition of multiple mutations in other genes affecting cell cycle, senescence, and apoptosis. By greatly accelerating the intrinsic mutation rate at several levels, c-Myc increases the likelihood that these additional mutational "hits" will occur. Among the types of genomic instability mediated by c-Myc are single nucleotide substitutions and double-stranded breaks arising via the induction of reactive oxygen species, gene amplification and the generation of extrachromosomal elements, and numerical chromosomal defects resulting from aberrant DNA synthesis and defects in the mitotic spindle checkpoint. These non-mutually exclusive activities ensure a constant and varied source of genotoxic insults and suggest that c-Myc over-expression imposes a "mutator phenotype". This may be an early and necessary requirement for the initial steps in chronic transformation as well as for subsequent evolutionary changes that produce important tumor behaviors such as invasiveness, metastasis, and acquisition of chemotherapy resistance.

Higher Intensity of Purifying Selection on >90% of the Human Genes Revealed by the Intrinsic Replacement Mutation Rates

For over 3 decades, the rate of replacement mutations has been assumed to be equal to, and estimated from, the rate of "strictly" neutral sequence divergence in noncoding regions and in silent-codon positions where mutations do not alter the amino acid encoded. This assumption is fundamental to estimating the fraction of harmful protein mutations and to identifying adaptive evolution at individual codons and proteins. We show that the assumption is not justifiable because a much larger fraction of codon positions is involved in hypermutable CpG dinucleotides as compared with the introns, leading to a higher expected replacement mutation rate per site in a vast majority of the genes. Consideration of this difference reveals a higher intensity of purifying natural selection than previously inferred in human genes. We also show that a much smaller number of genes are expected to be evolving with positive selection than that predicted using sequence divergence at intron and silent positions in the human genome. These patterns indicate the need for using new approaches for estimating rates of amino acid-altering mutations in order to find positively selected genes and codons in genomes that contain hypermutable CpG's.

Defects in mismatch repair occur after APC mutations in the pathogenesis of sporadic colorectal tumours.

The roles of the intrinsic mutation rate and genomic instability in tumorigenesis are currently controversial. In most colorectal tumours, it is generally supposed that the first mutations occur at the adenomatous polyposis coli (APC) locus; APC mutations are thought to provide cells with a selective advantage but have no known effect on the mutation rate. It has also been suggested that genomic instability is the initiating event in colorectal tumorigenesis and, if this is true, mutations of DNA mismatch repair (MMR) genes (or at similar loci) are the most likely candidates. If defective MMR precedes APC mutations, the APC mutations of colon tumours with defective MMR and hence replication errors (RER+) should differ from those of RER- tumours, in at least three specific ways: (1) a higher frequency of allele loss at APC in RER- tumours; (2) more frameshift than nonsense mutations in RER+ tumours; and (3) APC mutations in simple repeat sequences [(N)n, (N1N2)n, or (N1N2N3)n] in RER+ tumours. We found no evidence that sporadic RER+ and RER- colon cancers (including cell lines) differ in any of these three ways. Although it remains possible that MMR is abnormal in tumours from HNPCC families before APC mutations occur, it is likely that in sporadic colon tumours, APC mutations, rather than genomic instability, are the initiating events in tumorigenesis.

Very slightly deleterious mutations and the molecular clock.

The model of very slightly deleterious mutations was examined from the standpoint of population genetics in relation to the molecular evolutionary clock. The distribution of selection coefficients of mutants (in terms of amino acid changes) with small effect is thought to be continuous around zero, with an average negative value. The variance of selection coefficients depends upon environmental diversity and hence on total population size of a species. By considering various examples of amino acid substitutions, the average and standard error of selection coefficients and the reciprocal of population size are assumed to have similar values. The model predicts negative correlation between evolutionary rate and population size. This effect is expected to be partially cancelled with the generation time effect of intrinsic mutation rate. Implications of this prediction on the molecular evolutionary clock are discussed.