Ku86 deficiency leads to reduced intrachromosomal homologous recombination in vivo in mice

Ku70 and Ku86 together with DNA-PKcs form the DNA-dependent protein kinase (DNA-PK) complex that is involved in DNA double-strand break repair by nonhomologous end joining. We investigated the effect of Ku86 mutation on intrachromosomal homologous recombination (HR) resulting in deletions in vivo in mice. We quantified such deletion events using a phenotypic pigmentation assay. Deletion of one copy of a 70 kb DNA duplication in the pink-eyed unstable (pun) allele results in reversion to the wildtype pink-eyed dilution (p) gene, allowing black pigment accumulation in cells of the retinal pigment epithelium (RPE). We found that the frequency of homologous recombination was significantly reduced in Ku86 deficient mice. Furthermore, the proliferation of cells in which recombination events occurred was reduced and developmentally delayed in the Ku86 deficient mice. These data indicate a role for Ku86 directly or indirectly in homologous recombination in vivo.     

Detection of homologous recombination events in SARS-CoV-2

Biotechnology Letters - The COVID-19 disease with acute respiratory symptoms emerged in 2019. The causal agent of the disease, the SARS-CoV-2 virus, is classified into the Betacoronaviruses family....     

Inactivation of homologous recombination suppresses defects in topoisomerase III-deficient mutants

The Saccharomyces cerevisiae TOP3 gene encodes the type IA topoisomerase (Top3p) that is highly conserved in evolution. Deletion of TOP3 leads to a reduction in cell viability, hyper-recombination between repetitive DNA sequences, and abnormalities in both cell cycle progression and responses to DNA damaging agents. Deletion of SGS1, encoding the sole RecQ family helicase in S. cerevisiae, strongly suppresses the phenotypic effects of loss of TOP3 function. Here, we show that many of the adverse phenotypic effects of TOP3 deletion can also be partially alleviated by disruption of homologous recombination (HR) functions. This genetic interaction is seen both in strains deleted for TOP3 and in wild-type strains over-expressing a dominant-negative Top3p mutant form that confers a top3-like phenotype. Moreover, we show that this genetic interaction is conserved in the distantly-related fission yeast, Schizosaccharomyces pombe. Our results implicate topoisomerase III enzymes in recombination repair events required for cellular protection against DNA damaging agents and DNA replication inhibitors.     

Short homologies efficiently generate detectable homologous recombination events

When recombineering bacterial artificial chromosomes (BACs), it is common practice to design the ends of the donor molecule with 50 bp of homology specifying its insertion site. We demonstrate that desired recombinants can be produced using intermolecular homologies as short as 15 bp. Although the use of shorter donor end regions decreases total recombinants by several fold, the frequency of recombinants with correctly inserted donor molecules was high enough for easy detection by simple polymerase chain reaction (PCR) screening. This observation may have important implications for the design of oligonucleotides for recombineering, including significant cost savings, especially for high-throughput projects that use large quantities of primers.     

Extensive homologous recombination in classical swine fever virus: A re-evaluation of homologous recombination events in the strain AF407339

In this short report, the genome-wide homologous recombination events were re-evaluated for classical swine fever virus (CSFV) strain {"type":"entrez-nucleotide","attrs":{"text":"AF407339","term_id":"15488012","term_text":"AF407339"}}AF407339. We challenged a previous study which suggested only one recombination event in {"type":"entrez-nucleotide","attrs":{"text":"AF407339","term_id":"15488012","term_text":"AF407339"}}AF407339 based on 25 CSFV genomes. Through our re-analysis on the 25 genomes in the previous study and the 41 genomes used in the present study, we argued that there should be possibly at least two clear recombination events happening in {"type":"entrez-nucleotide","attrs":{"text":"AF407339","term_id":"15488012","term_text":"AF407339"}}AF407339 through genome-wide scanning. The reasons for identifying only one recombination event in the previous study might be due to the limited number of available CSFV genome sequences at that time and the limited usage of detection methods. In contrast, as identified by most detection methods using all available CSFV genome sequences, two major recombination events were found at the starting and ending zones of the genome {"type":"entrez-nucleotide","attrs":{"text":"AF407339","term_id":"15488012","term_text":"AF407339"}}AF407339, respectively. The first one has two parents {"type":"entrez-nucleotide","attrs":{"text":"AF333000","term_id":"13383931","term_text":"AF333000"}}AF333000 (minor) and {"type":"entrez-nucleotide","attrs":{"text":"AY554397","term_id":"49860681","term_text":"AY554397"}}AY554397 (major) with beginning and ending breakpoints located at 19 and 607 nt of the genome respectively. The second one has two parents {"type":"entrez-nucleotide","attrs":{"text":"AF531433","term_id":"22347661","term_text":"AF531433"}}AF531433 (minor) and {"type":"entrez-nucleotide","attrs":{"text":"GQ902941","term_id":"298113017","term_text":"GQ902941"}}GQ902941 (major) with beginning and ending breakpoints at 8397 and 11,078 nt of the genome respectively. Phylogenetic incongruence analysis using neighbor-joining algorithm with 1000 bootstrapping replicates further supported the existence of these two recombination events. In addition, we also identified additional 18 recombination events on the available CSFV strains. Some of them may be trivial and can be ignored. In conclusion, CSFV might have relatively high frequency of homologous recombination events. Genome-wide scanning of identifying recombination events should utilize multiple detection methods so as to reduce the risk of misidentification.     

BKM120 sensitizes glioblastoma to the PARP inhibitor rucaparib by suppressing homologous recombination repair

PARP inhibitors have been approved for the therapy of cancers with homologous recombination (HR) deficiency based on the concept of “synthetic lethality”. However, glioblastoma (GBM) patients have gained little benefit from PARP inhibitors due to a lack of BRCA mutations. Herein, we demonstrated that concurrent treatment with the PARP inhibitor rucaparib and the PI3K inhibitor BKM120 showed synergetic anticancer effects on GBM U251 and U87MG cells. Mechanistically, BKM120 decreased expression of HR molecules, including RAD51 and BRCA1/2, and reduced HR repair efficiency in GBM cells, therefore increasing levels of apoptosis induced by rucaparib. Furthermore, we discovered that the two compounds complemented each other in DNA damage response and drug accumulation. Notably, in the zebrafish U87MG-RFP orthotopic xenograft model, nude mouse U87MG subcutaneous xenograft model and U87MG-Luc orthotopic xenograft model, combination showed obviously increased antitumor efficacy compared to each monotherapy. Immunohistochemical analysis of tumor tissues indicated that the combination obviously reduced expression of HR repair molecules and increased the DNA damage biomarker γ-H2AX, consistent with the in vitro results. Collectively, our findings provide new insight into combined blockade of PI3K and PARP, which might represent a promising therapeutic approach for GBM.Subject terms: Targeted therapies, Drug development     

PLK1 depletion alters homologous recombination and synaptonemal complex disassembly events during mammalian spermatogenesis

Homologous recombination (HR) is an essential meiotic process that contributes to the genetic variation of offspring and ensures accurate chromosome segregation. Recombination is facilitated by the formation and repair of programmed DNA double-strand breaks. These DNA breaks are repaired via recombination between maternal and paternal homologous chromosomes and a subset result in the formation of crossovers. HR and crossover formation is facilitated by synapsis of homologous chromosomes by a proteinaceous scaffold structure known as the synaptonemal complex (SC). Recent studies in yeast and worms have indicated that polo-like kinases (PLKs) regulate several events during meiosis, including DNA recombination and SC dynamics. Mammals express four active PLKs (PLK1–4), and our previous work assessing localization and kinase function in mouse spermatocytes suggested that PLK1 coordinates nuclear events during meiotic prophase. Therefore, we conditionally mutated Plk1 in early prophase spermatocytes and assessed stages of HR, crossover formation, and SC processes. Plk1 mutation resulted in increased RPA foci and reduced RAD51/DMC1 foci during zygonema, and an increase of both class I and class II crossover events. Furthermore, the disassembly of SC lateral elements was aberrant. Our results highlight the importance of PLK1 in regulating HR and SC disassembly during spermatogenesis.     

Role of SIRT1 in homologous recombination

The class III histone deacetylase (HDAC) SIRT1 plays a role in the metabolism, aging, and carcinogenesis of organisms and regulates senescence and apoptosis in cells. Recent reports revealed that SIRT1 also deacetylates several DNA double-strand break (DSB) repair proteins. However, its exact functions in DNA repair remained elusive. Using nuclear foci analysis and fluorescence-based, chromosomal DSB repair reporter, we find that SIRT1 activity promotes homologous recombination (HR) in human cells. Importantly, this effect is unrelated to functions of poly(ADP-ribose) polymerase 1 (PARP1), another NAD(+)-catabolic protein, and does not correlate with cell cycle changes or apoptosis. Interestingly, we demonstrate that inactivation of Rad51 does not eliminate the effect of SIRT1 on HR. By epistasis-like analysis through knockdown and use of mutant cells of distinct SIRT1 target proteins, we show that the non-homologous end joining (NHEJ) factor Ku70 as well as the Nijmegen Breakage Syndrome protein (nibrin) are not needed for this SIRT1-mediated effect, even though a partial contribution of nibrin cannot be excluded. Strikingly however, the Werner helicase (WRN), which in its mutated form causes premature aging and cancer and which was linked to the Rad51-independent single-strand annealing (SSA) DSB repair pathway, is required for SIRT1-mediated HR. These results provide first evidence that links SIRT1's functions to HR with possible implications for genomic stability during aging and tumorigenesis.     

BCCIP regulates homologous recombination by distinct domains and suppresses spontaneous DNA damage

Homologous recombination (HR) is critical for maintaining genome stability through precise repair of DNA double-strand breaks (DSBs) and restarting stalled or collapsed DNA replication forks. HR is regulated by many proteins through distinct mechanisms. Some proteins have direct enzymatic roles in HR reactions, while others act as accessory factors that regulate HR enzymatic activity or coordinate HR with other cellular processes such as the cell cycle. The breast cancer susceptibility gene BRCA2 encodes a critical accessory protein that interacts with the RAD51 recombinase and this interaction fluctuates during the cell cycle. We previously showed that a BRCA2- and p21-interacting protein, BCCIP, regulates BRCA2 and RAD51 nuclear focus formation, DSB-induced HR and cell cycle progression. However, it has not been clear whether BCCIP acts exclusively through BRCA2 to regulate HR and whether BCCIP also regulates the alternative DSB repair pathway, non-homologous end joining. In this study, we found that BCCIP fragments that interact with BRCA2 or with p21 each inhibit DSB repair by HR. We further show that transient down-regulation of BCCIP in human cells does not affect non-specific integration of transfected DNA, but significantly inhibits homology-directed gene targeting. Furthermore, human HT1080 cells with constitutive down-regulation of BCCIP display increased levels of spontaneous single-stranded DNA (ssDNA) and DSBs. These data indicate that multiple BCCIP domains are important for HR regulation, that BCCIP is unlikely to regulate non-homologous end joining, and that BCCIP plays a critical role in resolving spontaneous DNA damage.     

Arabidopsis RecQI4A suppresses homologous recombination and modulates DNA damage responses

The DNA damage response and DNA recombination are two interrelated mechanisms involved in maintaining the integrity of the genome, but in plants they are poorly understood. RecQ is a family of genes with conserved roles in the regulation of DNA recombination in eukaryotes; there are seven members in Arabidopsis. Here we report on the functional analysis of the Arabidopsis RecQl4A gene. Ectopic expression of Arabidopsis RecQl4A in yeast RecQ-deficient cells suppressed their hypersensitivity to the DNA-damaging drug methyl methanesulfonate (MMS) and enhanced their rate of homologous recombination (HR). Analysis of three recQl4A mutant alleles revealed no obvious developmental defects or telomere deregulation in plants grown under standard growth conditions. Compared with wild-type Arabidopsis, the recQl4A mutant seedlings were found to be hypersensitive to UV light and MMS, and more resistant to mitomycin C. The average frequency of intrachromosomal HR in recQl4A mutant plants was increased 7.5-fold over that observed in wild-type plants. The data reveal roles for Arabidopsis RecQl4A in maintenance of genome stability by modulation of the DNA damage response and suppression of HR.     

A conditional mouse model for measuring the frequency of homologous recombination events in vivo in the absence of essential genes

The ability to detect and repair DNA damage is crucial to the prevention of various diseases. Loss of function of genes involved in these processes is known to result in significant developmental defects and/or predisposition to cancer. One such DNA repair mechanism, homologous recombination, has the capacity to repair a wide variety of lesions. Knockout mouse models of genes thought to be involved in DNA repair processes are frequently lethal, making in vivo studies very difficult, if not impossible. Therefore, we set out to develop an in vivo conditional mouse model system to facilitate investigations into the involvement of essential genes in homologous recombination. To test our model, we measured the frequency of spontaneous homologous recombination using the pink-eyed unstable mouse model, in which we conditionally excised either Blm or full-length Brca1 (breast cancer 1, early onset). These two genes are hypothesized to have opposing roles in homologous recombination. In summary, our in vivo data supports in vitro studies suggesting that BLM suppresses homologous recombination, while full-length BRCA1 promotes this process.     

Caffeine suppresses homologous recombination through interference with RAD51-mediated joint molecule formation

Caffeine is a widely used inhibitor of the protein kinases that play a central role in the DNA damage response. We used chemical inhibitors and genetically deficient mouse embryonic stem cell lines to study the role of DNA damage response in stable integration of the transfected DNA and found that caffeine rapidly, efficiently and reversibly inhibited homologous integration of the transfected DNA as measured by several homologous recombination-mediated gene-targeting assays. Biochemical and structural biology experiments revealed that caffeine interfered with a pivotal step in homologous recombination, homologous joint molecule formation, through increasing interactions of the RAD51 nucleoprotein filament with non-homologous DNA. Our results suggest that recombination pathways dependent on extensive homology search are caffeine-sensitive and stress the importance of considering direct checkpoint-independent mechanisms in the interpretation of the effects of caffeine on DNA repair.     

A BRCA1-interacting lncRNA regulates homologous recombination

Long non-coding RNAs (lncRNAs) are important players in diverse biological processes. Upon DNA damage, cells activate a complex signaling cascade referred to as the DNA damage response (DDR). Using a microarray screen, we identify here a novel lncRNA, DDSR1 (DNA damage-sensitive RNA1), which is induced upon DNA damage. DDSR1 induction is triggered in an ATM-NF-κB pathway-dependent manner by several DNA double-strand break (DSB) agents. Loss of DDSR1 impairs cell proliferation and DDR signaling and reduces DNA repair capacity by homologous recombination (HR). The HR defect in the absence of DDSR1 is marked by aberrant accumulation of BRCA1 and RAP80 at DSB sites. In line with a role in regulating HR, DDSR1 interacts with BRCA1 and hnRNPUL1, an RNA-binding protein involved in DNA end resection. Our results suggest a role for the lncRNA DDSR1 in modulating DNA repair by HR.     

Keeping homologous recombination in check

Pathway choice is a critical event in the repair of DNA double-strand breaks. In a recent paper published in Nature, Orthwein et al. define a mechanism by which homologous recombination is controlled in G1 cells to favor non-homologous end joining.Homologous recombination (HR) is an essential process that produces genetic variation during meiosis and protects the genome during mitotic cell division¹. Inherited mutations in various HR factors, including the BRCA1, BRCA2 and PALB2 tumor suppressors, predispose to the development of cancer. Although HR is generally beneficial for maintaining genome integrity, HR events between homologous chromosomes can also be deleterious and lead to loss of genetic information. HR is therefore suppressed during G1 phase and in non-dividing cells, yet, the exact mechanism behind this phenomenon has remained elusive. New work from the laboratory of Daniel Durocher describes a mechanism that is both necessary and sufficient for the suppression of HR in G1 cells².DNA double-strand breaks (DSBs) are one of the most dangerous types of DNA lesion and need to be eliminated to prevent the accumulation of mutations. DSB repair is carried out by two main pathways, HR and non-homologous end joining (NHEJ)¹. Whereas NHEJ is an error-prone process that simply fuses the two broken ends together, HR is essentially error-free as it uses the genetically identical sister chromatid as a template for repair. Due to the cell cycle-dependent availability of sister chromatids, HR is restricted to the S and G2 phases of the cell cycle.In the HR repair pathway, the DSB ends are first resected to produce extended single-stranded DNA (ssDNA) tails by the coordinated actions of a series of helicase and nuclease activities (e.g., MRN, CtIP and EXO1)¹. CtIP plays a particularly important role in regulating resection, which is mediated through its interaction with BRCA1³. In the following cascade of events, BRCA1 interacts directly with the BRCA2-PALB2 complex, which in turn is recruited to the ssDNA where it acts as a chaperone that stimulates the formation of RAD51 nucleoprotein filaments that drive homology-directed HR repair to restore the integrity of the DNA^4,5.Whereas most HR events take place between the newly replicated sister chromatids, recombination between homologous chromosomes can result in loss of heterozygosity, a potentially mutagenic event that can lead to the inactivation of tumor suppressors or activation of oncogenes. HR must therefore be tightly regulated and effectively suppressed in G1 phase, at the time when only homologous chromosomes are available for repair. At such times, NHEJ is the favored mechanism for DSB repair.A number of mechanisms regulate HR to a specific phase of the cell cycle. For example, CtIP is activated for interaction with BRCA1 by CDK-dependent phosphorylation, which occurs in the S and G2 phases of the cell cycle. Conversely, HR is suppressed in G1 phase by the pro-NHEJ factors 53BP1⁶, RIF1⁷ and REV7⁸, which impair the recruitment of BRCA1 and thereby inhibit DNA end resection. Consequently, disruption of 53BP1 leads to the recruitment of BRCA1 to DSBs in G1 phase. In the recent Nature paper from Durocher''s laboratory, Orthwein et al.² discovered that although BRCA1 is localized to DSBs during G1 phase in 53BP1-deficient cells, it fails to recruit the BRCA2-PALB2 complex, which is consistent with the lack of HR activity in these cells.Through immunoprecipitation experiments Orthwein et al. showed that while BRCA2 and PALB2 interact throughout the cell cycle, BRCA1 and PALB2 only interact efficiently in S phase, suggesting that there might be a mechanism that restricts their interaction to S and G2 phases, while also blocking it in G1 phase. The region of PALB2 that is responsible for its cell cycle-regulated interaction with BRCA1 was localized to its N-terminal domain, which corresponds to a known interaction site for KEAP1, a substrate adaptor for the CUL3-RING (CRL3) ubiquitin ligase. Remarkably, they found that deletion of the KEAP1 gene using CRISPR-Cas9 technology restored the BRCA1-PALB2 interaction in G1 cells, and led to the recruitment of BRCA2-PALB2 to sites of DNA damage in 53BP1-deficient G1 cells.Since KEAP1 is involved in protein ubiquitylation, Orthwein et al. hypothesized that ubiquitylation of PALB2 in the BRCA1-interacting region might block their interaction. Indeed, mutation of lysines in the interacting region of PALB2 restored its interaction with BRCA1 in G1 cells. Furthermore, pull-down experiments showed that ubiquitylation of PALB2 on Lysine-20 by KEAP1-CRL3 prevented its interaction with BRCA1. However, as neither the activity of the KEAP1-CRL3 ubiquitin ligase nor its interaction with BRCA1 is cell cycle regulated, Orthwein et al. reasoned that a deubiquitylation step could be the rate-limiting regulator of the BRCA1-PALB2 interaction. They highlighted the deubiquitylating enzyme USP11 as a potential candidate for this activity due to its interaction with BRCA1, BRCA2 and PALB2, and indeed found that USP11 disruption impaired the interaction between BRCA1 and PALB2. Moreover, they found that USP11 was unstable and interacted poorly with PALB2 in G1 cells, and that USP11 was rapidly lost by proteasomal degradation in G1 phase after DNA damage. By contrast, expression of USP11 in S-phase was high and insensitive to DNA damage. Taken together, these data led the authors to propose that the opposing activities of USP11 and KEAP1-CRL3 regulate cell cycle-dependent interactions between BRCA1 and PALB2 (Figure 1).Open in a separate windowFigure 1Schematic representation indicating how the opposing activities of USP11 and KEAP1-CRL3 regulate cell cycle-dependent interactions between BRCA1 and PALB2, and thereby mediate pathway choice in DSB repair.To extend these remarkable observations, Orthwein et al. disrupted this regulatory network to allow HR in G1 cells. They expected that depletion of KEAP1 in 53BP1-deficient cells might be sufficient for RAD51 foci formation following ionizing radiation (IR), but this was not the case because end resection remained a limiting factor. To counteract this, the authors expressed a constitutively active form of CtIP (T847E)⁹, which augmented resection and led to the efficient formation of IR-induced RAD51 foci in 53BP1- and KEAP1-deficient G1 cells. To address whether these RAD51 foci in G1 cells corresponded to productive HR events, they used a fluorescent-based gene-targeting assay. Whereas CtIP (T847E)expressed in 53BP1-deficient cells alone was insufficient to induce productive HR, depletion of KEAP1 or expression of a non-ubiquitylable version of PALB2 led to a robust increase in gene-targeting events. Collectively, this study therefore demonstrates that activation of DNA end resection, combined with the recruitment of BRCA2 to DSBs, are both necessary and sufficient to produce HR in G1 cells.Gene targeting has great potential for therapeutic purposes, but the fact that most cells in the body are non-dividing has so far limited its use¹⁰. We suspect that the new knowledge highlighted in this work will further improve gene-targeting therapies to help fight human diseases.     

Shuffled antibody libraries created by in vivo homologous recombination and yeast surface display

Homologous recombination in yeast can be exploited to reliably generate libraries of >10⁷ transformants from a pool of PCR products and a linearized plasmid vector. Homology in the PCR insertion products drives shuffling of these genes in vivo by yeast homologous recombination. Two scFvs that share 89.8% homology were shuffled in vivo by homologous recombination, and chimeric genes were generated regardless of whether or not one of the scFv PCR products lacked 5′ homology to the cut vector and the second scFv PCR product lacked 3′ homology to the cut vector, or both PCR products had both 5′ and 3′ homology to the cut vector. A majority of the chimeras had single crossovers; however, double and triple crossovers were isolated. Crossover points were evenly distributed in the hybrids created and homology of as little as two nucleotides was able to produce a chimeric clone. The numbers of clones isolated with a given number of crossovers was approximated well by a Poisson distribution. Transformation efficiencies for the chimeric libraries were of the order of 10⁴–10⁵ transformants per microgram of insert, which is the same order of magnitude as when a single PCR product is inserted alone into the display vector by homologous recombination. This method eliminates ligation and Escherichia coli transformation steps of previous methods for generating yeast-displayed libraries, requires fewer PCR cycles than in vitro DNA shuffling and, unlike site-specific recombination methods, allows for recombination anywhere that homology exists between the genes to be recombined. This simple technique should prove useful for protein engineering in general and antibody engineering, specifically in yeast.     

Innovation by homologous recombination

1. Download : Download high-res image (233KB)
2. Download : Download full-size image