Intrachromosomal Homologous Recombination Research Articles

Evidence for yeast artificial synthesis in SARS-CoV-2 and SARS-CoV-1 genomic sequences

Background: Knowledge about the origin of SARS-CoV-2 is necessary for both a biological and epidemiological understanding of the COVID-19 pandemic. Evidence suggests that a proximal evolutionary ancestor of SARS-CoV-2 belongs to the bat coronavirus family. However, as further evidence for a direct zoonosis remains limited, alternative modes of SARS-CoV-2 biogenesis should also be considered. Results: Here we show that the genomes of SARS-CoV-2 and SARS-CoV-1 significantly diverge from other SARS-like coronaviruses through short chromosomal sequences from the yeast S. cerevisiae at focal positions that are known to be critical for host cell invasion, virus replication, and host immune response. For SARS-CoV-1, we identify two sites: one at the start of the RNA dependent RNA polymerase gene, and the other at the start of the spike protein’s receptor binding domain; for SARS-CoV-2, one at the start of the viral replicase domain, and the other toward the end of the spike gene past its domain junction. At this junction, we detect a highly specific stretch of yeast origin covering the critical furin cleavage site insert PRRA, which has not been seen in other lineage b betacoronaviruses. As yeast is not a natural host for this virus family, we propose an artificial synthesis model for viral constructs in yeast cells based on co-transformation of virus DNA plasmids carrying yeast selectable genetic markers followed by intra-chromosomal homologous recombination through gene conversion. Highly differential yeast sequence patterns congruent with chromosomes harboring specific auxotrophic markers further support yeast artificial synthesis. Conclusions: These results provide evidence that the genomes of SARS-CoV-1 and SARS-CoV-2 contain sequence information that points to their artificial synthesis in genetically modified yeast cells. Our data specifically allow the identification of the yeast S. cerevisiae as a potential recombination donor for the critical furin cleavage site in SARS-CoV-2.

Combining cytogenetic and genomic technologies for deciphering challenging complex chromosomal rearrangements.

Complex chromosomal rearrangements (CCRs), a class of structural variants (SVs) involving more than two chromosome breaks, were classically thought to be extremely rare. As advanced technologies become more available, it has become apparent that CCRs are more common than formerly thought, and are a substantial cause of genetic disorders. We attempted a novel approach for solving the mechanism of challenging CCRs, which involve repetitive sequences, by precisely identifying sequence-level changes and their order. Chromosomal microarray (CMA) and FISH analyses were used for interpretation of SVs detected by whole exome sequencing (WES). Breakpoint junctions were analyzed by Nanopore sequencing, a novel long-read whole genome sequencing tool. A large deletion identified by WES, encompassing the FOXF1 enhancer, was the cause of alveolar capillary dysplasia and respiratory insufficiency, resulting in perinatal death. CMA analysis of the newborn's mother revealed two duplications encompassing the deleted region in theproband, raising our hypothesis that the deletion resulted from themother's CCR. Breakpoint junctions of complex SVs were determined at thenucleotide level using Nanopore long-read sequencing. According to sequencing results of breakpoint junctions, the CCR in the newborn was considered the consequence of at least one double-strand break during meiosis, and reassembly of DNA fragments by intra-chromosomal homologous recombination. Our comprehensive approach, combining cytogenetics and long-read sequencing, enabled delineation of the exact breakpoints in a challenging CCR, and proposal of a mechanism in which it arises. We suggest applying our integrative approach combining technologies for deciphering future challenging CCRs, enabling risk assessment in families.

Seamless Gene Correction in the Human Cystic Fibrosis Transmembrane Conductance Regulator Locus by Vector Replacement and Vector Insertion Events.

Background: Gene correction via homology directed repair (HDR) in patient-derived induced pluripotent stem (iPS) cells for regenerative medicine are becoming a more realistic approach to develop personalized and mutation-specific therapeutic strategies due to current developments in gene editing and iPSC technology. Cystic fibrosis (CF) is the most common inherited disease in the Caucasian population, caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Since CF causes significant multi-organ damage and with over 2,000 reported CFTR mutations, CF patients could be one prominent population benefiting from gene and cell therapies. When considering gene-editing techniques for clinical applications, seamless gene corrections of the responsible mutations, restoring native “wildtype” DNA sequence without remnants of drug selectable markers or unwanted DNA sequence changes, would be the most desirable approach. Result: The studies reported here describe the seamless correction of the W1282X CFTR mutation using CRISPR/Cas9 nickases (Cas9n) in iPS cells derived from a CF patient homozygous for the W1282X Class I CFTR mutation. In addition to the expected HDR vector replacement product, we discovered another class of HDR products resulting from vector insertion events that created partial duplications of the CFTR exon 23 region. These vector insertion events were removed via intrachromosomal homologous recombination (IHR) enhanced by double nicking with CRISPR/Cas9n which resulted in the seamless correction of CFTR exon 23 in CF-iPS cells. Conclusion: We show here the removal of the drug resistance cassette and generation of seamless gene corrected cell lines by two independent processes: by treatment with the PiggyBac (PB) transposase in vector replacements or by IHR between the tandemly duplicated CFTR gene sequences.

Evidence for yeast artificial synthesis in SARS-CoV-2 and SARS-CoV-1 genomic sequences

Background: Knowledge about the origin of SARS-CoV-2 is necessary for both a biological and epidemiological understanding of the COVID-19 pandemic. Evidence suggests that a proximal evolutionary ancestor of SARS-CoV-2 belongs to the bat coronavirus family. However, as further evidence for a direct zoonosis remains limited, alternative modes of SARS-CoV-2 biogenesis should be considered. Results: Here we show that the genomes from SARS-CoV-2 and from SARS-CoV-1 are differentially enriched with short chromosomal sequences from the yeast S. cerevisiae at focal positions that are known to be critical for host cell invasion, virus replication, and host immune response. For SARS-CoV-1, we identify two sites: one at the start of the RNA dependent RNA polymerase gene, and the other at the start of the spike protein’s receptor binding domain; for SARS-CoV-2, one at the start of the viral replicase domain, and the other toward the end of the spike gene past its domain junction. At this junction, we detect a highly specific stretch of yeast DNA origin covering the critical furin cleavage site insert PRRA, which has not been seen in other lineage b betacoronaviruses. As yeast is not a natural host for this virus family, we propose a passage model for viral constructs in yeast cells based on co-transformation of virus DNA plasmids carrying yeast selectable genetic markers followed by intra-chromosomal homologous recombination through gene conversion. Highly differential sequence homology data across yeast chromosomes congruent with chromosomes harboring specific auxotrophic markers further support this passage model. Conclusions: These results provide evidence that among SARS-like coronaviruses only the genomes of SARS-CoV-1 and SARS-CoV-2 contain information that points to a synthetic passage in genetically modified yeast cells. Our data specifically allow the identification of the yeast S. cerevisiae as a potential recombination donor for the critical furin cleavage site in SARS-CoV-2.

Differential enrichment of yeast DNA in SARS-CoV-2 and related genomes supports synthetic origin hypothesis

Background: Knowledge about the origin of SARS-CoV-2 is necessary for both a biological and epidemiological understanding of the COVID-19 pandemic. Evidence suggests that a proximal evolutionary ancestor of SARS-CoV-2 belongs to the bat coronavirus family. However, as further evidence for a direct zoonosis remains limited, alternative modes of SARS-CoV-2 biogenesis should be considered. Results: Here we show that the genomes from SARS-CoV-2 and from SARS-CoV-1 are differentially enriched with short chromosomal sequences from the yeast S. cerevisiae at focal positions that are known to be critical for host cell invasion, virus replication, and host immune response. For SARS-CoV-1, we identify two sites: one at the start of the RNA dependent RNA polymerase gene, and the other at the start of the spike protein’s receptor binding domain; for SARS-CoV-2, one at the start of the viral replicase domain, and the other toward the end of the spike gene past its critical domain junction. At this junction, we detect a highly specific stretch of yeast DNA encoding for the critical furin cleavage site insert PRRA, which has not been seen in other lineage b betacoronaviruses. As yeast is not a natural host for this virus family, we propose a passage model for viral constructs in yeast cells based on co-transformation of virus DNA plasmids carrying yeast selectable genetic markers followed by intra-chromosomal homologous recombination through gene conversion. Highly differential sequence homology data across yeast chromosomes congruent with chromosomes harboring specific auxotrophic markers further support this passage model. Conclusions: These results provide evidence that among SARS-like coronaviruses only the genomes of SARS-CoV-1 and SARS-CoV-2 contain information that points to a synthetic passage in genetically modified yeast cells. Our data specifically allow the identification of the yeast S. cerevisiae as a potential recombination donor for the critical furin cleavage site in SARS-CoV-2.

Differential enrichment of yeast DNA in SARS-CoV-2 and related genomes supports synthetic origin hypothesis

Background: Knowledge about the origin of SARS-CoV-2 is necessary for both a biological and epidemiological understanding of the COVID-19 pandemic. Evidence suggests that a proximal evolutionary ancestor of SARS-CoV-2 belongs to the bat coronavirus family. However, as further evidence for a direct zoonosis remains limited, alternative modes of SARS-CoV-2 biogenesis should be also considered. Results: Here we show that the genomes from SARS-CoV-2 and from SARS-CoV-1 are differentially enriched with short chromosomal sequences from the yeast S. cerevisiae at focal positions that are known to be critical for virus replication, host cell invasion, and host immune response. Specifically, for SARS-CoV-2, we identify two sites: one at the start of the viral replicase domain, and the other at the end of the spike gene past its critical domain junction; for SARS-CoV-1, one at the start of the RNA dependent RNA polymerase gene, and the other at the start of the spike protein’s receptor binding domain. As yeast is not a natural host for this virus family, we propose a directed passage model for viral constructs, including virus replicase, in yeast cells based on co-transformation of virus DNA plasmids carrying yeast selectable genetic markers followed by intra-chromosomal homologous recombination through gene conversion. Highly differential sequence homology data across yeast chromosomes congruent with chromosomes harboring specific auxotrophic markers further support this passage model. Model and data together allow us to infer a hypothetical tripartite genome assembly scheme for the synthetic biogenesis of SARS-CoV-2 and SARS-CoV-1. Conclusions: These results provide evidence that the genome sequences of SARS-CoV-1, SARS-CoV-2, but not that of RaTG13, BANAL-20-52 and all other closest SARS coronavirus family members identified, are carriers of distinct homology signals that might point to large-scale genomic editing during a passage of directed replication and chromosomal integration inside genetically modified yeast cells.

Differential enrichment of yeast DNA in SARS-CoV-2 and related genomes supports synthetic origin hypothesis

Background: Knowledge about the origin of SARS-CoV-2 is necessary for both a biological and epidemiological understanding of the COVID-19 pandemic. Evidence suggests that a proximal evolutionary ancestor of SARS-CoV-2 belongs to the bat coronavirus family. However, as further evidence for a direct zoonosis remains limited, alternative modes of SARS-CoV-2 biogenesis should be also considered. Results: Here we show that genomes from SARS-CoV-2 and from closely related coronaviruses are differentially enriched with short chromosomal sequences from the yeast S. cerevisiae at focal positions that are known to be critical for virus replication, host cell invasion, and host immune response. Specifically, for SARS-CoV-2, we identify two sites: one at the start of the viral replicase domain, and the other at the end of the spike gene past its critical domain junction; for SARS-CoV-1, one at the start of the RNA dependent RNA polymerase gene, and the other at the start of the spike protein’s receptor binding domain. As yeast is not a natural host for this virus family, we propose a directed passage model for viral constructs, including virus replicase, in yeast cells based on co-transformation of virus DNA plasmids carrying yeast selectable genetic markers followed by intra-chromosomal homologous recombination through gene conversion. Highly differential sequence homology data across yeast chromosomes congruent with chromosomes harboring specific auxotrophic markers further support this passage model. Model and data together allow us to infer a hypothetical tripartite genome assembly scheme for the synthetic biogenesis of SARS-CoV-2 and SARS-CoV-1. Conclusions: These results provide evidence that the genome sequences of SARS-CoV-1, SARS-CoV-2, but not that of RaTG13 and all other closest SARS coronavirus family members identified, are carriers of distinct homology signals that might point to large-scale genomic editing during a passage of directed replication and chromosomal integration inside genetically modified yeast cells.

Human shelterin protein POT1 prevents severe telomere instability induced by homology-directed DNA repair.

The evolutionarily conserved POT1 protein binds single-stranded G-rich telomeric DNA and has been implicated in contributing to telomeric DNA maintenance and the suppression of DNA damage checkpoint signaling. Here, we explore human POT1 function through genetics and proteomics, discovering that a complete absence of POT1 leads to severe telomere maintenance defects that had not been anticipated from previous depletion studies in human cells. Conditional deletion of POT1 in HEK293E cells gives rise to rapid telomere elongation and length heterogeneity, branched telomeric DNA structures, telomeric R-loops, and telomere fragility. We determine the telomeric proteome upon POT1-loss, implementing an improved telomeric chromatin isolation protocol. We identify a large set of proteins involved in nucleic acid metabolism that engage with telomeres upon POT1-loss. Inactivation of the homology-directed repair machinery suppresses POT1-loss-mediated telomeric DNA defects. Our results unravel as major function of human POT1 the suppression of telomere instability induced by homology-directed repair.

Stimulation of homologous recombination in plants expressing heterologous recombinases

BackgroundCurrent excitement about the opportunities for gene editing in plants have been prompted by advances in CRISPR/Cas and TALEN technologies. CRISPR/Cas is widely used to knock-out or modify genes by inducing targeted double-strand breaks (DSBs) which are repaired predominantly by error-prone non-homologous end-joining or microhomology-mediated end joining resulting in mutations that may alter or abolish gene function. Although such mutations are random, they occur at sufficient frequency to allow useful mutations to be routinely identified by screening. By contrast, gene knock-ins to replace entire genes with alternative alleles or copies with specific characterised modifications, is not yet routinely possible. Gene replacement (or gene targeting) by homology directed repair occurs at extremely low frequency in higher plants making screening for useful events unfeasible. Homology directed repair might be increased by inhibiting non-homologous end-joining and/or stimulating homologous recombination (HR). Here we pave the way to increasing gene replacement efficiency by evaluating the effect of expression of multiple heterologous recombinases on intrachromosomal homologous recombination (ICR) in Nicotiana tabacum plants.ResultsWe expressed several bacterial and human recombinases in different combinations in a tobacco transgenic line containing a highly sensitive β-glucuronidase (GUS)-based ICR substrate. Coordinated simultaneous expression of multiple recombinases was achieved using the viral 2A translational recoding system. We found that most recombinases increased ICR dramatically in pollen, where HR will be facilitated by the programmed DSBs that occur during meiosis. DMC1 expression produced the greatest stimulation of ICR in primary transformants, with one plant showing a 1000-fold increase in ICR frequency. Evaluation of ICR in homozygous T2 plant lines revealed increases in ICR of between 2-fold and 380-fold depending on recombinase(s) expressed. By comparison, ICR was only moderately increased in vegetative tissues and constitutive expression of heterologous recombinases also reduced plant fertility.ConclusionExpression of heterologous recombinases can greatly increase the frequency of HR in plant reproductive tissues. Combining such recombinase expression with the use of CRISPR/Cas9 to induce DSBs could be a route to radically improving gene replacement efficiency in plants.

In planta gene targeting can be enhanced by the use of CRISPR/Cas12a

The controlled change of plant genomes by homologous recombination (HR) is still difficult to achieve. We previously developed the in planta gene targeting (ipGT) technology which depends on the simultaneous activation of the target locus by a double-strand break and the excision of the target vector. Whereas the use of SpCas9 resulted in low ipGT frequencies in Arabidopsis, we were recently able to improve the efficiency by using egg cell-specific expression of the potent but less broadly applicable SaCas9 nuclease. In this study, we now tested whether we could improve ipGT further, by either performing it in cells with enhanced intrachromosomal HR efficiencies or by the use of Cas12a, a different kind of CRISPR/Cas nuclease with an alternative cutting mechanism. We could show before that plants possess three kinds of DNA ATPase complexes, which all lead to instabilities of homologous genomic repeats if lost by mutation. As these proteins act in independent pathways, we tested ipGT in double mutants in which intrachromosomal HR is enhanced 20-80-fold. However, we were not able to obtain higher ipGT frequencies, indicating that mechanisms for gene targeting (GT) and chromosomal repeat-induced HR differ. However, using LbCas12a, the GT frequencies were higher than with SaCas9, despite a lower non-homologous end-joining (NHEJ) induction efficiency, demonstrating the particular suitability of Cas12a to induce HR. As SaCas9 has substantial restrictions due to its longer GC rich PAM sequence, the use of LbCas12a with its AT-rich PAM broadens the range of ipGT drastically, particularly when targeting in CG-deserts like promoters and introns.

A Rational Combination Therapy Approach with Radiation Therapy in Ewing Sarcoma

Ewing sarcoma (EWS) is the second most common malignant bone tumor primarily occurring in adolescents and young adults. Radiation therapy (RT) is usually used in tumors not amenable to surgery or in those resected with compromised margins. It is also used in metastatic disease. Despite advances in the treatment of EWS that have improved the survival substantially to 70% in localized disease, outcome for metastatic, relapsed and recurrent disease remains dismal. Therefore, newer therapeutic approaches, in combination with conventional chemotherapy and RT are needed to improve outcome. Given known deficits in DNA damage repair in EWS, we hypothesize that adding poly-ADP ribose polymerase inhibitors (PARPis), which selectively target the DNA damage response, to the current treatment combinations may potentially improve the therapeutic outcome. We investigated primary and recurrent standard of care (SOC) chemotherapeutics and PARPis as single agents and in combination with RT in the treatment of several human EWS cell lines using colony forming assay. We also compared the result to a radioresistant osteosarcoma cell line, U2OS. DNA damage was assessed using immunofluorescence microscopy by quantifying γ-H2A.X foci. The effect of drugs on DNA repair was measured using a combined intra-chromosomal homologous recombination (HR) / mutagenic non-homologous end joining (mNHEJ) assay. To determine whether sensitivity of PARPis depends on the expression of PARP1, we established a stable cell line which PARP1 was knocked down when induced with doxycycline. The drug combinations were tested in vivo using orthotopic xenograft models of EWS with image-guided fractionated irradiation and tumor burden was monitored via bioluminescence. At physiologically-relevant concentrations of the PARPi talazoparib (TAL), the combination of RT+TAL showed the highest synergist effect and combined efficacy compared to the other PARPis and SOC chemotherapeutics in human EWS cell lines. Differences in radiosensitivity across systemic agents were partially correlated to their ability to induce DNA damage. Moreover, PARPis did not significantly affect HR or mNHEJ DNA repair pathways and inducible PARP knockdown affected EWS (ES8) cells’ sensitivity to TAL. The combinations of irinotecan (IRN)-TAL-RT was found to have equivalent tumor growth inhibition to IRN-TAL-temozolomide (TMZ), a combination currently under clinical investigation in pediatric patients with recurrent/refractory sarcoma (NCT02392793) in EWS mouse xenografts. Taking together, our work suggested the combination of TAL and IRN may be a useful therapeutic strategy when RT is used in the progressive disease setting.

Review of molecular mechanisms at distal Xq28 leading to balanced or unbalanced genomic rearrangements and their phenotypic impacts on hemophilia.

The distal Xq28 region is very gene-rich, comprising a relatively large number of low-copy repeats (LCRs) predisposing to genomic rearrangements. The best-known rearrangement at this locus is the F8 intron 22 inversion, responsible for up to 45% of severe hemophilia A (HA) cases. An additional inversion of intron 1 of F8 has more recently been described, affecting 2%-5% of patients with severe HA. These "balanced" rearrangements are mediated by intrachromosomal homologous recombination between inversely oriented LCRs located in intron 1 or 22 and other extragenic copies positioned more telomerically outside the F8 gene. The successive innovations of semi-quantitative technologies like multiplex ligation-dependent probe amplification (MLPA) and array comparative genomic hybridization (array CGH) have rendered it possible to highlight a significant number of "unbalanced" rearrangements associated or not with these inversions. Some rearrangements are generated by the non-allelic homologous recombination (NAHR) pathway between directly oriented LCRs. Others are probably the result of unequal crossing-over or U-loop exchanges during female meiosis. This review sought to provide an overview of the mechanisms underlying rearrangements at the distal Xq28 locus and discuss their clinical impacts other than HA, such as risks of developing high inhibitor levels and spontaneous abortion, as well as other pathologies like cardiovascular disease or potentially X-linked intellectual disease.

RPA Stabilization of Single-Stranded DNA Is Critical for Break-Induced Replication

DNA double-strand breaks (DSBs) are cytotoxic lesions that must be accurately repaired to maintain genome stability. Replication protein A (RPA) plays an important role in homology-dependent repair ofDSBs by protecting the single-stranded DNA (ssDNA) intermediates formed by end resection and by facilitating Rad51 loading. We found that hypomorphic mutants of RFA1 that support intra-chromosomal homologous recombination are profoundly defective for repair processes involving long tracts of DNA synthesis, in particular break-induced replication (BIR). The BIR defects of the rfa1 mutants could be partially suppressed by eliminating the Sgs1-Dna2 resection pathway, suggesting that Dna2 nuclease attacks the ssDNA formed during end resection when not fully protected by RPA. Overexpression of Rad51 was also found to suppress the rfa1 BIR defects. We suggest that Rad51 binding to the ssDNA formed by excessive end resection and during D-loop migration can partially compensate for dysfunctional RPA.

Mobilization of LINE-1 in irradiated mammary gland tissue may potentially contribute to low dose radiation-induced genomic instability.

It is known that cellular stresses such as ionizing radiation activate LINE-1 (long interspersed nuclear element type 1, L1), but the molecular mechanisms of LINE-1 activation have not been fully elucidated. There is a possibility that DNA methylation changes induced by genotoxic stresses might contribute to LINE-1 activation in mammalian cells. L1 insertions usually cause major genomic rearrangements, such as deletions, transductions, the intrachromosomal homologous recombination between L1s, and the generation of pseudogenes, which could lead to genomic instability. The purpose of this study was to evaluate the effects of low and high doses of ionizing radiation on the DNA methylation status of LINE-1 transposable elements in rat mammary glands. Here we describe radiation-induced hypomethylation and activation of LINE-1 ORF1 in rat mammary gland tissues. We show that radiation exposure has also led to the translation of the LINE-1 element, whereby the 148 kDa LINE-1 protein level was increased 96 hours after treatment with a low dose and low energy level radiation and remained elevated for 24 weeks after treatment. The mobilization of LINE-1 in irradiated tissue may potentially contribute to genomic instability. The observed activation of mobile elements in response to radiation exposure is consistently discussed as a plausible mechanism of cancer etiology and development.

FVIII intron 22 homologous region mediated recombinations and genotyping methods of hemophilia A

The intron 22 inversion is caused by intrachromosomal homologous recombination of the Int22h (intron 22 homologous region) repeats in opposite orientation, accounting for almost 45%-50% of all cases of severe hemophilia A. By contrast, recombinations between similarly oriented int22h repeats in the same chromosome, homologous chromosomes or chromatids may cause deletion or duplication of the intermediate region between the two int22h copies, resulting in false-positive or false-negative possibility that obscure the characterization of intron 22 inversion. The modified long distance PCR(LD-PCR) and inverse shifting PCR(IS-PCR) would distinguish all the possible int22h-mediated rearrangements.

Identification and characterisation of a novel aberrant pattern of intron 1 inversion with concomitant large insertion and deletion within the F8 gene

Intron 1 inversion (Inv1) is a recurrent causative mutation of haemophilia A (HA) and is responsible for 1-5% of severe HA. Inv1 occurs as a result of intra-chromosomal homologous recombination between int1h-1 within intron 1 and int1h-2 located in approximately 125 kb telomeric to the F8 gene. In this report, we presented a previously undescribed aberrant type of Inv1 with complex genomic rearrangement in a pedigree with severe HA. The breakpoints of the rearrangement were identified by the genome walking technique; copy number variations (CNVs) of the F8 gene and X chromosome were detected by AccuCopy technique, Affymetrix CytoScan HD CNV assay and quantitative PCR (qPCR); the F8 transcripts related to the aberrant Inv1 were analysed by reverse transcription PCR (RT-PCR). We have characterised the exact breakpoints of the complex rearrangement, and determined the location and size of the insertion and deletion. The rearrangements can be summarised as an aberrant pattern of Inv1 with a deletion of 2.56 kb and a duplication of 227.3 kb inserted in the rejoining junction within the F8 gene. Our results suggested that this complex genomic rearrangement was generated by two distinct repair mechanisms of fork stalling and template switching/microhomology-mediated break-induced replication (FoSTeS/MMBIR) and nonallelic homologous recombination (NAHR).

Double-Strand Break Repair by Interchromosomal Recombination: An In Vivo Repair Mechanism Utilized by Multiple Somatic Tissues in Mammals

Homologous recombination (HR) is essential for accurate genome duplication and maintenance of genome stability. In eukaryotes, chromosomal double strand breaks (DSBs) are central to HR during specialized developmental programs of meiosis and antigen receptor gene rearrangements, and form at unusual DNA structures and stalled replication forks. DSBs also result from exposure to ionizing radiation, reactive oxygen species, some anti-cancer agents, or inhibitors of topoisomerase II. Literature predicts that repair of such breaks normally will occur by non-homologous end-joining (in G1), intrachromosomal HR (all phases), or sister chromatid HR (in S/G2). However, no in vivo model is in place to directly determine the potential for DSB repair in somatic cells of mammals to occur by HR between repeated sequences on heterologs (i.e., interchromosomal HR). To test this, we developed a mouse model with three transgenes—two nonfunctional green fluorescent protein (GFP) transgenes each containing a recognition site for the I-SceI endonuclease, and a tetracycline-inducible I-SceI endonuclease transgene. If interchromosomal HR can be utilized for DSB repair in somatic cells, then I-SceI expression and induction of DSBs within the GFP reporters may result in a functional GFP+ gene. Strikingly, GFP+ recombinant cells were observed in multiple organs with highest numbers in thymus, kidney, and lung. Additionally, bone marrow cultures demonstrated interchromosomal HR within multiple hematopoietic subpopulations including multi-lineage colony forming unit–granulocyte-erythrocyte-monocyte-megakaryocte (CFU-GEMM) colonies. This is a direct demonstration that somatic cells in vivo search genome-wide for homologous sequences suitable for DSB repair, and this type of repair can occur within early developmental populations capable of multi-lineage differentiation.

Zinc finger artificial transcription factor–based nearest inactive analogue/nearest active analogue strategy used for the identification of plant genes controlling homologous recombination

In previous work, we selected a particular transcription factor, designated VP16-HRU, from a pool of zinc finger artificial transcription factors (ZF-ATFs) used for genome interrogation. When expressed in Arabidopsis thaliana under control of the ribosomal protein S5A promoter, the RPS5A::VP16-HRU construct led to a 200- to 300-fold increase in the frequency of somatic intrachromosomal homologous recombination (iHR). Because the expression of each ZF-ATF leads to a large number of transcriptional changes, we designed a strategy employing a collection of structurally similar ZF-ATFs to filter out the transcriptional changes relevant to the phenotype by deep sequencing. In that manner, 30 transcripts were found to be consistently induced in plants with enhanced homologous recombination (HR). For 25 of the cognate genes, their effect on the HR process was assessed using cDNA/gDNA expression constructs. For three genes, ectopic expression indeed led to enhanced iHR frequencies, albeit much lower than the frequency observed when a HR-inducing ZF-ATF was present. Altogether, our data demonstrate that despite the large number of transcriptional changes brought about by individual ZF-ATFs, causal changes can be identified. In our case, the picture emerged that a natural regulatory switch for iHR does not exist but that ZF-ATFs-like VP16-HRU act as an ectopic master switch, orchestrating the timely expression of a set of plant genes that each by themselves only have modest effects, but when acting together support an extremely high iHR frequency.

Intrachromosomal homologous recombination between inverted amplicons on opposing Y-chromosome arms

Amplicons – large, nearly identical repeats in direct or inverted orientation – are abundant in the male-specific region of the human Y chromosome (MSY) and provide targets for intrachromosomal non-allelic homologous recombination (NAHR). Thus far, NAHR events resulting in deletions, duplications, inversions, or isodicentric chromosomes have been reported only for amplicon pairs located exclusively on the short arm (Yp) or the long arm (Yq). Here we report our finding of four men with Y chromosomes that evidently formed by intrachromosomal NAHR between inverted repeat pairs comprising one amplicon on Yp and one amplicon on Yq. In two men with spermatogenic failure, sister-chromatid crossing-over resulted in pseudoisoYp chromosome formation and loss of distal Yq. In two men with normal spermatogenesis, intrachromatid crossing-over generated pericentric inversions. These findings highlight the recombinogenic nature of the MSY, as intrachromosomal NAHR occurs for nearly all Y-chromosome amplicon pairs, even those located on opposing chromosome arms.

Stress Activated Protein Kinase Pathway Modulates Homologous Recombination in Fission Yeast

Rad52 is a key player in homologous recombination (HR), a DNA repair pathway that is dedicated to double strand breaks repair and recovery of perturbed replication forks. Here we show that fission yeast Rad52 homologue is phosphorylated when S phase cells are exposed to ROS inducers such as ultraviolet A radiation or hydrogen peroxide, but not to ultraviolet C or camptothecin. Phosphorylation does not depend on kinases Chk1, Rad3, Tel1 or Cdc2, but depends on a functional stress activated protein kinase (SAPK) pathway and can be partially prevented by anti-oxidant treatment. Indeed, cells lacking Sty1, the major fission yeast MAP kinase of the SAPK pathway, do not display Rad52 phosphorylation and have UVA induced Rad52 foci that persist longer if compared to wild type cells. In addition, spontaneous intrachromosomal HR is diminished in cells lacking Sty1 and, more precisely, gene conversion is affected. Moreover, HR induced by site-specific arrest of replication forks is twice less efficient in cells that do not express Sty1. Importantly, impairing HR by deletion of the gene encoding the recombinase Rhp51 leads to Sty1 dependent Rad52 phosphorylation. Thus, SAPK pathway impinges on early step of HR through phosphorylation of Rad52 in cells challenged by oxidative stress or lacking Rhp51 and is required to promote spontaneous gene conversion and recovery from blocked replication forks.