Proofreading activity of DNA polymerase Pol2 mediates 3'-end processing during nonhomologous end joining in yeast.

Genotoxic agents that cause double-strand breaks (DSBs) often generate damage at the break termini. Processing enzymes, including nucleases and polymerases, must remove damaged bases and/or add new bases before completion of repair. Artemis is a nuclease involved in mammalian nonhomologous end joining (NHEJ), but in Saccharomyces cerevisiae the nucleases and polymerases involved in NHEJ pathways are poorly understood.

Kinetic pathway of pyrophosphorolysis by a retrotransposon reverse transcriptase.

DNA and RNA polymerases use a common phosphoryl transfer mechanism for base addition that requires two or three acidic amino acid residues at their active sites. We previously showed, for the reverse transcriptase (RT) encoded by the yeast retrotransposon Ty1, that one of the three conserved active site aspartates (D(211)) can be substituted by asparagine and still retain in vitro polymerase activity, although in vivo transposition is lost. Transposition is partially restored by second site suppressor mutations in the RNAse H domain.

Capture of linear fragments at a double-strand break in yeast.

Double-strand breaks (DSBs) are dangerous chromosomal lesions that must be efficiently repaired in order to avoid loss of genetic information or cell death. In all organisms studied to date, two different mechanisms are used to repair DSBs: homologous recombination (HR) and non-homologous end joining (NHEJ). Previous studies have shown that during DSB repair, non-homologous exogenous DNA (also termed 'filler DNA') can be incorporated at the site of a DSB.

Global mapping of transposon location.

Transposable genetic elements are ubiquitous, yet their presence or absence at any given position within a genome can vary between individual cells, tissues, or strains. Transposable elements have profound impacts on host genomes by altering gene expression, assisting in genomic rearrangements, causing insertional mutations, and serving as sources of phenotypic variation. Characterizing a genome's full complement of transposons requires whole genome sequencing, precluding simple studies of the impact of transposition on interindividual variation.

Widespread RNA editing of embedded alu elements in the human transcriptome.

More than one million copies of the approximately 300-bp Alu element are interspersed throughout the human genome, with up to 75% of all known genes having Alu insertions within their introns and/or UTRs. Transcribed Alu sequences can alter splicing patterns by generating new exons, but other impacts of intragenic Alu elements on their host RNA are largely unexplored. Recently, repeat elements present in the introns or 3'-UTRs of 15 human brain RNAs have been shown to be targets for multiple adenosine to inosine (A-to-I) editing.

Insights into the role of an active site aspartate in Ty1 reverse transcriptase polymerization.

Long terminal repeat-containing retrotransposons encode reverse transcriptases (RTs) that replicate their RNA into integratable, double-stranded DNA. A mutant version of the RT from Saccharomyces cerevisiae retrotransposon Ty1, in which one of the three active site aspartates has been changed to asparagine (D211N), is still capable of in vitro polymerization, although it is blocked for in vivo transposition. We generated recombinant WT and D211N Ty1 RTs to study RT function and determine specific roles for the Asp(211) residue.

Reciprocal translocations in Saccharomyces cerevisiae formed by nonhomologous end joining.

Reciprocal translocations are common in cancer cells, but their creation is poorly understood. We have developed an assay system in Saccharomyces cerevisiae to study reciprocal translocation formation in the absence of homology. We induce two specific double-strand breaks (DSBs) simultaneously on separate chromosomes with HO endonuclease and analyze the subsequent chromosomal rearrangements among surviving cells.

Extension and cleavage of the polypurine tract plus-strand primer by Ty1 reverse transcriptase.

Using hybrid RNA/DNA substrates containing the polypurine tract (PPT) plus-strand primer, we have examined the interaction between the Ty1 reverse transcriptase (RT) and the plus-strand initiation complex. We show here that, although the PPT sequence is relatively resistant to RNase H cleavage, it can be cleaved internally by the polymerase-independent RNase H activity of Ty1 RT. Alternatively, this PPT can be used to initiate plus-strand DNA synthesis.

Ku-dependent and Ku-independent end-joining pathways lead to chromosomal rearrangements during double-strand break repair in Saccharomyces cerevisiae.

Chromosomal double-strand breaks (DSBs) can be repaired by either homology-dependent or homology-independent pathways. Nonhomologous repair mechanisms have been relatively less well studied, despite their potential importance in generating chromosomal rearrangements. We have developed a Saccharomyces cerevisiae-based assay to identify and characterize homology-independent chromosomal rearrangements associated with repair of a unique DSB generated within an engineered URA3 gene.