Joint Inference of Microsatellite Mutation Models, Population History and Genealogies Using Transdimensional Markov Chain Monte Carlo

We provide a framework for Bayesian coalescent inference from microsatellite data that enables inference of population history parameters averaged over microsatellite mutation models. To achieve this we first implemented a rich family of microsatellite mutation models and related components in the software package BEAST. BEAST is a powerful tool that performs Bayesian MCMC analysis on molecular data to make coalescent and evolutionary inferences. Our implementation permits the application of existing nonparametric methods to microsatellite data. The implemented microsatellite models are based on the replication slippage mechanism and focus on three properties of microsatellite mutation: length dependency of mutation rate, mutational bias toward expansion or contraction, and number of repeat units changed in a single mutation event. We develop a new model that facilitates microsatellite model averaging and Bayesian model selection by transdimensional MCMC. With Bayesian model averaging, the posterior distributions of population history parameters are integrated across a set of microsatellite models and thus account for model uncertainty. Simulated data are used to evaluate our method in terms of accuracy and precision of θ estimation and also identification of the true mutation model. Finally we apply our method to a red colobus monkey data set as an example

Characterization of a replicative DNA polymerase mutant with reduced fidelity and increased translesion synthesis capacity

Changing a highly conserved amino acid in motif A of any of the four yeast family B DNA polymerases, DNA polymerase α, δ, ɛ or ζ, results in yeast strains with elevated mutation rates. In order to better understand this phenotype, we have performed structure–function studies of homologous mutants of RB69 DNA polymerase (RB69 pol), a structural model for family B members. When Leu415 in RB69 pol is replaced with phenylalanine or glycine, the mutant polymerases retain high-catalytic efficiency for correct nucleotide incorporation, yet have increased error rates due to increased misinsertion, increased mismatch extension and inefficient proofreading. The Leu415Phe mutant also has increased dNTP insertion efficiency opposite a template 8-oxoG and opposite an abasic site. The 2.5 Å crystal structure of a ternary complex of RB69 L415F pol with a correctly base-paired incoming dTTP reveals that the phenylalanine ring is accommodated within a cavity seen in the wild-type enzyme, without steric clash or major change in active site geometry, consistent with retention of high-catalytic efficiency for correct incorporation. In addition, slight structural differences were observed that could be relevant to the reduced fidelity of L415F RB69 pol

The nucleotide composition of microsatellites impacts both replication fidelity and mismatch repair in human colorectal cells

Microsatellite instability is a key mechanism of colon carcinogenesis. We have previously studied mutations within a (CA)13 microsatellite using an enhanced green fluorescent protein (EGFP)-based reporter assay that allows the distinction of replication errors and mismatch repair (MMR) activity. Here we utilize this assay to compare mutations of mono- and dinucleotide repeats in human colorectal cells. HCT116 and HCT116+chr3 cells were stably transfected with EGFP-based plasmids harboring A10, G10, G16, (CA)13 and (CA)26 repeats. EGFP-positive mutant fractions were quantitated by flow cytometry, mutation rates were calculated and the mutant spectrum was analyzed by cycle sequencing. EGFP fluorescence pattern changed with the microsatellite's nucleotide sequence and cell type and clonal variations were observed in mononucleotide repeats. Replication errors (as calculated in HCT116) at A10 repeats were 5–10-fold higher than in G10, G16 were 30-fold higher than G10 and (CA)26 were 10-fold higher than (CA)13. The mutation rates in hMLH1-proficient HCT116+chr3 were 30–230-fold lower than in HCT116. MMR was more efficient in G16 than in A10 clones leading to a higher stability of poly-G tracts. Mutation spectra revealed predominantly 1-unit deletions in A10, (CA)13 and G10 and 2-unit deletions or 1-unit insertion in (CA)26. These findings indicate that both replication fidelity and MMR are affected by the microsatellite's nucleotide composition

Cloning and Characterization of zRICH, a 2′,3′-Cyclic-Nucleotide 3′-Phosphodiesterase Induced During Zebrafish Optic Nerve Regeneration

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65941/1/j.1471-4159.1999.721362.x.pd

Use of RecA fusion proteins to induce genomic modifications in zebrafish

The bacterial recombinase RecA forms a nucleic acid-protein filament on single-stranded (ss) DNA during the repair of double-strand breaks (DSBs) that efficiently undergoes a homology search and engages in pairing with the complementary DNA sequence. We utilized the pairing activity of RecA–DNA filaments to tether biochemical activities to specific chromosomal sites. Different filaments with chimeric RecA proteins were tested for the ability to induce loss of heterozygosity at the golden locus in zebrafish after injection at the one-cell stage. A fusion protein between RecA containing a nuclear localization signal (NLS) and the DNA-binding domain of Gal4 (NLS-RecA-Gal4) displayed the most activity. Our results demonstrate that complementary ssDNA filaments as short as 60 nucleotides coated with NLS-RecA-Gal4 protein are able to cause loss of heterozygosity in ∼3% of the injected embryos. We demonstrate that lesions in ∼9% of the F0 zebrafish are transmitted to subsequent generations as large chromosomal deletions. Co-injection of linear DNA with the NLS-RecA-Gal4 DNA filaments promotes the insertion of the DNA into targeted genomic locations. Our data support a model whereby NLS-RecA-Gal4 DNA filaments bind to complementary target sites on chromatin and stall DNA replication forks, resulting in a DNA DSB

Alkylation damage causes MMR-dependent chromosomal instability in vertebrate embryos

SN1-type alkylating agents, like N-methyl-N-nitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU), are potent mutagens. Exposure to alkylating agents gives rise to O6-alkylguanine, a modified base that is recognized by DNA mismatch repair (MMR) proteins but is not repairable, resulting in replication fork stalling and cell death. We used a somatic mutation detection assay to study the in vivo effects of alkylation damage on lethality and mutation frequency in developing zebrafish embryos. Consistent with the damage-sensing role of the MMR system, mutant embryos lacking the MMR enzyme MSH6 displayed lower lethality than wild-type embryos after exposure to ENU and MNU. In line with this, alkylation-induced somatic mutation frequencies were found to be higher in wild-type embryos than in the msh6 loss-of-function mutants. These mutations were found to be chromosomal aberrations that may be caused by chromosomal breaks that arise from stalled replication forks. As these chromosomal breaks arise at replication, they are not expected to be repaired by non-homologous end joining. Indeed, Ku70 loss-of-function mutants were found to be equally sensitive to ENU as wild-type embryos. Taken together, our results suggest that in vivo alkylation damage results in chromosomal instability and cell death due to aberrantly processed MMR-induced stalled replication forks

An Integrin-Dependent Role of Pouch Endoderm in Hyoid Cartilage Development

Pharyngeal endoderm is essential for and can reprogram development of the head skeleton. Here we investigate the roles of specific endodermal structures in regulating craniofacial development. We have isolated an integrinα5 mutant in zebrafish that has region-specific losses of facial cartilages derived from hyoid neural crest cells. In addition, the cranial muscles that normally attach to the affected cartilage region and their associated nerve are secondarily reduced in integrinα5(−) animals. Earlier in development, integrinα5 mutants also have specific defects in the formation of the first pouch, an outpocketing of the pharyngeal endoderm. By fate mapping, we show that the cartilage regions that are lost in integrinα5 mutants develop from neural crest cells directly adjacent to the first pouch in wild-type animals. Furthermore, we demonstrate that Integrinα5 functions in the endoderm to control pouch formation and cartilage development. Time-lapse recordings suggest that the first pouch promotes region-specific cartilage development by regulating the local compaction and survival of skeletogenic neural crest cells. Thus, our results reveal a hierarchy of tissue interactions, at the top of which is the first endodermal pouch, which locally coordinates the development of multiple tissues in a specific region of the vertebrate face. Lastly, we discuss the implications of a mosaic assembly of the facial skeleton for the evolution of ray-finned fish

DNA models of trinucleotide frameshift deletions: the formation of loops and bulges at the primer–template junction

Although mechanisms of single-nucleotide residue deletion have been investigated, processes involved in the loss of longer nucleotide sequences during DNA replication are poorly understood. Previous reports have shown that in vitro replication of a 3′-TGC TGC template sequence can result in the deletion of one 3′-TGC. We have used low-energy circular dichroism (CD) and fluorescence spectroscopy to investigate the conformations and stabilities of DNA models of the replication intermediates that may be implicated in this frameshift. Pyrrolocytosine or 2-aminopurine residues, site-specifically substituted for cytosine or adenine in the vicinity of extruded base sequences, were used as spectroscopic probes to examine local DNA conformations. An equilibrium mixture of four hybridization conformations was observed when template bases looped-out as a bulge, i.e. a structure flanked on both sides by duplex DNA. In contrast, a single-loop structure with an unusual unstacked DNA conformation at its downstream edge was observed when the extruded bases were positioned at the primer–template junction, showing that misalignments can be modified by neighboring DNA secondary structure. These results must be taken into account in considering the genetic and biochemical mechanisms of frameshift mutagenesis in polymerase-driven DNA replication