Experiments on broken chromosomes in budding yeast as well as in mammalian cells have shown that DNA damage elicits a change such that the volume of the nucleus explored by the broken chromosome more than doubles up to as much as 10 times from that seen in the absence of DSBs Dion et al. In yeast, the chromosomes were marked by multiple tandem arrays, whereas in mammalian cells, the movement was measured by tracking 53BP1, a protein that binds to broken ends to promote DNA repair.
The increase in both global and local mobility in haploid and diploid budding yeast cells is genetically controlled. For example, both depend on the Rad51 recombinase Dion et al. In haploid budding yeast, the increase in local mobility depends on Rad54 as well as two checkpoint proteins, Rad9 and Mec1 Dion et al.
In diploid cells, deletion of SAE2 , which likely causes a delayed appearance of ssDNA, also delays the increase in local chromosome mobility Mine-Hattab and Rothstein Unlike the increase in mobility seen after IR or enzymatically induced DSBs, spontaneous Rad52 foci are constrained in haploid cells, possibly reflecting recombination between sister chromatids in the context of DNA replication Dion et al.
In diploid yeast cells, after a DSB, homologous pairing takes approximately 20 min before the repair center disassembles and the loci separate again Mine-Hattab and Rothstein Thus, increased chromosomal mobility likely facilitates the homology search, which is otherwise restricted by the proximity of donor and recipient loci Goldman and Lichten ; Agmon et al. Whole genome approaches to study proteins involved in the DNA damage response have come to the fore in recent years. Two common kinds of genome-wide cell biology screens have been used to examine the cellular response to DNA damage.
In the first, a single tagged protein is introduced into the entire nonessential S. In the second, a genome-wide library of GFP-tagged yeast proteins is examined for changes in subcellular localization in response to DNA damage treatment. In , Alvaro et al. The Rad52 protein in S.
The genes governing Rad52 focus formation and maintenance were not well known when this screen was initiated. To find new genes, the formation of spontaneous subnuclear RadYFP foci was monitored in the mutant background of over nonessential gene disruptions using epifluorescence microscopy.
To avoid the potential problem of additional recessive traits that can accumulate in individual strains of the haploid yeast gene disruption library, hybrid diploid strains that are homozygous for each deletion were made by using systematic hybrid loss of heterozygosity SHyLOH [ Alvaro et al.
Image analysis was performed manually and all images were uploaded into the JCB DataViewer, which allows anyone to access and examine the primary data from that screen Thorpe et al. In this screen, more than 80 gene disruptions resulted in increased spontaneous Rad52 foci, including mutations in many genes involved in DNA metabolism and cell-cycle regulation.
The identified genes also included 22 uncharacterized open reading frames, IRC2—11 , 13—16 , 18—25 increased recombination centers , providing new leads to genes that control the cellular response to DNA damage. In mammalian cell studies, two laboratories used a similar approach to identify genes that regulate the DNA damage response. Similarly, the Cortez group performed a shRNA screen that targeted almost genes preselected for protein domains associated with nuclear regulatory activities Lovejoy et al.
In that study, HeLa cells were treated both with and without aphidicolin to induce replication stress and were assayed for KAP1 phosphorylation, a substrate of the apical ATM kinase.
In the Cortez study, many of the genes identified were involved in DNA metabolism and repair. Only a few genes overlap between these screens as well as with the yeast results using Rad52 foci as the reporter. For the second approach, Brown and colleagues measured global changes in the localization and levels of the S.
For their experiments, they tested two drug treatments for the induction of different types of DNA damage. They compared the effects of these two drug treatments for each protein with untreated controls by monitoring changes in protein levels of all images using the freely available Cellprofiler software Kamentsky et al.
They also determined changes in localization by visually comparing each fluorescent strain. Of the more than tagged proteins screened, they found proteins whose abundance changed significantly after DNA damage, and that changed their localization.
Surprisingly, only 35 of these proteins showed changes in both abundance and localization. Furthermore they found that proteins that share a location are also enriched for physical and genetic interactions, which predict common functions, encouraging even more global studies of proteome relocalization in response to other cellular stresses.
One of the surprises of their study was that cytoplasmic P-body components, which are important for mRNA turnover in the cytoplasm, become elevated in HU-treated cells. To explore this finding, they next used a genome-wide GFP microscopy assay similar to the Alvaro et al.
Another surprise is that some proteins Cmr1, Hos2, Apj1, and Pph21 form DNA damage-induced foci in the nucleus that do not colocalize with repair foci of Rad A genome-wide screen of GFP-tagged proteins has also been conducted in Schizosaccharomyces pombe Yu et al. Eight of these proteins were previously uncharacterized open reading frames ORFs. All in all, these high-throughput, cell biology-based screens for increased spontaneous DNA damage foci show that repair proteins respond to a variety of genome stress conditions and reveal other pathways not usually associated with the DNA damage response.
Studies on the cell biology of recombination are still in their infancy. We can look forward to a myriad of technical advances in protein tagging as well as in microscopy e. These advances when combined with genetic studies will allow a more in-depth look at the architecture of foci as well as provide further insight into the regulation of focus assembly and disassembly.
Expansion of genome-wide screens promises to uncover new genes and pathways that impact the cellular response to DNA damage. In the future, more multiple mutant analyses will be undertaken to define the epistasis groups involved. It will be especially important to uncover the genes and regulatory circuitry involved in controlling DNA repair dynamics. Because most live cell—imaging studies only look at a single DNA end, it is important to visualize both ends of a DSB to understand how the homology search is coordinated.
Furthermore, DSBs are not the only lesions leading to HR and there is a need to analyze the repair of single-strand nicks and gaps. In addition, methods need to be developed to visualize the underlying changes to the recombining DNA molecules. Another challenge for the future will be to understand which nuclear components are responsible for chromosome territories as well as those that confine diffusion and the mobility of chromosomes.
Finally, we can look forward to new insights into the cellular response of recombination processes, especially as they relate to oncogenesis, aging, and other human health issues. Figure 1. View this table: In this window In a new window. Table 1. Previous Section Next Section. Figure 2. Figure 3. Figure 4. Previous Section. Effect of nuclear architecture on the efficiency of double-strand break repair.
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Cell 91 : 35 — Mitotic recombination MR results from somatic crossing over between homologous sequences during mitosis. MR is a term used inconsistently among geneticists, molecular biologists and biochemists. Herein, MR will be used to describe both exchange of genetic material between sister chromatids SCEs — which is an effective, common repair mechanism usually without genetic or phenotypic consequence — and exchange between homologous chromosomes.
GC and MXO are rare compared with meiotic crossing-over because homologues do not normally pair at mitosis. GC and MXO lead to homozygosity in a daughter cell for markers distal to the crossover site for a variable length Skip to main content Skip to table of contents. This service is more advanced with JavaScript available. Contents Search. Mitotic Recombination.
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