Identifying the mechanisms of eukaryotic genome evolution by comparative genomics is often complicated by the multiplicity of events that have taken place throughout the history of individual lineages, leaving only distorted and superimposed traces in the genome of each living organism. The hemiascomycete yeasts, with their compact genomes, similar lifestyle and distinct sexual and physiological properties, provide a unique opportunity to explore such mechanisms. We present here the complete, assembled genome sequences of four yeast species, selected to represent a broad evolutionary range within a single eukaryotic phylum, that after analysis proved to be molecularly as diverse as the entire phylum of chordates. A total of approximately 24,200 novel genes were identified, the translation products of which were classified together with Saccharomyces cerevisiae proteins into about 4,700 families, forming the basis for interspecific comparisons. Analysis of chromosome maps and genome redundancies reveal that the different yeast lineages have evolved through a marked interplay between several distinct molecular mechanisms, including tandem gene repeat formation, segmental duplication, a massive genome duplication and extensive gene loss.

Homing endonucleases stimulate gene conversion by generating site-specific DNA double-strand breaks that are repaired by homologous recombination. These enzymes are potentially valuable tools for targeted gene correction and genome engineering. We have engineered a variant of the I-AniI homing endonuclease that nicks its cognate target site. This variant contains a mutation of a basic residue essential for proton transfer and solvent activation in one active site. The cleavage mechanism, DNA-binding affinity, and substrate specificity profile of the nickase are similar to the wildtype enzyme. I-AniI nickase stimulates targeted gene correction in human cells, in cis and in trans, at Ϸ1/4 the efficiency of the wild-type enzyme. The development of sequence-specific nicking enzymes like the I-AniI nickase will facilitate comparative analyses of DNA repair and mutagenesis induced by single-or double-strand breaks.protein engineering ͉ recombination ͉ single strand breaks ͉ gene therapy ͉ gene repair H oming endonucleases generate sequence-specific DNA double-strand breaks (DSBs) that are eventually invaded by their own open reading frames, usually in concert with surrounding intron or intein sequences (1, 2; reviewed in ref.3). Repair of the break by homologous recombination results in genetic transmission and persistence of these mobile elements (4). Homing endonucleases are promising reagents for catalyzing targeted gene correction or modification because they recognize long DNA target sites (spanning 14-40 bp) with great sequence specificity (5-7; reviewed in refs. 8 and 9). Members of one particular family, the LAGLIDADG homing endonucleases (LHEs), are especially promising because they exhibit the greatest sequence specificity, cleaving as few as 1 in 10 8 -10 9 random DNA sequences (10, 11).LHEs contain two similar core folds of mixed ␣/␤ topology. The conserved LAGLIDADG amino acid sequence motifs form two ␣-helices that are packed together at the domain or subunit interface, where each contributes a catalytic residue to an active site (12). Enzymes containing a single LAGLIDADG motif per protein chain form homodimers that recognize palindromic and pseudopalindromic DNA target sites, whereas proteins containing two motifs form asymmetric monomers that recognize correspondingly asymmetric DNA target sites.To use LHEs as therapeutic gene correction reagents, it is essential that endonuclease-induced breaks be conservatively repaired. Naturally occurring LHEs create double-strand breaks that can be repaired by either homologous recombination (HR), which uses a homologous donor sequence as a template to repair the damage, or by nonhomologous end joining (NHEJ), which directly rejoins the two free DNA ends (13-16). Homologous recombination occurs without loss of sequence information, whereas NHEJ usually results in sequence loss at the repair junction (15, 17) and can also promote chromosome translocations at DSBs, leading to genomic instability. Several homing endonucleases have been shown to cause such genomic in...

Homing endonucleases (HEs) cut long DNA target sites with high specificity to initiate and target the lateral transfer of mobile introns or inteins. This high site specificity of HEs makes them attractive reagents for gene targeting to promote DNA modification or repair. We have generated several hundred catalytically active, monomerized versions of the well-characterized homodimeric I-CreI and I-MsoI LAGLIDADG family homing endonuclease (LHE) proteins. Representative monomerized I-CreI and I-MsoI proteins (collectively termed mCreIs or mMsoIs) were characterized in detail by using a combination of biochemical, biophysical and structural approaches. We also demonstrated that both mCreI and mMsoI proteins can promote cleavage-dependent recombination in human cells. The use of single chain LHEs should simplify gene modification and targeting by requiring the expression of a single small protein in cells, rather than the coordinate expression of two separate protein coding genes as is required when using engineered heterodimeric zinc finger or homing endonuclease proteins.

This study identified 35 new sites for targeted transgene insertion that have the potential to serve as new human genomic ''safe harbor'' sites (SHS). SHS potential for these 35 sites, located on 16 chromosomes, including both arms of the human X chromosome, and for the existing human SHS AAVS1, hROSA26, and CCR5 was assessed using eight different desirable, widely accepted criteria for SHS verifiable with human genomic data. Three representative newly identified sites on human chromosomes 2 and 4 were then experimentally validated by in vitro and in vivo cleavage-sensitivity tests, and analyzed for populationlevel and cell line-specific sequence variants that might confound site targeting. The highly ranked site on chromosome 4 (SHS231) was further characterized by targeted homology-dependent and-independent transgene insertion and expression in different human cell lines. The structure and fidelity of transgene insertions at this site were confirmed, together with analyses that demonstrated stable expression and function of transgene-encoded proteins, including fluorescent protein markers, selectable marker cassettes, and Cas9 protein variants. SHS-integrated transgene-encoded Cas9 proteins were shown to be capable of introducing a large (17 kb) gRNA-specified deletion in the PAX3/FOXO1 fusion oncogene in human rhabdomyosarcoma cells and as a Cas9-VPR fusion protein to upregulate expression of the muscle-specific transcription factor MYF5 in human rhabdomyosarcoma cells. An engineering ''toolkit'' was developed to enable easy use of the most extensively characterized of these new human sites, SHS231, located on the proximal long arm of chromosome 4. The target sites identified here have the potential to serve as additional human SHS to enable basic and clinical gene editing and genome-engineering applications.

The first group I intron in the cox1 gene (cox1I1b ) of the mitochondrial genome of the fission yeast Schizosaccharomyces pombe is a mobile DNA element. The mobility is dependent on an endonuclease protein that is encoded by an intronic open reading frame (ORF). The intron-encoded endonuclease is a typical member of the LAGLIDADG protein family of endonucleases with two consensus motifs. In addition to this, analysis of several intron mutants revealed that this protein is required for intron splicing. However, this protein is one of the few group I intron-encoded proteins that functions in RNA splicing simultaneously with its DNA endonuclease activity. We report here on the biochemical characterization of the endonuclease activity of this protein artificially expressed in Escherichia coli. Although the intronic ORF is expressed as a fusion protein with the upstream exon in vivo, the experiments showed that a truncated translation product consisting of the C-terminal 304 codons of the cox1I1b ORF restricted to loop 8 of the intron RNA secondary structure is sufficient for the specific endonuclease activity in vitro. Based on the results, we speculate on the evolution of site-specific homing endonucleases encoded by group I introns in eukaryotes.