Project description:We used a two-component transgene system to study the RNA-dependent DNA methylation (RdDM) and transcriptional gene silencing (TGS) in Arabidopsis. By profiling the small RNA population in mutants defected in RdDM or RNA polymerase II-transcribed trigger for generating silencing siRNA, we investigated how repetitive loci such as tandem repeats were regulated transcriptionally through the action of RNA polymerase IV. Genome-wide small RNA profiling was done by Illumina TruSeq sample preparation followed by high-throughput sequencing with the Illumina HiSeq 2000 platform. The six samples represent mutants and their corresponding control lines.
Project description:We used a two-component transgene system to study the RNA-dependent DNA methylation (RdDM) and transcriptional gene silencing (TGS) in Arabidopsis. By profiling the small RNA population in mutants defected in RdDM or RNA polymerase II-transcribed trigger for generating silencing siRNA, we investigated how repetitive loci such as tandem repeats were regulated transcriptionally through the action of RNA polymerase IV.
Project description:Short tandem repeats (STRs) significantly contribute to de novo mutagenesis, driving phenotypic diversity and genetic disease. Although highly diverse, their low complexity and repetitive nature induces DNA polymerase slippage and stalling, leading to length variation and base substitutions. However, the characterisation of DNA synthesis through STR loci has been restricted to a handful of selected sequences, limiting our broader understanding of their evolutionary behaviour. In order to understand the interplay between the ability of a given STR to impair DNA synthesis and its genomic stability, we developed a high-throughput polymerase extension assay that allows monitoring the kinetics of DNA synthesis at all STR permutations in different lengths in parallel. We have used the assay to map at single-nucleotide resolution the movement of a prototypical A-family replicative DNA polymerase (T7 DNA polymerase) through the repeats over time. From this data we can infer the secondary structure adopted by a given STR from the precise manner in which it stalls polymerase and link this to slippage and point mutation during DNA synthesis.