Suzanne E. Hile

Defective Mismatch Repair, Microsatellite Mutation Bias, and Variability in Clinical Cancer Phenotypes

Microsatellite instability is associated with 10% to 15% of colorectal, endometrial, ovarian, and gastric cancers, and has long been used as a diagnostic tool for hereditary nonpolyposis colorectal carcinoma-related cancers. Tumor-specific length alterations within microsatellites are generally accepted to be a consequence of strand slippage events during DNA replication, which are uncorrected due to a defective postreplication mismatch repair (MMR) system. Mutations arising within microsatellites associated with critical target genes are believed to play a causative role in the evolution of MMR-defective tumors. In this review, we summarize current evidence of mutational biases within microsatellites arising as a consequence of intrinsic DNA sequence effects as well as variation in MMR efficiency. Microsatellite mutational biases are generally not considered during clinical testing; however, we suggest that such biases may be clinically significant as a factor contributing to phenotypic variation among microsatellite instability-positive tumors.

What Is a Microsatellite: A Computational and Experimental Definition Based upon Repeat Mutational Behavior at A/T and GT/AC Repeats

Microsatellites are abundant in eukaryotic genomes and have high rates of strand slippage-induced repeat number alterations. They are popular genetic markers, and their mutations are associated with numerous neurological diseases. However, the minimal number of repeats required to constitute a microsatellite has been debated, and a definition of a microsatellite that considers its mutational behavior has been lacking. To define a microsatellite, we investigated slippage dynamics for a range of repeat sizes, utilizing two approaches. Computationally, we assessed length polymorphism at repeat loci in ten ENCODE regions resequenced in four human populations, assuming that the occurrence of polymorphism reflects strand slippage rates. Experimentally, we determined the in vitro DNA polymerase-mediated strand slippage error rates as a function of repeat number. In both approaches, we compared strand slippage rates at tandem repeats with the background slippage rates. We observed two distinct modes of mutational behavior. At small repeat numbers, slippage rates were low and indistinguishable from background measurements. A marked transition in mutability was observed as the repeat array lengthened, such that slippage rates at large repeat numbers were significantly higher than the background rates. For both mononucleotide and dinucleotide microsatellites studied, the transition length corresponded to a similar number of nucleotides (approximately 10). Thus, microsatellite threshold is determined not by the presence/absence of strand slippage at repeats but by an abrupt alteration in slippage rates relative to background. These findings have implications for understanding microsatellite mutagenesis, standardization of genome-wide microsatellite analyses, and predicting polymorphism levels of individual microsatellite loci.

Every microsatellite is different: Intrinsic DNA features dictate mutagenesis of common microsatellites present in the human genome.

Microsatellite sequences are ubiquitous in the human genome and are important regulators of genome function. Here, we examine the mutational mechanisms governing the stability of highly abundant mono‐, di‐, and tetranucleotide microsatellites. Microsatellite mutation rate estimates from pedigree analyses and experimental models range from a low of ∼10−6 to a high of ∼10−2 mutations per locus per generation. The vast majority of observed mutational variation can be attributed to features intrinsic to the allele itself, including motif size, length, and sequence composition. A greater than linear relationship between motif length and mutagenesis has been observed in several model systems. Motif sequence differences contribute up to 10‐fold to the variation observed in human cell mutation rates. The major mechanism of microsatellite mutagenesis is strand slippage during DNA synthesis. DNA polymerases produce errors within microsatellites at a frequency that is 10‐ to 100‐fold higher than the frequency of frameshifts in coding sequences. Motif sequence significantly affects both polymerase error rate and specificity, resulting in strand biases within complementary microsatellites. Importantly, polymerase errors within microsatellites include base substitutions, deletions, and complex mutations, all of which produced interrupted alleles from pure microsatellites. Postreplication mismatch repair efficiency is affected by microsatellite motif size and sequence, also contributing to the observed variation in microsatellite mutagenesis. Inhibition of DNA synthesis within common microsatellites is highly sequence‐dependent, and is positively correlated with the production of errors. DNA secondary structure within common microsatellites can account for some DNA polymerase pause sites, and may be an important factor influencing mutational specificity.

DNA polymerase kappa produces interrupted mutations and displays polar pausing within mononucleotide microsatellite sequences

Microsatellites are ubiquitously present in eukaryotic genomes and are implicated as positive factors in evolution. At the nucleotide level, microsatellites undergo slippage events that alter allele length and base changes that interrupt the repetitive tract. We examined DNA polymerase errors within a [T]11 microsatellite using an in vitro assay that preferentially detects mutations other than unit changes. We observed that human DNA polymerase kappa (Pol κ) inserts dGMP and dCMP within the [T]11 mononucleotide repeat, producing an interrupted 12-bp allele. Polymerase β produced such interruptions at a lower frequency. These data demonstrate that DNA polymerases are capable of directly producing base interruptions within microsatellites. At the molecular level, expanded microsatellites have been implicated in DNA replication fork stalling. Using an in vitro primer extension assay, we observed sequence-specific synthesis termination by DNA polymerases within mononucleotides. Quantitatively, intense, polar pausing was observed for both pol κ and polymerase α-primase within a [T]11 allele. A mechanism is proposed in which pausing results from DNA bending within the duplex stem of the nascent DNA. Our data support the concept of a microsatellite life-cycle, and are consistent with the models in which DNA sequence or secondary structures contributes to non-uniform rates of replication fork progression.

Mutation rate and specificity analysis of tetranucleotide microsatellite DNA alleles in somatic human cells

We have systematically varied microsatellite sequence composition to determine the effects of repeat unit size, G+C content, and DNA secondary structure on microsatellite stability in human cells. The microsatellites were inserted in frame within the 5′ region of the herpes simplex virus thymidine kinase (HSV‐tk) gene. The polypyrimidine/polypurine microsatellites displayed enhanced S1 nuclease sensitivity in vitro, consistent with the formation of non–B‐form DNA structures. Microsatellite mutagenesis studies were performed with a shuttle vector system in which inactivating HSV‐tk mutations are measured after replication in a nontumorigenic cell line. A significant increase in the HSV‐tk mutation frequency per cell generation was observed after insertion of [TTCC/AAGG]9, [TTTC/AAAG]9, or [TCTA/AGAT]9 sequences (P ≤ 0.0002), relative to the HSV‐tk gene control. We observed that the G + C content of the microsatellite may affect mutagenesis, as the mean microsatellite mutation rates of the [TTTC/AAAG]9 and [TCTA/AGAT]9 alleles were sevenfold and 11‐fold higher, respectively, than the [TTCC/AAGG]9 allele. A bias toward expansion mutations was noted for the majority of clones bearing the [TTCC/AAGG]9 allele as well as a [TC/AG]17 microsatellite of similar allele length. The mean microsatellite mutation rate of the [TTCC/AAGG]9 allele did not differ significantly from that for a [TC/AG]11 allele, demonstrating that these tetranucleotide and dinucleotide alleles are of equivalent stability. It is known that microsatellite mutagenesis is affected by the number of repeat units within an allele. Our data suggest that additional biochemical factors may regulate both the rate and specificity of somatic cell microsatellite mutagenesis.

Accurate typing of short tandem repeats from genome-wide sequencing data and its applications

Short tandem repeats (STRs) are implicated in dozens of human genetic diseases and contribute significantly to genome variation and instability. Yet profiling STRs from short-read sequencing data is challenging because of their high sequencing error rates. Here, we developed STR-FM, short tandem repeat profiling using flank-based mapping, a computational pipeline that can detect the full spectrum of STR alleles from short-read data, can adapt to emerging read-mapping algorithms, and can be applied to heterogeneous genetic samples (e.g., tumors, viruses, and genomes of organelles). We used STR-FM to study STR error rates and patterns in publicly available human and in-house generated ultradeep plasmid sequencing data sets. We discovered that STRs sequenced with a PCR-free protocol have up to ninefold fewer errors than those sequenced with a PCR-containing protocol. We constructed an error correction model for genotyping STRs that can distinguish heterozygous alleles containing STRs with consecutive repeat numbers. Applying our model and pipeline to Illumina sequencing data with 100-bp reads, we could confidently genotype several disease-related long trinucleotide STRs. Utilizing this pipeline, for the first time we determined the genome-wide STR germline mutation rate from a deeply sequenced human pedigree. Additionally, we built a tool that recommends minimal sequencing depth for accurate STR genotyping, depending on repeat length and sequencing read length. The required read depth increases with STR length and is lower for a PCR-free protocol. This suite of tools addresses the pressing challenges surrounding STR genotyping, and thus is of wide interest to researchers investigating disease-related STRs and STR evolution.

Tumor-Specific Microsatellite Instability: Do Distinct Mechanisms Underlie the MSI-L and EMAST Phenotypes?

Microsatellite DNA sequences display allele length alterations or microsatellite instability (MSI) in tumor tissues, and MSI is used diagnostically for tumor detection and classification. We discuss the known types of tumor-specific MSI patterns and the relevant mechanisms underlying each pattern. Mutation rates of individual microsatellites vary greatly, and the intrinsic DNA features of motif size, sequence, and length contribute to this variation. MSI is used for detecting mismatch repair (MMR)-deficient tumors, which display an MSI-high phenotype due to genome-wide microsatellite destabilization. Because several pathways maintain microsatellite stability, tumors that have undergone other events associated with moderate genome instability may display diagnostic MSI only at specific di- or tetranucleotide markers. We summarize evidence for such alternative MSI forms (A-MSI) in sporadic cancers, also referred to as MSI-low and EMAST. While the existence of A-MSI is not disputed, there is disagreement about the origin and pathologic significance of this phenomenon. Although ambiguities due to PCR methods may be a source, evidence exists for other mechanisms to explain tumor-specific A-MSI. Some portion of A-MSI tumors may result from random mutational events arising during neoplastic cell evolution. However, this mechanism fails to explain the specificity of A-MSI for di- and tetranucleotide instability. We present evidence supporting the alternative argument that some A-MSI tumors arise by a distinct genetic pathway, and give examples of DNA metabolic pathways that, when altered, may be responsible for instability at specific microsatellite motifs. Finally, we suggest that A-MSI in tumors could be molecular signatures of environmental influences and DNA damage. Importantly, A-MSI occurs in several pre-neoplastic inflammatory states, including inflammatory bowel diseases, consistent with a role of oxidative stress in A-MSI. Understanding the biochemical basis of A-MSI tumor phenotypes will advance the development of new diagnostic tools and positively impact the clinical management of individual cancers.

The human gastric cancer-associated DNA polymerase β variant D160N is a mutator that induces cellular transformation.

Approximately 30% of human tumors sequenced to date harbor mutations in the POLB gene that are not present in matched normal tissue. Many mutations give rise to enzymes that contain non-synonymous single amino acid substitutions, several of which have been found to have aberrant activity or fidelity and transform cells when expressed. The DNA Polymerase β (Pol β) variant Asp160Asn (D160N) was first identified in a gastric tumor. Expression of D160N in cells induces cellular transformation as measured by hyperproliferation, focus formation, anchorage-independent growth and invasion. Here, we show that D160N is an active mutator polymerase that induces complex mutations. Our data support the interpretation that complex mutagenesis is the underlying mechanism of the observed cellular phenotypes, all of which are linked to tumorigenesis or tumor progression.

The In Vitro Fidelity Of Yeast DNA Polymerase δ And Polymerase ε Holoenzymes During Dinucleotide Microsatellite DNA Synthesis

Elucidating the sources of genetic variation within microsatellite alleles has important implications for understanding the etiology of human diseases. Mismatch repair is a well described pathway for the suppression of microsatellite instability. However, the cellular polymerases responsible for generating microsatellite errors have not been fully described. We address this gap in knowledge by measuring the fidelity of recombinant yeast polymerase δ (Pol δ) and ɛ (Pol ɛ) holoenzymes during synthesis of a [GT/CA] microsatellite. The in vitro HSV-tk forward assay was used to measure DNA polymerase errors generated during gap-filling of complementary GT(10) and CA(10)-containing substrates and ∼90 nucleotides of HSV-tk coding sequence surrounding the microsatellites. The observed mutant frequencies within the microsatellites were 4 to 30-fold higher than the observed mutant frequencies within the coding sequence. More specifically, the rate of Pol δ and Pol ɛ misalignment-based insertion/deletion errors within the microsatellites was ∼1000-fold higher than the rate of insertion/deletion errors within the HSV-tk gene. Although the most common microsatellite error was the deletion of a single repeat unit, ∼ 20% of errors were deletions of two or more units for both polymerases. The differences in fidelity for wild type enzymes and their exonuclease-deficient derivatives were ∼2-fold for unit-based microsatellite insertion/deletion errors. Interestingly, the exonucleases preferentially removed potentially stabilizing interruption errors within the microsatellites. Since Pol δ and Pol ɛ perform not only the bulk of DNA replication in eukaryotic cells but also are implicated in performing DNA synthesis associated with repair and recombination, these results indicate that microsatellite errors may be introduced into the genome during multiple DNA metabolic pathways.

Alkylation-induced frameshift mutagenesis during in vitro DNA synthesis by DNA polymerases α and β

Abstract We have analyzed the mutational spectra produced during in vitro DNA synthesis by DNA polymerase α-primase and DNA polymerase β. The polymerase mutation frequency as measured in the in vitro herpes simplex virus thymidine kinase (HSV- tk ) forward assay was increased when reactions utilized single-stranded DNA templates randomly modified by 20 mM N -ethyl- N -nitrosourea (ENU), relative to solvent-treated templates. A 20- to 50-fold increase in the frequency of G→A transition mutations was observed for both polymerases, as expected due to mispairing by O 6 -ethylguanine lesions. Strikingly, ENU treatment of the template also resulted in a five- to 12-fold increased frequency of frameshift errors at heteropolymeric (non-repetitive) template sequences produced by polymerase β and polymerase α-primase, respectively. The increased proportion of frameshift mutations at heteropolymeric sequences relative to homopolymeric (repetitive) sequences produced by each polymerase in response to ENU damage was statistically significant. For polymerase α-primase, one-base deletion errors at template guanine residues was the second most frequent mutational event, observed at a frequency only four-fold lower than the G→A transition frequency. In the polymerase β reactions, the frequency of insertion errors at homopolymeric (repetitive) sequences was increased six-fold using alkylated templates, relative to solvent controls. The frequency of such insertion errors was only three-fold lower than the frequency of G→A transition errors by polymerase β. Although ENU is generally regarded as a potent base substitution mutagen, these data show that monofunctional alkylating agents are capable of inducing frameshift mutations in vitro. Alkylation-induced frameshift mutations occur in both repetitive and non-repetitive DNA sequences; however, the mutational specificity is dependent upon the DNA polymerase.