Induction Of Gene Conversion Research Articles

DNA Damage Tolerance Mechanisms Revealed from the Analysis of Immunoglobulin V Gene Diversification in Avian DT40 Cells.

DNA replication is an essential biochemical reaction in dividing cells that frequently stalls at damaged sites. Homologous/homeologous recombination (HR)-mediated template switch and translesion DNA synthesis (TLS)-mediated bypass processes release arrested DNA replication forks. These mechanisms are pivotal for replication fork maintenance and play critical roles in DNA damage tolerance (DDT) and gap-filling. The avian DT40 B lymphocyte cell line provides an opportunity to examine HR-mediated template switch and TLS triggered by abasic sites by sequencing the constitutively diversifying immunoglobulin light-chain variable gene (IgV). During IgV diversification, activation-induced deaminase (AID) converts dC to dU, which in turn is excised by uracil DNA glycosylase and yields abasic sites within a defined window of around 500 base pairs. These abasic sites can induce gene conversion with a set of homeologous upstream pseudogenes via the HR-mediated template switch, resulting in templated mutagenesis, or can be bypassed directly by TLS, resulting in non-templated somatic hypermutation at dC/dG base pairs. In this review, we discuss recent works unveiling IgV diversification mechanisms in avian DT40 cells, which shed light on DDT mode usage in vertebrate cells and tolerance of abasic sites.

Highly Efficient Editing of the Beta-Globin Gene in Patient Derived Hematopoietic Stem and Progenitor Cells to Treat Sickle Cell Disease

Sickle cell disease (SCD) is an inherited blood disorder associated with a debilitating chronic illness. SCD is caused by a point mutation in the β-globin gene (HBB). A single nucleotide substitution converts glutamic acid to a valine that leads to the production of sickle hemoglobin (HbS), which impairs the function of red blood cells. Here we show that delivery of Streptococcus pyogenes (Sp) Cas9 protein and CRISPR guide RNA as a ribonucleoprotein complex (RNP) together with a short single-stranded DNA donor (ssODN) template into CD34+ hematopoietic stem and progenitor cells (HSPCs) from SCD patients' bone marrow (BM) was able to correct the sickling HBB mutation, with up to 33% homology directed repair (HDR) without selection. Further, CRISPR/Cas9 cutting of HBB in SCD HSPCs induced gene conversion between the HBB sequences in the vicinity of the target locus and the homologous region in δ-globin gene (HBD), with up to 4.4% additional gene correction mediated by the HBD conversion in cells with Cas9 cutting only. The erythrocytes derived from gene-edited cells showed a marked reduction of the HbS level, increased expression of normal adult hemoglobin (HbA), and a complete loss of cell sickling, demonstrating the potential in curing SCD.We performed extensive off-target analysis of gene-edited SCD HSPCs using the in-silico prediction tool COSMID and unbiased, genome-wide assay Guide-Seq, revealing a gross intrachromosomal rearrangement event between the on- and off-target Cas9 cutting sites. We used a droplet digital PCR assay to quantify deletion and inversion events from Day 2 to Day 12 after RNP delivery, and found that large chromosomal deletion decreased from 1.8% to 0.2%, while chromosomal inversion maintained at 3.3%. We demonstrated that the use of high-fidelity SpCas9 (HiFi Cas9 by IDT) significantly reduced off-target effects and completely eliminated the intrachromosome rearrangement events, while maintaining the same level of on-target gene editing, leading to high-efficiency gene correction with increased specificity.In order to determine if gene-corrected SCD HSPCs retain the ability to engraft, CD34+ cells from the BM of SCD patients were treated with Cas9/gRNA RNP and ssODN donor for HBB gene correction, cryopreserved at Day 2 post genome editing, then intravenously transplanted into NSG mice shortly after thawing. These mice were euthanized at Week 16 after transplantation, and the BM was harvested to determine the engraftment potential. An average of 7.5 ±5.4% of cells were double positive for HLA and hCD45 in mice injected with gene-edited CD34+ cells, compared to 16.8 ±9.3% with control CD34+ cells, indicating a good level of engraftment of gene-corrected SCD HSPCs. A higher fraction of human cells were positive for CD19 (66 ±28%), demonstrating lymphoid lineage bias. DNA was extracted from unsorted cells, CD19 or CD33 sorted cells for gene-editing analysis; the HBB editing rates were respectively 29.8% HDR, 2.4% HBD conversion, and 42.8% non-homologous end joining (NHEJ) pretransplantation, and editing rates at Week 16 posttransplantation were respectively 8.8 ±12% HDR, 1.8 ±1.7% HBD conversion, and 24.5 ±12% NHEJ. The highly variable editing rate and indel diversity in gene-edited cells at Week 16 in all four transplanted mice suggest clonal dominance of a limited number of HSPCs after transplantation.Taken together, our results demonstrate highly efficient gene and phenotype correction of the sickling mutation in BM HSPCs from SCD patients mediated by HDR and HBD conversion, and the ability of gene-edited SCD HSPCs to engraft in vivo. We also demonstrate the importance of genome-wide analysis for off-target analysis and the use of HiFi Cas9. Our results provide further evidence for the potential of moving genome editing-based SCD treatment into clinical practice.Acknowledgments: This work was supported by the Cancer Prevention and Research Institute of Texas grants RR140081 and RP170721 (to G. B.), and the National Heart, Lung and Blood Institute of NIH (1K08DK110448 to V.S.) DisclosuresPorteus:CRISPR Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees.

A Conserved DNA Repeat Promotes Selection of a Diverse Repertoire of Trypanosoma brucei Surface Antigens from the Genomic Archive

African trypanosomes are mammalian pathogens that must regularly change their protein coat to survive in the host bloodstream. Chronic trypanosome infections are potentiated by their ability to access a deep genomic repertoire of Variant Surface Glycoprotein (VSG) genes and switch from the expression of one VSG to another. Switching VSG expression is largely based in DNA recombination events that result in chromosome translocations between an acceptor site, which houses the actively transcribed VSG, and a donor gene, drawn from an archive of more than 2,000 silent VSGs. One element implicated in these duplicative gene conversion events is a DNA repeat of approximately 70 bp that is found in long regions within each BES and short iterations proximal to VSGs within the silent archive. Early observations showing that 70-bp repeats can be recombination boundaries during VSG switching led to the prediction that VSG-proximal 70-bp repeats provide recombinatorial homology. Yet, this long held assumption had not been tested and no specific function for the conserved 70-bp repeats had been demonstrated. In the present study, the 70-bp repeats were genetically manipulated under conditions that induce gene conversion. In this manner, we demonstrated that 70-bp repeats promote access to archival VSGs. Synthetic repeat DNA sequences were then employed to identify the length, sequence, and directionality of repeat regions required for this activity. In addition, manipulation of the 70-bp repeats allowed us to observe a link between VSG switching and the cell cycle that had not been appreciated. Together these data provide definitive support for the long-standing hypothesis that 70-bp repeats provide recombinatorial homology during switching. Yet, the fact that silent archival VSGs are selected under these conditions suggests the 70-bp repeats also direct DNA pairing and recombination machinery away from the closest homologs (silent BESs) and toward the rest of the archive.

Assessment of Anti-recombination and Double-strand Break-induced Gene Conversion in Human Cells by a Chromosomal Reporter

Gene conversion is one of the frequent end results of homologous recombination, and it often underlies the inactivation of tumor suppressor genes in cancer cells. Here, we have developed an integrated assay system that allows simultaneous examination of double-strand break (DSB)-induced gene conversion events at the site of a DSB (proximal region) and at a surrounding region ~1 kb away from the break (distal region). Utilizing this assay system, we find that gene conversion events at the proximal and distal regions are relatively independent of one another. The results also indicate that synthesis-dependent strand annealing (SDSA) plays a major role in DSB-induced gene conversion. In addition, our current study has demonstrated that hMLH1 plays an essential role in anti-recombination and gene conversion. Specifically, the anti-recombination activity of hMLH1 is partially dependent on its interaction with hMRE11. Our data suggests that the role of hMLH1 and hMRE11 in the process of gene conversion is complex, and these proteins play different roles in DSB-induced proximal and distal gene conversions. In particular, the involvement of hMLH1 and hMRE11 in the distal gene conversion requires both hMSH2 and heteroduplex formation.

Mutagenic effect of freezing on nuclear DNA of Saccharomyces cerevisiae

Although fragmentation of DNA has been observed in cells undergoing freezing procedures, a mutagenic effect of sub-zero temperature treatment has not been proved by induction and isolation of mutants in nuclear DNA (nDNA). In this communication we supply evidence for mutagenicity of freezing on nDNA of Saccharomyces cerevisiae cells. In the absence of cryoprotectors, cooling for 2 h at +4°C and freezing for 1 h at -10°C and 16 h at -20°C, with a cooling rate of 3°C/min, resulted in induction of frame-shift and reverse mutations in microsatellite and coding regions of nDNA. The sub-zero temperature exposure also has a strong recombinogenic effect, evidenced by induction of gene-conversion and crossing-over events. Freezing induces mutations and enhances recombination with a frequency equal to or higher than that of methylmethanesulphonate at comparable survival rates. The signals for the appearance of nDNA lesions induced by freezing are detected and transduced by the DNA damage pathway. Extracellular cryoprotectors did not prevent the mutagenic effect of freezing, while accumulation of trehalose inside cells reduced nDNA cryodamage. Freezing of cells is accompanied by generation of high ROS levels, and the oxidative stress raised during the freeze-thaw process is the most likely reason for the DNA damaging effect. Experiments with mitochondrial rho⁻ mutants or scavengers of ROS indicated that mutagenic and recombinogenic effects of sub-zero temperatures can be decreased but not eliminated by reduction of ROS level. The complete protection against cryodamage in nDNA required simultaneous usage of intracellular cryoprotector and ROS scavenger during the freeze-thaw process.

Suppression of the Double-Strand-Break-Repair Defect of the Saccharomyces cerevisiae rad57 Mutant

The Rad51 paralogs Rad55 and Rad57 form a heterodimer required to mediate the formation and/or stabilization of the Rad51 filament. To further characterize the function of Rad55-Rad57, we used a combination of rad57 partial suppressors to determine whether the DNA repair and recombination defects of the rad57 mutant could be completely suppressed. The combination of all suppressors, elevated temperature, srs2, rad51-I345T, and mating-type (MAT) heterozygosity resulted in almost complete suppression of the rad57 mutant defect in the recruitment of Rad51 to DNA-damaged sites, as well as survival in response to ionizing radiation and camptothecin. In a physical assay to monitor the kinetics of double-strand-break (DSB)-induced gene conversion, the rad57 mutant defect was effectively suppressed by srs2 and MAT heterozygosity, but these same suppressors failed to suppress the spontaneous recombination defect. Thus the Rad55-Rad57 heterodimer appears to have a unique function in spontaneous recombination that is not essential for DSB repair. Furthermore, we investigated the currently unknown mechanism of rad57 suppression by MAT heterozygosity and found that it is independent of DNL4.

Incarnation of classical pro- and eukaryotic mechanisms of mutagenesis in hypermutagenesis and immunity of vertebrates

M.E. Lobashev has brilliantly postulated in 1947 that error-prone repair contribute to mutations in cells. This was shown to be true once the mechanisms of UV mutagenesis in Escherichia coli were deciphered. Induced mutations are generated during error-prone SOS DNA repair with the involvement of inaccurate DNA polymerases belonging to the Y family. Currently, several distinct mutator enzymes participating in spontaneous and induced mutagenesis have been identified. Upon induction of these proteins, mutation rates increase by several orders of magnitude. These proteins regulate the mutation rates in evolution and in ontogeny during immune response. In jawed vertebrates, somatic hypermutagenesis occurs in the variable regions of immunoglobulin genes, leading to affinity maturation of antibodies. The process is initiated by cytidine deamination in DNA to uracil by AID (Activation-Induced Deaminase). Further repair of uracil-containing DNA through proteins that include the Y family DNA polymerases causes mutations, induce gene conversion, and class switch recombination. In jawless vertebrates, the variable lymphocyte receptors (VLR) serve as the primary molecules for adaptive immunity. Generation of mature VLRs most likely depends on agnathan AID-like deaminases. AID and its orthologs in lamprey (PmCDA1 and PMCDA2) belong to the AID/APOBEC family of RNA/DNA editing cytidine deaminases. This family includes enzymes with different functions: APOBEC1 edits RNA, APOBEC3 restricts retroviruses. The functions of APOBEC2 and APOBEC4 have not been yet determined. Here, we report a new member of the AID/APOBEC family, APOBEC5, in the bacterium Xanthomonas oryzae. The widespread presence of RNA/DNA editing deaminases suggests that they are an ancient means of generating genetic diversity.

Intermediate frequency magnetic fields do not have mutagenic, co-mutagenic or gene conversion potentials in microbial genotoxicity tests

We used bacterial mutation and yeast genotoxicity tests to evaluate the effects of intermediate frequency (IF; 2 kHz, 20 kHz and 60 kHz) magnetic fields (MFs) on mutagenicity, co-mutagenicity and gene conversion. We constructed a Helmholtz type exposure system that generated vertical and sinusoidal IF MFs, such as 0.91 mT at 2 kHz, 1.1 mT at 20 kHz and 0.11 mT at 60 kHz. Mutagenicity, co-mutagenicity and gene conversion assays were performed for each of the three MF exposure conditions. Mutagenicity testing was performed in four strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and two strains of Escherichia coli (WP2 uvrA and WP2 uvrA/pKM101) to cover a wide spectrum of point mutations. For co-mutagenicity tests, we used four sensitive test strains (TA98, TA100, WP2 uvrA and WP2 uvrA/pKM101) with five chemical mutagens ( t-butyl hydroperoxide (BH, a hydroxyl free radical precursor), 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF2) and N-ethyl- N′-nitro- N-nitrosoguanidine (ENNG, DNA reactive reagents), benz[a]pyrene (BaP) and 2-aminoanthracene (2AA, DNA reactive promutagens). Gene conversion testing was performed in the yeast test strain, Saccharomyces cerevisiae XD83. We also examined the effects on the repair process of DNA damage by UV irradiation. No statistically significant effects were observed between exposed and control groups in any of the genotoxicity tests, indicating that the IF MFs (0.91 mT at 2 kHz, 1.1 mT at 20 kHz or 0.11 mT at 60 kHz) do not have mutagenic or co-mutagenic potentials for the chemical mutagens tested under these experimental conditions. Our findings also indicate that these IF MFs do not induce gene conversion or affect the repair process of DNA damage in eukaryotic cells.

A review of the genotoxicity of ethylbenzene

Ethylbenzene is an important industrial chemical that has recently been classified as a possible human carcinogen (IARC class 2B). It induces tumours in rats and mice, but neither the relevance of these tumours to humans nor their mechanism of induction is clear. Considering the carcinogenic potential of ethylbenzene, it is of interest to determine whether there is sufficient data to characterize its mode of action as either genotoxic or non-genotoxic. A review of the currently available genotoxicity data is assessed. Ethylbenzene is not a bacterial mutagen, does not induce gene conversion or mutations in yeast and does not induce sister chromatid exchanges in CHO cells. Ethylbenzene is not clastogenic in CHO or rat liver cell lines but was reported to induce micronuclei in SHE cells in vitro. No evidence for genotoxicity has been seen in humans exposed to relatively high levels of ethylbenzene. Mouse lymphoma gene mutation studies produced a mixed series of responses that have proved difficult to interpret. An increase in morphological transformation of SHE cells was also found. Results from a more relevant series of in vivo genotoxicity studies, including acute and sub-chronic micronucleus tests and the mouse liver UDS assay, indicate a lack of in vivo genotoxic activity. The composite set of results from both in vitro and in vivo tests known to assess direct damage to DNA have been predominantly negative in the absence of excessive toxicity. The available data from the standard battery of genotoxicity assays do not support a genotoxic mechanism for ethylbenzene-induced kidney, liver or lung tumors in rats and mice.

Effect of Tumour Promoter Anthralin on Gamma Radiation and 4-nitroquinoline 1-oxide Induced Genotoxicity in Diploid Yeast

KEYWORDS Anthralin, radiation, 4-Nitroquinoline1-oxide, yeast, recombination, mutation ABSTRACT The potentiating effect of tumour promoter anthralin on gamma radiation and 4-Nitroquinoline 1- oxide (4-NQO) induced gene conversion and back mutation was studied using diploid yeast Saccharomyces cerevisiae D7. Cells were exposed to 20-400 Gy of gamma radiation and plated on media without anthralin or containing 0.05- 10 mg/ml of anthralin. In another set of experiments, cells were treated with 0.15-0.5 µM 4-NQO and plated on media without anthralin or containing 0.05-0.5 µg/ml of anthralin. The results indicated that anthralin did not modify gamma radiation induced gene conversion or back mutation. Anthralin, however, was found to enhance 4-NQO induced back mutation but not gene conversion. The enhancement was highest with the lowest concentration of anthralin and it decreased subsequently as anthralin concentration was increased.

Role of DNA replication proteins in double-strand break-induced recombination in Saccharomyces cerevisiae.

Mitotic double-strand break (DSB)-induced gene conversion involves new DNA synthesis. We have analyzed the requirement of several essential replication components, the Mcm proteins, Cdc45p, and DNA ligase I, in the DNA synthesis of Saccharomyces cerevisiae MAT switching. In an mcm7-td (temperature-inducible degron) mutant, MAT switching occurred normally when Mcm7p was degraded below the level of detection, suggesting the lack of the Mcm2-7 proteins during gene conversion. A cdc45-td mutant was also able to complete recombination. Surprisingly, even after eliminating both of the identified DNA ligases in yeast, a cdc9-1 dnl4 Delta strain was able to complete DSB repair. Previous studies of asynchronous cultures carrying temperature-sensitive alleles of PCNA, DNA polymerase alpha (Pol alpha), or primase showed that these mutations inhibited MAT switching (A. M. Holmes and J. E. Haber, Cell 96:415-424, 1999). We have reevaluated the roles of these proteins in G(2)-arrested cells. Whereas PCNA was still essential for MAT switching, neither Pol alpha nor primase was required. These results suggest that arresting cells in S phase using ts alleles of Pol alpha-primase, prior to inducing the DSB, sequesters some other component that is required for repair. We conclude that DNA synthesis during gene conversion is different from S-phase replication, involving only leading-strand polymerization.

Marker structure and recombination substrate environment influence conversion preference of broken and unbroken alleles in Saccharomyces cerevisiae.

Double-strand break (DSB)-induced gene conversion was investigated using plasmid x chromosome (P x C) and chromosomal direct-repeat recombination substrates with markers arranged such that functional (selected) products could not arise by longpatch mismatch repair initiated from the DSB. As seen previously with analogous substrates, these substrates yield products with discontinuous conversion tracts, albeit at low frequency. Most conversion tracts were of minimum length, suggesting that heteroduplex DNA (hDNA) is limiting, or that co-repair imposes selective pressure against products with more extensive hDNA. When functional products can arise by long-patch mismatch repair, the broken allele is converted in nearly all products. In contrast, in the absence of long-patch mismatch repair, unbroken alleles are frequently converted, and we show that such conversion depends on both marker structure (i.e., long palindromic vs. nonpalindromic insertions) and the chromosomal environment of the recombination substrate. We propose that conversion of unbroken alleles is largely a consequence of the segregation of unrepaired markers, and that differences in mismatch repair efficiency underlie the observed effects of marker structure and chromosome environment on allele conversion preference.

Effect of Caffeine on the Genotoxic Effects of Gamma Radiation and 4-NQO in Diploid Yeast

Caffeine is an environmental agent to which people are commonly exposed through medicines, drinks, food items, etc. It has been shown to be mutagenic in a number of test systems. In addition, it has also been shown to modify the mutagenic response of ionizing radiation, UV, and several chemical mutagens in a number of test systems. We have studied the effect of caffeine on gamma radiation and 4-Nitroquinoline 1-oxide (4-NQO)-induced gene conversion in the yeast Saccharomyces cerevisiae D7. Stationary phase cells were either exposed to 100-600 Gy of 60Co gamma radiation or treated with 0.15-0.3 microM 4-NQO (30 degrees C, 1 hour), after which they were plated on synthetic complete or minimal media with or without caffeine. Caffeine concentrations ranged from 5 to 15 mM. The results indicated that caffeine at 5 and 10 mM decreased gamma radiation-induced gene conversion frequencies significantly at 400 and 600 Gy. At 600 Gy, the decrease was about 30% and 50% with caffeine concentrations of 5 and 10 mM, respectively. In contrast, caffeine was found to increase the induced gene conversion frequency when cells treated with 0.15, 0.225, and 0.3 microM 4-NQO were plated on media containing caffeine. The increase with 5, 10, and 15 mM caffeine was approximately 1.5, 2, and 2.5, respectively, times the value of 4-NQO alone. The results indicate that the posttreatment repair processes following gamma irradiation or 4-NQO treatment are modified via different pathways.

Involvement of the essential yeast DNA polymerases in induced gene conversion.

In the yeast Saccharomyces cerevisiae three different DNA polymerases alpha, delta and epsilon are involved in DNA replication. DNA polymerase alpha is responsible for initiation of DNA synthesis and polymerases delta and epsilon are required for elongation of DNA strand during replication. DNA polymerases delta and epsilon are also involved in DNA repair. In this work we studied the role of these three DNA polymerases in the process of recombinational synthesis. Using thermo-sensitive heteroallelic mutants in genes encoding DNA polymerases we studied their role in the process of induced gene conversion. Mutant strains were treated with mutagens, incubated under permissive or restrictive conditions and the numbers of convertants obtained were compared. A very high difference in the number of convertants between restrictive and permissive conditions was observed for polymerases alpha and delta, which suggests that these two polymerases play an important role in DNA synthesis during mitotic gene conversion. Marginal dependence of gene conversion on the activity of polymerase epsilon indicates that this DNA polymerase may be involved in this process but rather as an auxiliary enzyme.

Development of B cells expressing surface immunoglobulin molecules that lack V(D)J-encoded determinants in the avian embryo bursa of fabricius.

Immunoglobulin gene rearrangement in avian B cell precursors generates surface Ig receptors of limited diversity. It has been proposed that specificities encoded by these receptors play a critical role in B lineage development by recognizing endogenous ligands within the bursa of Fabricius. To address this issue directly we have introduced a truncated surface IgM, lacking variable region domains, into developing B precursors by retroviral gene transfer in vivo. Cells expressing this truncated receptor lack endogenous surface IgM, and the low level of endogenous Ig rearrangements that have occurred within this population of cells has not been selected for having a productive reading frame. Such cells proliferate rapidly within bursal epithelial buds of normal morphology. In addition, despite reduced levels of endogenous light chain rearrangement, those light chain rearrangements that have occurred have undergone variable region diversification by gene conversion. Therefore, although surface expression of an Ig receptor is required for bursal colonization and the induction of gene conversion, the specificity encoded by the prediversified receptor is irrelevant and, consequently, there is no obligate ligand for V(D)J-encoded determinants of prediversified avian cell surface IgM receptor.

Two mechanisms of antimutagenicity of the aminothiols cysteamine and WR-1065 in Saccharomycescerevisiae

The aminothiols cysteamine (CSM) and 2-[(aminopropyl)amino] ethanethiol (WR-1065) are radioprotectors, in that they reduce the mutagenic and potentially lethal consequences of ionizing radiation. We have studied the effects of these compounds on the induction of gene conversion at the trp5 locus in Saccharomyces cerevisiae strain D7 by two chemical mutagens: bleomycin (BLM) and β-propiolactone ( β-PL). Both mutagens are potent recombinagens in this assay, giving rise to trp5 convertant frequencies greater than 10 −3. CSM and WR-1065 are effective antimutagens, reducing the recombinagenic effect of β-PL by about 95%. The maximum reduction in the recombinagenic activity of BLM was 91% with CSM and 89% with WR-1065. Although the antimutagenic effects of the aminothiols against β-PL and BLM appear to be similar, they stem from different underlying mechanisms. Aminothiols protect against the recombinagenicity of β-PL through direct inactivation of the electrophilic mutagen by the nucleophilic antimutagen. They protect against that of BLM through modification of physiological conditions, most notably by depletion of oxygen required for BLM activity.

Double-Strand Break Repair in Yeast Requires Both Leading and Lagging Strand DNA Polymerases

Mitotic double-strand break (DSB)-induced gene conversion at MAT in Saccharomyces cerevisiae was analyzed molecularly in mutant strains thermosensitive for essential replication factors. The processivity cofactors PCNA and RFC are essential even to synthesize as little as 30 nucleotides following strand invasion. Both PCNA-associated DNA polymerases δ and ε are important for gene conversion, though a temperature-sensitive Pol ε mutant is more severe than one in Pol δ. Surprisingly, mutants of lagging strand replication, DNA polymerase α (pol1-17), DNA primase (pri2-1), and Rad27p (rad27 Δ) also greatly inhibit completion of DSB repair, even in G1-arrested cells. We propose a novel model for DSB-induced gene conversion in which a strand invasion creates a modified replication fork, involving leading and lagging strand synthesis from the donor template. Replication is terminated by capture of the second end of the DSB.

DNA damage caused by etoposide and γ-irradiation induces gene conversion of the MHC in a mouse non-germline testis cell line

We have explored the effects of γ-irradiation and etoposide on the gene conversion frequency between the endogenous major histocompatibility complex class II genes Abk and Ebd in a mouse testis cell line of non-germline origin with a polymerase chain reaction assay. Both γ-rays and etoposide were shown to increase the gene conversion frequency with up to 15-fold compared to untreated cells. Etoposide, which is an agent that stabilise a cleavable complex between DNA and DNA topoisomerase II, shows an increased induction of gene conversion events with increased dose of etoposide. Cells treated with γ-rays, which induce strand breaks, had an increased gene conversion frequency when they were subjected to low doses of irradiation, but increasing doses of irradiation did not lead to an increase of gene conversion events, which might reflect differences in the repair process depending on the extent and nature of the DNA damage. These results where DNA damage was shown to be able to induce gene conversion of endogenous genes in mouse testis cells suggests that the DNA repair system could be involved in the molecular genetic mechanism that results in gene conversion in higher eukaryotes like mammals.

Gene-conversion tract directionality is influenced by the chromosome environment.

Spontaneous and double-strand break (DSB)-induced gene conversion in Saccharomyces cerevisiae was assayed using non-tandem chromosomal direct repeat crosses and plasmid x chromosome crosses. Each cross involved identical ura3 alleles marked with phenotypically silent restriction fragment length polymorphic (RFLP) mutations at approximately 100-bp intervals. DSBs introduced in vivo at HO sites in one allele stimulated recombination to Ura+ by more than two orders of magnitude. Spontaneous gene-conversion products were isolated from a related strain lacking a functional HO nuclease gene. The multiple markers did not appear to influence the frequency of direct repeat deletions for spontaneous or DSB-induced events. DSB-induced conversion reflected efficient mismatch repair of heteroduplex DNA. Conversion frequencies of equidistant markers on opposites sides of the DSB were similar in the direct repeat cross. In contrast, markers 5' of the DSB (promoter-proximal) converted more often than 3' markers in plasmid x chromosome crosses, a possible consequence of crossing-over associated with long conversion tracts. With direct repeats, bidirectional tracts (extending 5' and 3' of the DSB) occurred twice as often as in a plasmid x chromosome cross in which DSBs were introduced into the plasmid-borne allele. A key difference between the direct-repeat and plasmidxchromosome crosses is that the ends of a broken plasmid are linked, whereas the ends of a broken chromosome are unlinked. We tested whether linkage of ends influenced tract directionality using a second plasmid x chromosome cross in which DSBs were introduced into the chromosomal allele and found few bidirectional tracts. Thus, chromosome environment, but not linkage of ends, influences tract directionality. The similar tract spectra of the two plasmid x chromosome crosses suggest that similar mechanisms are involved whether recombination is initiated by DSBs in plasmid or chromosomal alleles.

Evidence for independent mismatch repair processing on opposite sides of a double-strand break in Saccharomyces cerevisiae.

Double-strand break (DSB) induced gene conversion in Saccharomyces cerevisiae during meiosis and MAT switching is mediated primarily by mismatch repair of heteroduplex DNA (hDNA). We used nontandem ura3 duplications containing palindromic frameshift insertion mutations near an HO nuclease recognition site to test whether mismatch repair also mediates DSB-induced mitotic gene conversion at a non-MAT locus. Palindromic insertions included in hDNA are expected to produce a stem-loop mismatch, escape repair, and segregate to produce a sectored (Ura+/-) colony. If conversion occurs by gap repair, the insertion should be removed on both strands, and converted colonies will not be sectored. For both a 14-bp palindrome, and a 37-bp near-palindrome, approximately 75% of recombinant colonies were sectored, indicating that most DSB-induced mitotic gene conversion involves mismatch repair of hDNA. We also investigated mismatch repair of well-repaired markers flanking an unrepaired palindrome. As seen in previous studies, these additional markers increased loop repair (likely reflecting corepair). Among sectored products, few had additional segregating markers, indicating that the lack of repair at one marker is not associated with inefficient repair at nearby markers. Clear evidence was obtained for low levels of short tract mismatch repair. As seen with full gene conversions, donor alleles in sectored products were not altered. Markers on the same side of the DSB as the palindrome were involved in hDNA less often among sectored products than nonsectored products, but markers on the opposite side of the DSB showed similar hDNA involvement among both product classes. These results can be explained in terms of corepair, and they suggest that mismatch repair on opposite sides of a DSB involves distinct repair tracts.