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1.
Mol Cell ; 72(2): 222-238.e11, 2018 10 18.
Artículo en Inglés | MEDLINE | ID: mdl-30293786

RESUMEN

DNA polymerase stalling activates the ATR checkpoint kinase, which in turn suppresses fork collapse and breakage. Herein, we describe use of ATR inhibition (ATRi) as a means to identify genomic sites of problematic DNA replication in murine and human cells. Over 500 high-resolution ATR-dependent sites were ascertained using two distinct methods: replication protein A (RPA)-chromatin immunoprecipitation (ChIP) and breaks identified by TdT labeling (BrITL). The genomic feature most strongly associated with ATR dependence was repetitive DNA that exhibited high structure-forming potential. Repeats most reliant on ATR for stability included structure-forming microsatellites, inverted retroelement repeats, and quasi-palindromic AT-rich repeats. Notably, these distinct categories of repeats differed in the structures they formed and their ability to stimulate RPA accumulation and breakage, implying that the causes and character of replication fork collapse under ATR inhibition can vary in a DNA-structure-specific manner. Collectively, these studies identify key sources of endogenous replication stress that rely on ATR for stability.


Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteínas de la Ataxia Telangiectasia Mutada/genética , Replicación del ADN/genética , Repeticiones de Microsatélite/genética , Animales , Proteínas de Ciclo Celular/genética , Cromatina/genética , Inmunoprecipitación de Cromatina/métodos , Roturas del ADN de Doble Cadena , Daño del ADN/genética , Femenino , Inestabilidad Genómica/genética , Humanos , Ratones , Proteína de Replicación A/genética
2.
Genome Res ; 25(5): 736-49, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-25823460

RESUMEN

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.


Asunto(s)
Algoritmos , Genoma Humano , Técnicas de Genotipaje/métodos , Repeticiones de Microsatélite , Análisis de Secuencia de ADN/métodos , Secuencia de Bases , Humanos , Datos de Secuencia Molecular , Sensibilidad y Especificidad
3.
PLoS Genet ; 10(7): e1004498, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-25033203

RESUMEN

Interruptions of microsatellite sequences impact genome evolution and can alter disease manifestation. However, human polymorphism levels at interrupted microsatellites (iMSs) are not known at a genome-wide scale, and the pathways for gaining interruptions are poorly understood. Using the 1000 Genomes Phase-1 variant call set, we interrogated mono-, di-, tri-, and tetranucleotide repeats up to 10 units in length. We detected ∼26,000-40,000 iMSs within each of four human population groups (African, European, East Asian, and American). We identified population-specific iMSs within exonic regions, and discovered that known disease-associated iMSs contain alleles present at differing frequencies among the populations. By analyzing longer microsatellites in primate genomes, we demonstrate that single interruptions result in a genome-wide average two- to six-fold reduction in microsatellite mutability, as compared with perfect microsatellites. Centrally located interruptions lowered mutability dramatically, by two to three orders of magnitude. Using a biochemical approach, we tested directly whether the mutability of a specific iMS is lower because of decreased DNA polymerase strand slippage errors. Modeling the adenomatous polyposis coli tumor suppressor gene sequence, we observed that a single base substitution interruption reduced strand slippage error rates five- to 50-fold, relative to a perfect repeat, during synthesis by DNA polymerases α, ß, or η. Computationally, we demonstrate that iMSs arise primarily by base substitution mutations within individual human genomes. Our biochemical survey of human DNA polymerase α, ß, δ, κ, and η error rates within certain microsatellites suggests that interruptions are created most frequently by low fidelity polymerases. Our combined computational and biochemical results demonstrate that iMSs are abundant in human genomes and are sources of population-specific genetic variation that may affect genome stability. The genome-wide identification of iMSs in human populations presented here has important implications for current models describing the impact of microsatellite polymorphisms on gene expression.


Asunto(s)
Inestabilidad Genómica , Repeticiones de Microsatélite/genética , Polimorfismo de Nucleótido Simple/genética , Primates/genética , Alelos , Animales , Secuencia de Bases , Regulación de la Expresión Génica , Genoma Humano , Humanos , Población/genética
4.
Nucleic Acids Res ; 40(4): 1636-47, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22021378

RESUMEN

Microsatellite DNA synthesis represents a significant component of human genome replication that must occur faithfully. However, yeast replicative DNA polymerases do not possess high fidelity for microsatellite synthesis. We hypothesized that the structural features of Y-family polymerases that facilitate accurate translesion synthesis may promote accurate microsatellite synthesis. We compared human polymerases κ (Pol κ) and η (Pol η) fidelities to that of replicative human polymerase δ holoenzyme (Pol δ4), using the in vitro HSV-tk assay. Relative polymerase accuracy for insertion/deletion (indel) errors within 2-3 unit repeats internal to the HSV-tk gene concurred with the literature: Pol δ4 >> Pol κ or Pol η. In contrast, relative polymerase accuracy for unit-based indel errors within [GT](10) and [TC](11) microsatellites was: Pol κ ≥ Pol δ4 > Pol η. The magnitude of difference was greatest between Pols κ and δ4 with the [GT] template. Biochemically, Pol κ displayed less synthesis termination within the [GT] allele than did Pol δ4. In dual polymerase reactions, Pol κ competed with either a stalled or moving Pol δ4, thereby reducing termination. Our results challenge the ideology that pol κ is error prone, and suggest that DNA polymerases with complementary biochemical properties can function cooperatively at repetitive sequences.


Asunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , Inestabilidad de Microsatélites , Repeticiones de Microsatélite , Alelos , ADN/biosíntesis , Daño del ADN , ADN Polimerasa III/metabolismo , Humanos , Mutación INDEL
5.
Mutat Res ; 743-744: 67-77, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23206442

RESUMEN

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.


Asunto(s)
Inestabilidad de Microsatélites , Neoplasias/genética , Reparación de la Incompatibilidad de ADN , Humanos , Mutación , Fenotipo
6.
DNA Repair (Amst) ; 119: 103402, 2022 11.
Artículo en Inglés | MEDLINE | ID: mdl-36116264

RESUMEN

G-quadruplexes (G4s), a type of non-B DNA, play important roles in a wide range of molecular processes, including replication, transcription, and translation. Genome integrity relies on efficient and accurate DNA synthesis, and is compromised by various stressors, to which non-B DNA structures such as G4s can be particularly vulnerable. However, the impact of G4 structures on DNA polymerase fidelity is largely unknown. Using an in vitro forward mutation assay, we investigated the fidelity of human DNA polymerases delta (δ4, four-subunit), eta (η), and kappa (κ) during synthesis of G4 motifs representing those in the human genome. The motifs differ in sequence, topology, and stability, features that may affect DNA polymerase errors. Polymerase error rate hierarchy (δ4 < κ < Î·) is largely maintained during G4 synthesis. Importantly, we observed unique polymerase error signatures during synthesis of VEGF G4 motifs, stable G4s which form parallel topologies. These statistically significant errors occurred within, immediately flanking, and encompassing the G4 motif. For pol δ4, the errors were deletions, insertions and complex errors within the G4 or encompassing the G4 motif and surrounding sequence. For pol η, the errors occurred in 3' sequences flanking the G4 motif. For pol κ, the errors were frameshift mutations within G-tracts of the G4. Because these error signatures were not observed during synthesis of an antiparallel G4 and, to a lesser extent, a hybrid G4, we suggest that G4 topology and/or stability could influence polymerase fidelity. Using in silico analyses, we show that most polymerase errors are predicted to have minimal effects on predicted G4 stability. Our results provide a unique view of G4s not previously elucidated, showing that G4 motif heterogeneity differentially influences polymerase fidelity within the motif and flanking sequences. Thus, our study advances the understanding of how DNA polymerase errors contribute to G4 mutagenesis.


Asunto(s)
G-Cuádruplex , ADN/genética , Replicación del ADN , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Humanos , Factor A de Crecimiento Endotelial Vascular/genética
7.
Nucleic Acids Res ; 36(2): 688-96, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-18079151

RESUMEN

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 kappa) inserts dGMP and dCMP within the [T](11) mononucleotide repeat, producing an interrupted 12-bp allele. Polymerase beta 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 kappa and polymerase alpha-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.


Asunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , Repeticiones de Microsatélite , Mutagénesis , Alelos , Secuencia de Bases , ADN/biosíntesis , Humanos , Mutación , Nucleótidos , Nucleótidos de Timina/análisis
8.
Mol Carcinog ; 48(4): 379-88, 2009 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-19306292

RESUMEN

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 approximately 10(-6) to a high of approximately 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.


Asunto(s)
ADN/genética , Genoma Humano , Repeticiones de Microsatélite/genética , Mutagénesis , Mutación/genética , Humanos
9.
Cell Rep ; 27(4): 1151-1164.e5, 2019 04 23.
Artículo en Inglés | MEDLINE | ID: mdl-31018130

RESUMEN

Common fragile sites (CFSs) are genomic regions that display gaps and breaks in human metaphase chromosomes under replication stress and are often deleted in cancer cells. We studied an ∼300-bp subregion (Flex1) of human CFS FRA16D in yeast and found that it recapitulates characteristics of CFS fragility in human cells. Flex1 fragility is dependent on the ability of a variable-length AT repeat to form a cruciform structure that stalls replication. Fragility at Flex1 is initiated by structure-specific endonuclease Mus81-Mms4 acting together with the Slx1-4/Rad1-10 complex, whereas Yen1 protects Flex1 against breakage. Sae2 is required for healing of Flex1 after breakage. Our study shows that breakage within a CFS can be initiated by nuclease cleavage at forks stalled at DNA structures. Furthermore, our results suggest that CFSs are not just prone to breakage but also are impaired in their ability to heal, and this deleterious combination accounts for their fragility.


Asunto(s)
Rotura Cromosómica , Sitios Frágiles del Cromosoma/genética , Cromosomas Humanos Par 16/genética , Replicación del ADN , Endonucleasas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , ADN Polimerasa III/genética , ADN Polimerasa III/metabolismo , Humanos , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Secuencias Repetidas en Tándem
10.
DNA Repair (Amst) ; 57: 1-11, 2017 09.
Artículo en Inglés | MEDLINE | ID: mdl-28605669

RESUMEN

Common fragile sites (CFSs) are inherently unstable genomic loci that are recurrently altered in human tumor cells. Despite their instability, CFS are ubiquitous throughout the human genome and associated with large tumor suppressor genes or oncogenes. CFSs are enriched with repetitive DNA sequences, one feature postulated to explain why these loci are inherently difficult to replicate, and sensitive to replication stress. We have shown that specialized DNA polymerases (Pols) η and κ replicate CFS-derived sequences more efficiently than the replicative Pol δ. However, we lacked an understanding of how these enzymes cooperate to ensure efficient CFS replication. Here, we designed a model of lagging strand replication with RFC loaded PCNA that allows for maximal activity of the four-subunit human Pol δ holoenzyme, Pol η, and Pol κ in polymerase mixing assays. We discovered that Pol η and κ are both able to exchange with Pol δ stalled at repetitive CFS sequences, enhancing Normalized Replication Efficiency. We used this model to test the impact of PCNA mono-ubiquitination on polymerase exchange, and found no change in polymerase cooperativity in CFS replication compared with unmodified PCNA. Finally, we modeled replication stress in vitro using aphidicolin and found that Pol δ holoenzyme synthesis was significantly inhibited in a dose-dependent manner, preventing any replication past the CFS. Importantly, Pol η and κ were still proficient in rescuing this stalled Pol δ synthesis, which may explain, in part, the CFS instability phenotype of aphidicolin-treated Pol η and Pol κ-deficient cells. In total, our data support a model wherein Pol δ stalling at CFSs allows for free exchange with a specialized polymerase that is not driven by PCNA.


Asunto(s)
Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Repeticiones de Microsatélite , Antígeno Nuclear de Célula en Proliferación/metabolismo , Sitios Frágiles del Cromosoma , ADN/metabolismo , ADN Polimerasa III/metabolismo , Inestabilidad Genómica , Humanos , Proteína de Replicación C/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitinación
11.
J Mol Biol ; 335(3): 745-59, 2004 Jan 16.
Artículo en Inglés | MEDLINE | ID: mdl-14687571

RESUMEN

Microsatellite DNA sequences are ubiquitous in the human genome, and mutation rates of these repetitive sequences vary with respect to DNA sequence as well as length. We have analyzed polymerase-DNA interactions as a function of microsatellite sequence, using polypyrimidine/polypurine di- and tetranucleotide alleles representative of those found in the human genome. Using an in vitro primer extension assay and the mammalian DNA polymerase alpha-primase complex, we have observed a polymerase termination profile for each microsatellite that is unique to that allele. Interestingly, a periodic termination profile with an interval size (9-11 nucleotides) unrelated to microsatellite unit length was observed for the [TC](20) and [TTCC](9) templates. In contrast, a unit-punctuated polymerase termination profile was found for the longer polypurine templates. We detected strong polymerase pauses within the [TC](20) allele at low reaction pH which were eliminated by the addition of deaza-dGTP, consistent with these specific pauses being a consequence of triplex DNA formation during DNA synthesis. Quantitatively, a strand bias was observed in the primer extension assay, in that polymerase synthesis termination is more intense when the polypurine sequence serves as the template, relative to its complementary polypyrimidine sequence. The HSV-tk forward mutation assay was utilized to determine the corresponding polymerase alpha-primase error frequencies and specificities at the microsatellite alleles. A higher microsatellite polymerase error frequency (50x10(-4) to 60x10(-4)) was measured when polypurine sequences serve as templates for DNA synthesis, relative to the polypyrimidine template (18x10(-4)). Thus, a positive correlation exists between polymerase alpha-primase pausing and mutagenesis within microsatellite DNA alleles.


Asunto(s)
ADN Polimerasa I/genética , ADN Primasa/genética , Repeticiones de Microsatélite/genética , Mutagénesis , Moldes Genéticos , Animales , Secuencia de Bases , Bovinos , ADN , Análisis Mutacional de ADN , Cartilla de ADN , Replicación del ADN , Cinética , Nucleótidos de Purina , Nucleótidos de Pirimidina
12.
DNA Repair (Amst) ; 11(4): 381-90, 2012 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-22341651

RESUMEN

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 ß (Pol ß) 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.


Asunto(s)
Sustitución de Aminoácidos , Transformación Celular Neoplásica/genética , ADN Polimerasa beta/genética , Mutación , Neoplasias Gástricas/enzimología , Neoplasias Gástricas/patología , Animales , Secuencia de Bases , Línea Celular Tumoral , Proliferación Celular , ADN/metabolismo , ADN Ligasa (ATP) , ADN Ligasas/metabolismo , ADN Polimerasa beta/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Inestabilidad Genómica/genética , Humanos , Ratones , Datos de Secuencia Molecular , Invasividad Neoplásica , Fenotipo , Proteínas de Unión a Poli-ADP-Ribosa , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X , Proteínas de Xenopus
13.
DNA Repair (Amst) ; 10(5): 497-505, 2011 May 05.
Artículo en Inglés | MEDLINE | ID: mdl-21429821

RESUMEN

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.


Asunto(s)
ADN Polimerasa III/metabolismo , ADN Polimerasa II/metabolismo , Repeticiones de Microsatélite/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Secuencia de Bases , Reparación de la Incompatibilidad de ADN/genética , ADN Polimerasa II/genética , ADN Polimerasa II/aislamiento & purificación , ADN Polimerasa III/genética , ADN Polimerasa III/aislamiento & purificación , Replicación del ADN/genética , Holoenzimas , Humanos , Datos de Secuencia Molecular , Mutación/genética , Simplexvirus/genética , Timidina Quinasa/genética
14.
Cancer Res ; 70(2): 431-5, 2010 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-20068152

RESUMEN

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.


Asunto(s)
Reparación de la Incompatibilidad de ADN , Inestabilidad de Microsatélites , Neoplasias/genética , Animales , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Neoplasias Colorrectales Hereditarias sin Poliposis/patología , Humanos , Neoplasias/patología
15.
Genome Biol Evol ; 2: 620-35, 2010.
Artículo en Inglés | MEDLINE | ID: mdl-20668018

RESUMEN

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.


Asunto(s)
Biología Computacional/métodos , Repeticiones de Microsatélite/genética , Secuencia Rica en At , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Humanos , Mutación , Polimorfismo Genético/genética
16.
Biochemistry ; 44(48): 15664-73, 2005 Dec 06.
Artículo en Inglés | MEDLINE | ID: mdl-16313169

RESUMEN

Studies show that 30% of 189 tumors sequenced to date express variants of the polymerase beta (pol beta) protein that are not present in normal tissue. This raises the possibility that variants of pol beta might be linked to the etiology of cancer. Here, we characterize the I260M prostate-cancer-associated variant of pol beta. Ile260 is a key residue of the hydrophobic hinge that is important for the closing of the polymerase. In this study, we demonstrate that the I260M variant is a sequence context-dependent mutator polymerase. Specifically, I260M is a mutator for misalignment-mediated errors in dipyrimidine sequences. I260M is also a low-fidelity polymerase with regard to the induction of transversions within specific sequence contexts. Our results suggest that the hinge influences the geometry of the DNA within the polymerase active site that is important for accurate DNA synthesis. Importantly, characterization of the I260M variant shows that it has a functional phenotype that could be linked to the etiology or malignant progression of human cancer.


Asunto(s)
Transformación Celular Neoplásica/genética , ADN Polimerasa beta/genética , Reparación del ADN/fisiología , Mutación/genética , Neoplasias de la Próstata/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Dicroismo Circular , ADN Polimerasa beta/metabolismo , ADN Circular , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Neoplasias de la Próstata/enzimología , Pliegue de Proteína , Moldes Genéticos
17.
Mol Carcinog ; 34(3): 140-50, 2002 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-12112308

RESUMEN

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

Asunto(s)
Repeticiones de Microsatélite , Mutación , Alelos , Células Cultivadas , Análisis Mutacional de ADN/métodos , Femenino , Vectores Genéticos/metabolismo , Humanos , Mutagénesis , Simplexvirus/genética , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/genética , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/metabolismo , Timidina Quinasa/genética
18.
Biochemistry ; 41(33): 10490-8, 2002 Aug 20.
Artículo en Inglés | MEDLINE | ID: mdl-12173936

RESUMEN

Mutations arising in microsatellite DNA are associated with neurological diseases and cancer. To elucidate the molecular basis of microsatellite mutation, we have determined the in vitro polymerase error frequencies at microsatellite sequences representative of those found in the human genome: [GT/CA](10), [TC/AG](11), and [TTCC/AAGG](9). DNA templates contained the microsatellites inserted in-frame into the 5' region of the herpes simplex virus thymidine kinase (HSV-tk) gene. Polymerase beta (polbeta) error frequencies were quantitated in microsatellite sequences, relative to frame-shift error frequencies in coding sequences, from the same DNA synthesis reaction. The polbeta error frequencies within the dinucleotide sequences were (2-9) x 10(-3), 14-72-fold higher than the ssDNA template frequencies. The polbeta error frequencies within the tetranucleotide sequences were (4-6) x 10(-3), a 4-13-fold increase over background. Strand biases were observed for the [TC/AG](11) and [TTCC/AAGG](9) alleles, in which more errors were produced when the purine strand served as a template. Mutations within each microsatellite included noncanonical base substitution events and single nucleotide deletions as well as the expected unit length changes. An exponential relationship was observed between the polymerase error frequency per site and both the number of repetitive units and total length of the allele. Our observations are consistent with the strand slippage model of microsatellite mutagenesis and demonstrate that DNA sequence and/or structural differences result in mutational strand biases. To our knowledge, this is the first direct quantitation of DNA polymerase errors in vitro using template microsatellite sequences.


Asunto(s)
Disparidad de Par Base/genética , ADN Polimerasa beta/genética , ADN Polimerasa beta/metabolismo , Mutación del Sistema de Lectura , Repeticiones de Microsatélite/genética , Secuencia de Bases , Análisis Mutacional de ADN/métodos , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Repeticiones de Dinucleótido/genética , Vectores Genéticos/síntesis química , Herpesvirus Humano 1/enzimología , Herpesvirus Humano 1/genética , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Eliminación de Secuencia , Moldes Genéticos , Timidina Quinasa/genética , Proteínas Virales/genética
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