Luther Davis

Meiotic recombination and chromosome segregation in Schizosaccharomyces pombe.

In most organisms homologous recombination is vital for the proper segregation of chromosomes during meiosis, the formation of haploid sex cells from diploid precursors. This review compares meiotic recombination and chromosome segregation in the fission yeast Schizosaccharomyces pombe and the distantly related budding yeast Saccharomyces cerevisiae, two especially tractable microorganisms. Certain features, such as the occurrence of DNA breaks associated with recombination, appear similar, suggesting that these features may be common in eukaryotes. Other features, such as the role of these breaks and the ability of chromosomes to segregate faithfully in the absence of recombination, appear different, suggesting multiple solutions to the problems faced in meiosis.

Generation of a nicking enzyme that stimulates site-specific gene conversion from the I-AniI LAGLIDADG homing endonuclease

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 wild-type enzyme. I-AniI nickase stimulates targeted gene correction in human cells, in cis and in trans, at ≈1/4 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.

A Large-Scale Screen in S. pombe Identifies Seven Novel Genes Required for Critical Meiotic Events

Meiosis is a specialized form of cell division by which sexually reproducing diploid organisms generate haploid gametes. During a long prophase, telomeres cluster into the bouquet configuration to aid chromosome pairing, and DNA replication is followed by high levels of recombination between homologous chromosomes (homologs). This recombination is important for the reductional segregation of homologs at the first meiotic division; without further replication, a second meiotic division yields haploid nuclei. In the fission yeast Schizosaccharomyces pombe, we have deleted 175 meiotically upregulated genes and found seven genes not previously reported to be critical for meiotic events. Three mutants (rec24, rec25, and rec27) had strongly reduced meiosis-specific DNA double-strand breakage and recombination. One mutant (tht2) was deficient in karyogamy, and two (bqt1 and bqt2) were deficient in telomere clustering, explaining their defects in recombination and segregation. The moa1 mutant was delayed in premeiotic S phase progression and nuclear divisions. Further analysis of these mutants will help elucidate the complex machinery governing the special behavior of meiotic chromosomes.

Homology-directed repair of DNA nicks via pathways distinct from canonical double-strand break repair

Significance Nicks are a very common form of DNA damage, but their threat to genomic integrity has been neglected because it is assumed that all nicks are repaired by simple religation. Here we challenge that assumption. We identify a robust pathway for homology-directed repair (HDR) that is active at DNA nicks. This alternative HDR pathway is stimulated upon down-regulation of key factors in canonical HDR at double-strand breaks. Alternative HDR at targeted nicks has immediate practical applications to genome engineering. Alternative HDR can promote repair of a nicked target by a nicked donor and may thereby contribute to loss of heterozygosity, a common form of genomic instability in tumors. DNA nicks are the most common form of DNA damage, and if unrepaired can give rise to genomic instability. In human cells, nicks are efficiently repaired via the single-strand break repair pathway, but relatively little is known about the fate of nicks not processed by that pathway. Here we show that homology-directed repair (HDR) at nicks occurs via a mechanism distinct from HDR at double-strand breaks (DSBs). HDR at nicks, but not DSBs, is associated with transcription and is eightfold more efficient at a nick on the transcribed strand than at a nick on the nontranscribed strand. HDR at nicks can proceed by a pathway dependent upon canonical HDR factors RAD51 and BRCA2; or by an efficient alternative pathway that uses either ssDNA or nicked dsDNA donors and that is strongly inhibited by RAD51 and BRCA2. Nicks generated by either I-AniI or the CRISPR/Cas9D10A nickase are repaired by the alternative HDR pathway with little accompanying mutagenic end-joining, so this pathway may be usefully applied to genome engineering. These results suggest that alternative HDR at nicks may be stimulated in physiological contexts in which canonical RAD51/BRCA2-dependent HDR is compromised or down-regulated, which occurs frequently in tumors.

DNA Nicks Promote Efficient and Safe Targeted Gene Correction

Targeted gene correction employs a site-specific DNA lesion to promote homologous recombination that eliminates mutation in a disease gene of interest. The double-strand break typically used to initiate correction can also result in genomic instability if deleterious repair occurs rather than gene correction, possibly compromising the safety of targeted gene correction. Here we show that single-strand breaks (nicks) and double-strand breaks both promote efficient gene correction. However, breaks promote high levels of inadvertent but heritable genomic alterations both locally and elsewhere in the genome, while nicks are accompanied by essentially no collateral local mutagenesis, and thus provide a safer approach to gene correction. Defining efficacy as the ratio of gene correction to local deletion, nicks initiate gene correction with 70-fold greater efficacy than do double-strand breaks (29.0±6.0% and 0.42±0.03%, respectively). Thus nicks initiate efficient gene correction, with limited local mutagenesis. These results have clear therapeutic implications, and should inform future design of meganucleases for targeted gene correction.

Targeted gene therapies: tools, applications, optimization.

Many devastating human diseases are caused by mutations in a single gene that prevent a somatic cell from carrying out its essential functions, or by genetic changes acquired as a result of infectious disease or in the course of cell transformation. Targeted gene therapies have emerged as potential strategies for treatment of such diseases. These therapies depend upon rare-cutting endonucleases to cleave at specific sites in or near disease genes. Targeted gene correction provides a template for homology-directed repair, enabling the cell’s own repair pathways to erase the mutation and replace it with the correct sequence. Targeted gene disruption ablates the disease gene, disabling its function. Gene targeting can also promote other kinds of genome engineering, including mutation, insertion, or gene deletion. Targeted gene therapies present significant advantages compared to approaches to gene therapy that depend upon delivery of stably expressing transgenes. Recent progress has been fueled by advances in nuclease discovery and design, and by new strategies that maximize efficiency of targeting and minimize off-target damage. Future progress will build on deeper mechanistic understanding of critical factors and pathways.

Rec25 and Rec27, Novel Linear-Element Components, Link Cohesin to Meiotic DNA Breakage and Recombination

Meiosis is a specialized nuclear division by which sexually reproducing diploid organisms generate haploid gametes. Recombination between homologous chromosomes facilitates accurate meiotic chromosome segregation and is initiated by DNA double-strand breaks (DSBs) made by the conserved topoisomerase-like protein Spo11 (Rec12 in fission yeast), but DSBs are not evenly distributed across the genome. In Schizosaccharomyces pombe, proteinaceous structures known as linear elements (LinEs) are formed during meiotic prophase. The meiosis-specific cohesin subunits Rec8 and Rec11 are essential for DSB formation in some regions of the genome, as well as for formation of LinEs or the related synaptonemal complex (SC) in other eukaryotes. Proteins required for DSB formation decorate LinEs, and mutants lacking Rec10, a major component of LinEs, are completely defective for recombination. Although recombination may occur in the context of LinEs, it is not well understood how Rec10 is loaded onto chromosomes. We describe two novel components of LinEs in fission yeast, Rec25 and Rec27. Comparisons of rec25Delta, rec27Delta, and rec10Delta mutants suggest multiple pathways to load Rec10. In the major pathway, Rec10 is loaded, together with Rec25 and Rec27, in a Rec8-dependent manner with subsequent region-specific effects on recombination.

G4 DNA: at risk in the genome

Regions bearing G‐quadruplex (G4) DNA motifs can be sites of genomic instability and are frequently depleted in streamlined genomes, but are nevertheless maintained in many other genomes. Whether G4 secondary structures form in vivo , and how they are maintained or eliminated, remains little known. In this issue of The EMBO Journal , Lopes et al (2011) provide new evidence that G4 structures form and contribute to genomic instability in living cells, and identify specific mechanisms that minimize the risks posed by G4 motifs.

Assaying Repair at DNA Nicks

Nicks are the most common form of DNA damage, but they have only recently been shown to initiate damage that requires repair. Analysis of the pathways of nick repair in human cells has benefited from the development of enzymes that target nicks to specific sites in the genome and of reporters that enable rapid analysis of homology-directed repair and mutagenic end joining. Nicks undergo efficient repair by single-stranded oligonucleotide donors complementary to either the nicked or intact DNA strand, via pathways that are normally suppressed by RAD51. Here we discuss the details of reporter assays that take advantage of the convenience and sensitivity of flow cytometry to analyze pathways of repair at targeted DNA nicks. These assays are readily carried out in 96-well format cell culture plates, enabling mechanistic questions to be addressed by determining the contributions of specific factors by depletion and/or ectopic expression.

Initiation of homologous recombination at DNA nicks

Abstract Discontinuities in only a single strand of the DNA duplex occur frequently, as a result of DNA damage or as intermediates in essential nuclear processes and DNA repair. Nicks are the simplest of these lesions: they carry clean ends bearing 3′-hydroxyl groups that can undergo ligation or prime new DNA synthesis. In contrast, single-strand breaks also interrupt only one DNA strand, but they carry damaged ends that require clean-up before subsequent steps in repair. Despite their apparent simplicity, nicks can have significant consequences for genome stability. The availability of enzymes that can introduce a nick almost anywhere in a large genome now makes it possible to systematically analyze repair of nicks. Recent experiments demonstrate that nicks can initiate recombination via pathways distinct from those active at double-strand breaks (DSBs). Recombination at targeted DNA nicks can be very efficient, and because nicks are intrinsically less mutagenic than DSBs, nick-initiated gene correction is useful for genome engineering and gene therapy. This review revisits some physiological examples of recombination at nicks, and outlines experiments that have demonstrated that nicks initiate homology-directed repair by distinctive pathways, emphasizing research that has contributed to our current mechanistic understanding of recombination at nicks in mammalian cells.