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Mitotic recombination

Mitotic recombination is a form of homologous recombination that occurs between homologous chromosomes or sister chromatids during the mitotic cell cycle in eukaryotic cells, primarily facilitating the repair of DNA double-strand breaks (DSBs) and other lesions to maintain genomic integrity.^[1] Unlike meiotic recombination, which promotes genetic diversity, mitotic recombination is typically suppressed to avoid chromosomal rearrangements but is essential for error-free DNA repair, often resulting in gene conversion or loss of heterozygosity (LOH).^[2] This process is conserved across eukaryotes, from yeast like Saccharomyces cerevisiae to mammals, and is most active in the S and G2 phases when homologous templates are available.^[3] The initiation of mitotic recombination usually begins with the formation of DSBs, which can arise from replication fork collapse, exogenous damage (e.g., ionizing radiation), or endogenous sources like reactive oxygen species.^[1] These breaks undergo 5' to 3' end resection by nucleases such as the MRN complex (Mre11-Rad50-Nbs1 in mammals) and Exo1, generating single-stranded DNA (ssDNA) tails coated by RPA, which are then invaded by Rad51 filaments to search for homologous sequences.^[2] Key pathways include synthesis-dependent strand annealing (SDSA), which favors non-crossover outcomes through strand displacement, and double-strand break repair (DSBR) via double Holliday junctions that can resolve into crossovers or non-crossovers.^[3] In cases of one-ended breaks, such as at telomeres, break-induced replication (BIR) extends DNA synthesis semi-conservatively, though it risks mutagenesis.^[2] Central proteins like Rad52 mediate Rad51 loading, while antirecombinases (e.g., Srs2 in yeast) limit excessive recombination to prevent instability.^[3] Mitotic recombination plays a critical role in genome stability by enabling high-fidelity repair that non-homologous end joining (NHEJ) cannot achieve, reducing mutations and chromosomal aberrations.^[1] However, aberrant events can lead to LOH, promoting tumorigenesis in heterozygous cells (e.g., via BRCA2 mutations), or gross rearrangements like translocations.^[3] In yeast, spontaneous recombination rates are low (~10⁻⁶ events per cell per generation), but increase with DNA damage, highlighting its responsive nature.^[2] Regulation involves cell cycle checkpoints (e.g., CDK1), chromatin remodelers (e.g., RSC complex), and post-translational modifications like sumoylation of Rad52, ensuring recombination occurs only when appropriate templates are present.^[2] Dysregulation is implicated in cancers and genetic disorders, underscoring its dual role in repair and potential pathology.^[3]

Introduction

Definition

Mitotic recombination refers to the exchange, which can be reciprocal or nonreciprocal, of genetic material between homologous chromosomes or sister chromatids that occurs during the mitotic cell cycle in eukaryotic cells.^[1] This process typically takes place after DNA replication, enabling the repair of genetic damage or the resolution of replication issues through homologous sequences.^[4] Unlike meiotic recombination, which is programmed and essential for gamete formation, mitotic recombination is generally infrequent and arises sporadically, often in response to DNA damage such as double-strand breaks.^[5] The event primarily occurs in the G2 phase of the cell cycle, following S-phase replication when sister chromatids are available as templates. During this stage, a double-strand break or lesion on one chromatid can invade a homologous sequence on the sister or non-sister chromatid, leading to strand exchange and repair. If the recombinant chromatids segregate to different daughter cells during mitosis, this can result in cells with altered genotypes, such as loss of heterozygosity at specific loci.^[5] Consequently, mitotic recombination contributes to genetic diversity within somatic tissues, fostering somatic mosaicism where genetically distinct cell populations coexist in the same individual.^[6] Mitotic recombination can produce two main outcomes: crossover and non-crossover events. In a crossover event, there is a physical exchange of flanking chromosomal segments, producing two fully recombinant chromatids with swapped arms distal to the exchange point; this may lead to homozygous regions if the crossover occurs between heterozygous markers. Non-crossover events, by contrast, involve limited gene conversion where a short segment of DNA is copied from the donor chromatid to the recipient without exchanging flanking regions, resulting in partial allelic changes but no overall chromosome arm swap. These outcomes are illustrated conceptually as follows: for crossover, imagine two homologous chromosomes aligned post-replication, with breakage and rejoining creating intertwined chromatids that separate with exchanged segments; for non-crossover, the repair restores the original chromatid structure except for the converted allele tract.^[7]

Comparison with meiotic recombination

Mitotic recombination takes place in eukaryotic cells during or around the mitotic cell cycle, primarily serving to repair DNA damage and maintain genomic stability, whereas meiotic recombination occurs in germ cells during the specialized meiotic divisions to facilitate proper chromosome segregation and generate genetic diversity in gametes.^[8] In mitosis, recombination events are typically sporadic and confined to interphase or specific stages, involving homologous chromosomes as substrates without the programmed pairing seen in meiosis.^[8] A key distinction lies in their frequency and initiation mechanisms: mitotic recombination is far less frequent, occurring at rates orders of magnitude lower than in meiosis, due to the absence of deliberate double-strand breaks (DSBs); instead, it is often triggered by exogenous damage or replication errors.^[8] In contrast, meiotic recombination is highly regulated and initiated by hundreds of programmed DSBs generated by the Spo11 protein, ensuring at least one crossover per chromosome pair for segregation fidelity.^[9] Mitosis lacks this enzymatic induction, relying on opportunistic repair pathways that do not guarantee recombination.^[8] Structurally, mitotic recombination proceeds without the synaptonemal complex, a meiosis-specific protein scaffold that stabilizes homologous chromosome pairing and promotes crossover formation during prophase I; instead, mitotic events depend on transient, ad hoc alignment of homologs facilitated by cohesion and repair proteins. This absence contributes to the more variable and less controlled nature of mitotic exchanges compared to the orchestrated synapsis in meiosis. The outcomes of these processes also diverge significantly: mitotic recombination frequently results in loss of heterozygosity (LOH) in one of the two daughter cells, potentially leading to homozygous regions that can exacerbate mutations or contribute to diseases like cancer, while the other daughter retains the original heterozygosity.^[8] Meiotic recombination, however, yields gametes with balanced genetic diversity through reciprocal crossovers and gene conversions, ensuring allelic shuffling without net loss of genetic material across the population. These differences underscore the repair-oriented role of mitosis versus the evolutionary adaptation in meiosis.^[8]

History

Discovery

Mitotic recombination was first identified in the 1930s through genetic studies in Drosophila melanogaster, where researchers observed unusual mosaic patterns in somatic tissues that could not be explained by simple mutation or nondisjunction. In 1936, Curt Stern published seminal observations of "twin spots"—adjacent patches of tissue with complementary mutant phenotypes—in flies heterozygous for linked X-chromosome markers such as yellow (y, causing yellow body color) and singed (sn, causing curled bristles). These twin spots consisted of one clone homozygous for y (yellow tissue adjacent to wild-type) and another homozygous for sn (singed tissue adjacent to wild-type), arising from a single recombination event proximal to the markers during mitosis. Stern's work built on Alfred H. Sturtevant's earlier 1913 genetic linkage map of the X chromosome, which positioned y and sn relative to the centromere and allowed him to deduce that the twin spots resulted from crossing over between homologous chromosomes during the four-chromatid (two homolog pairs) stage post-DNA replication in mitosis. This interpretation was supported by evidence from X-ray irradiation experiments, which increased the frequency of such mosaics; for instance, sectoring in eye tissues—patches of mutant pigmentation amid wild-type—emerged in flies treated with X-rays during larval stages, as earlier noted by J.T. Patterson in 1929 for inducing somatic mosaics generally. Stern's analysis showed these sectors formed via mitotic recombination rather than direct mutation, with the proximal location of crossovers relative to the centromere determining the twin-spot pattern. These findings challenged the prevailing view of mitosis as a strictly vegetative process for clonal propagation without genetic variation, instead revealing it as capable of homologous recombination akin to meiosis. Stern proposed that such events occur post-DNA replication, leading to segregation of recombinant chromatids into daughter cells and producing homozygous clones in heterozygous backgrounds. This discovery, detailed in Stern's 1936 paper, established mitotic recombination as a fundamental genetic mechanism observable in somatic cells.

Key developments

During the 1950s and 1960s, research on mitotic recombination shifted toward Saccharomyces cerevisiae as a key model organism due to its eukaryotic features and amenability to genetic analysis in diploid strains.^[10] Pioneering studies by Herschel Roman demonstrated elevated rates of mitotic gene conversion and recombination in yeast diploids, facilitating the dissection of recombination events through sectoring assays and heterozygous marker analysis.^[10] By the 1970s, this work extended to tetrad-like dissections of mitotic products in diploids, revealing non-random segregation patterns and establishing yeast as a primary system for studying mitotic crossing-over. In the 1980s, mitotic recombination was increasingly integrated with DNA double-strand break (DSB) repair models, with experiments in mammalian cells providing evidence for homologous recombination as a DSB repair pathway. Studies using Chinese hamster ovary cell hybrids demonstrated genetic recombination between homologous chromosomes during mitosis, linking it to DSB-induced events and distinguishing it from non-homologous repair.^[11] These findings paralleled yeast-based DSB models and highlighted mitotic recombination's role in maintaining genomic integrity across eukaryotes. From the 1990s to the 2000s, investigations identified loss of heterozygosity (LOH) as a critical outcome of mitotic recombination in cancer, particularly in inactivating tumor suppressor genes.^[12] In retinoblastoma tumors, mitotic recombination was implicated in 46% of LOH events at the RB1 locus, leading to biallelic inactivation and tumor progression.^[12] Similar patterns emerged in colorectal cancers, where recombination-mediated LOH extended over large chromosomal regions, underscoring its contribution to oncogenesis beyond simple deletions.^[13] Post-2010 advances in high-throughput sequencing and live-cell imaging have illuminated mitotic recombination between homologous chromosomes, revealing its dynamics and prevalence in genome evolution. Next-generation sequencing of haplotypes in yeast and human cells quantified crossovers between homologous chromosomes, showing they drive rapid adaptive changes and LOH without whole-chromosome loss.^[14] Concurrently, fluorescence microscopy techniques visualized recombination intermediates in real time, demonstrating Rad52 foci formation during mitotic recombination repair in mammalian cells.^[3]

Occurrence

Triggers

Mitotic recombination is primarily triggered by DNA double-strand breaks (DSBs), which arise from various exogenous and endogenous sources. Exogenous agents such as ionizing radiation induce DSBs by directly ionizing DNA molecules, with a dose of 4 Gy typically generating 50–100 breaks per mammalian cell, prompting repair via homologous recombination to maintain genomic integrity. Similarly, chemical mutagens like methyl methanesulfonate (MMS) alkylate DNA bases, leading to replication fork collapse and subsequent DSB formation during S-phase. Replication stress, often induced by hydroxyurea (HU) treatment that depletes nucleotide pools, causes stalled forks that convert into one-ended DSBs, further stimulating recombination events. Spontaneous triggers also contribute significantly to mitotic recombination, originating from endogenous cellular processes. Reactive oxygen species (ROS), produced as metabolic byproducts, oxidize DNA bases and generate spontaneous DSBs or nicks that evolve into breaks during replication.^[15] Stalled replication forks during normal S-phase progression, due to natural obstacles like secondary structures or nucleotide imbalances, similarly initiate DSBs and associated recombination foci, as observed through Rad52 recruitment in yeast. These endogenous events underscore the role of mitotic recombination in routine genome maintenance without external insult. Cell cycle regulation tightly controls the timing of mitotic recombination, confining it predominantly to the G2/M phases where sister chromatids serve as templates. In G2/M, extensive 5' to 3' resection of DSB ends enables strand invasion and repair, whereas in G1, homologous recombination is suppressed, with non-homologous end joining (NHEJ) repairing approximately 80% of ionizing radiation-induced DSBs due to limited resection and absence of sister chromatids. This G1 suppression is mediated by checkpoint proteins such as p53, which inhibit recombination to prevent error-prone outcomes in the absence of suitable templates, alongside factors like 53BP1 that favor NHEJ. The frequency of mitotic recombination increases markedly in repair-deficient mutants, highlighting the interplay between DSB repair and recombination pathways. In yeast strains lacking Rad52, a key mediator of homologous recombination, HR is impaired, leading to reliance on alternative pathways and increased genomic instability.^[16]

Frequency and sites

Mitotic recombination occurs at a basal frequency of approximately 10^{-5} to 10^{-6} per locus per cell division in the yeast Saccharomyces cerevisiae, as measured by spontaneous loss-of-heterozygosity (LOH) events in diploid strains.^[17] In standard assays, such as those detecting red-white sectored colonies, the rate is around 3 × 10^{-5} events per cell, reflecting genome-wide interhomolog crossover probabilities estimated at 2.8 × 10^{-3}.^[17] These rates can vary slightly depending on the chromosomal locus and assay conditions but establish a low baseline under normal growth. In mammalian cells, basal mitotic recombination is generally rarer than in yeast, particularly in somatic tissues, where it contributes minimally to genomic variation outside of pathological contexts. However, under environmental stresses such as DNA hypomethylation or oxidative damage, frequencies can increase up to 10^{-4} per locus, as observed in DNA methyltransferase-deficient mouse embryonic stem cells where centromeric recombination rises from 0.149 to 0.243 events per chromosome.^[18] For instance, stressors like hydrogen peroxide or heavy metals elevate interhomolog recombination in model systems, though direct human somatic measurements remain challenging due to the infrequency of detectable events. Preferential sites of mitotic recombination cluster at centromeres and telomeres, driven by their structural features including repetitive DNA sequences and heterochromatin organization, which promote double-strand break formation and repair templating.^[18] In mammalian cells, centromeric recombination occurs at rates 6-fold higher than telomeric and up to 175-fold higher than arm regions, while in yeast, hotspots often align with inverted retrotransposon repeats near these structures.^[18]^[19] Intra-chromosomal events, involving sister chromatids, predominate over inter-homolog recombination, which is suppressed to maintain heterozygosity but can be detected at subtelomeric or pericentromeric loci.^[17] Across organisms, mitotic recombination frequency varies markedly: it is frequent and well-characterized in yeast under laboratory conditions, enabling genetic analysis, but rare in human somatic cells, where homologous recombination is a rare event and it primarily manifests in aging or cancer-associated mosaicism.^[17]^[20] Influencing factors include ploidy level, with polyploids (e.g., triploids at 2.2 × 10^{-2} and tetraploids at 8.4 × 10^{-2} LOH events per division) exhibiting higher rates due to increased DNA damage susceptibility, and heterozygosity, where elevated SNP density correlates with greater recombination propensity across genomic backgrounds.^[21]^[21]

Mechanisms

Homologous recombination pathway

Mitotic homologous recombination (HR) serves as a primary mechanism for repairing DNA double-strand breaks (DSBs) during the cell cycle, utilizing a homologous template—typically the sister chromatid—to restore genetic integrity with high fidelity. This pathway is particularly active in S and G2 phases when sister chromatids are available, initiating upon DSB detection and proceeding through a series of conserved enzymatic steps that can yield either crossover or non-crossover products.^[22]^[23] The process begins with DSB end resection, where the MRN complex (Mre11-Rad50-Nbs1 in mammals or Mre11-Rad50-Xrs2 in yeast) collaborates with CtIP (Sae2 in yeast) to perform initial end processing, followed by extensive 5'-to-3' resection mediated by Exo1 exonuclease and the Dna2 nuclease in conjunction with BLM helicase (Sgs1 in yeast). This generates long 3' single-stranded DNA (ssDNA) overhangs coated by replication protein A (RPA), which are essential for downstream homology search.^[22]^[24] Subsequently, RPA is displaced by the recombinase Rad51, forming a nucleoprotein filament on the ssDNA that facilitates strand invasion into the homologous duplex DNA, creating a displacement loop (D-loop). In eukaryotes, Rad51 loading is mediated by Rad52 in yeast and BRCA2 (with BRCA1 and PALB2 assistance) in mammals, ensuring filament stability through mediators like Rad55-Rad57 or the SHU complex; this step is highly conserved across species, underscoring HR's evolutionary preservation for genomic maintenance.^[25]^[26] The invaded strand then serves as a primer for DNA synthesis, copying information from the template. The second DSB end can be captured, leading to double Holliday junction (dHJ) formation. These junctions are resolved by structure-specific endonucleases such as GEN1 or MUS81-EME1, which cleave the junctions to produce crossover products that exchange flanking genetic material, an outcome generally suppressed in mitotic HR compared to meiotic contexts, where crossovers are essential.^[27]^[28]^[29] Alternatively, HR can proceed via synthesis-dependent strand annealing (SDSA), a non-crossover pathway where the extended invading strand dissociates from the template and anneals to the other resected DSB end, followed by gap filling and ligation. This mechanism, mediated by Rad51 and RPA, predominates in mitosis to avoid potentially deleterious crossovers between sister chromatids.^[30]

Break-induced replication

Break-induced replication (BIR) is a specialized form of homologous recombination that repairs one-ended double-strand breaks (DSBs) during mitosis, particularly those arising from collapsed replication forks or telomere dysfunction.^[31] Unlike bidirectional repair of two-ended DSBs, BIR initiates with the invasion of a single resected DSB end into a homologous DNA template, typically on the sister chromatid or homologous chromosome, forming a displacement loop (D-loop).^[32] This process is initiated approximately one hour after 5' to 3' end resection of the break, enabling Rad51-mediated strand invasion to prime unidirectional DNA synthesis.^[32] In yeast models, such as budding yeast, this mechanism has been extensively characterized using HO endonuclease-induced breaks, demonstrating its efficiency in restoring chromosome continuity.^[33] The core of BIR involves extensive, conservative DNA synthesis that proceeds from the D-loop via a migrating bubble structure, where leading-strand synthesis is continuous and lagging-strand synthesis occurs asynchronously.^[31] DNA polymerases δ and ε drive this replication, with polymerase δ synthesizing both strands and polymerase ε potentially contributing later; the Pol32 subunit of polymerase δ is essential for stable, long-tract extension beyond short patches (up to 15 kb without it).^[32] The synthesis rate is approximately 0.5 kb/min, significantly slower than S-phase replication, and relies on helicases like Pif1 for processivity.^[32] This unidirectional replication leads to loss of heterozygosity (LOH) distal to the breakpoint, as the entire segment beyond the invasion site is copied from the donor template, replacing the original sequence.^[33] BIR's conservative nature—where both newly synthesized strands are inherited together—distinguishes it from semi-conservative replication and heightens risks of genomic instability, including copy number variations through microhomology-mediated BIR (MMBIR) and template switching.^[32] Key proteins include Rad51 for initial strand invasion and D-loop formation, Rad52 for facilitating recombination, and Pol32 for BIR-specific synthesis extension; in some contexts, RAD51-independent pathways involve Rad59 and the MRX complex.^[31] In mitotic cells, BIR is suppressed compared to meiosis, partly due to transcription interference, limited end resection, and preferential use of alternative pathways like mitotic DNA synthesis (MiDAS) in G2/M phase, reducing its frequency to prevent excessive rearrangements.^[32]

Consequences

Genetic outcomes

Mitotic recombination can produce two primary genetic outcomes: crossover and non-crossover events. In crossover recombination, a reciprocal exchange occurs between homologous chromosomes during the G2 phase of the cell cycle, leading to the segregation of recombinant chromatids into daughter cells. One daughter cell inherits a chromosome that is homozygous for all markers distal to the crossover breakpoint toward the telomere, resulting in loss of heterozygosity (LOH) across that chromosomal arm. This terminal LOH typically extends from the recombination site to the chromosome end, with tract lengths averaging tens of kilobases in model systems like Saccharomyces cerevisiae.^[34] In contrast, non-crossover events involve gene conversion without associated chromosomal exchange, where a segment of DNA from one homolog is copied onto the other, altering the sequence without gross structural changes to the chromosomes.^[35] The LOH from crossovers is often observed as runs of homozygosity (ROH) in genomic analyses, particularly in organisms with heterozygous backgrounds such as the diploid fungus Candida albicans, where such events uncover recessive mutations and maintain diploidy. Break-induced replication (BIR), a specialized form of recombination, can also generate long terminal LOH tracts extending to the telomere, though it is mechanistically distinct from standard crossovers. Gene conversion tracts in non-crossover outcomes are unidirectional or bidirectional, with median lengths of 6.1 kb for unidirectional and 11.6 kb for bidirectional events in yeast, typically not exceeding tens of kilobases and confined to localized regions without affecting distant chromosomal segments. These tracts repair double-strand breaks (DSBs) by non-reciprocal transfer, preserving overall heterozygosity beyond the converted area.^[36]^[35]^[37] In multicellular organisms, mitotic recombination contributes to somatic mosaicism, where recombinant cells expand clonally within tissues, creating a patchwork of genetically distinct cell populations. For instance, in the adult Drosophila midgut, aging intestinal stem cells accumulate LOH clones spanning up to 20 Mb due to recombination, leading to heterogeneous tissue composition with homozygous segments replacing heterozygous ones in affected lineages. This clonal expansion amplifies the genetic patchiness, especially for markers distal to recombination sites.^[38] If mitotic recombination events remain unresolved or involve unequal exchanges, they can potentially lead to aneuploidy, such as chromosomal gains or losses, as seen in fungal pathogens like Phytophthora ramorum where recombination associates with copy number variants alongside LOH. In yeast mutation accumulation lines, aneuploidy events occur at rates of about 1 × 10⁻⁴ per cell division, sometimes co-occurring with recombination-induced instability.^[39]^[37]

Biological significance

Mitotic recombination plays a crucial role in DNA repair by enabling accurate resolution of double-strand breaks (DSBs) during the S and G2 phases of the cell cycle, where a sister chromatid template is available.^[40] This process, mediated by homologous recombination (HR), contrasts with the error-prone non-homologous end joining (NHEJ) pathway predominant in G1 and helps prevent mutations, chromosomal aberrations, and cell death by faithfully restoring genetic information.^[41] In proliferating cells, such as those in tissues with high turnover, this mechanism maintains genomic stability by minimizing indel formation or translocations that could arise from alternative repair routes.^[42] In cancer, mitotic recombination contributes to tumorigenesis by promoting loss of heterozygosity (LOH), which inactivates the remaining wild-type allele of tumor suppressor genes like BRCA1.^[43] Whole-genome sequencing analyses have shown that somatic LOH events in tumors primarily arise through mitotic recombination, leading to homozygous loss of protective alleles and accelerated tumor evolution.^[44] Recent studies from 2021 onward have linked such recombination-driven LOH to therapy resistance, particularly in BRCA-associated cancers, where reversion mutations restore HR proficiency and confer resistance to PARP inhibitors.^[45] From an evolutionary perspective, mitotic recombination generates adaptive genetic variants in clonal populations by reshuffling alleles and uncovering hidden diversity, thereby facilitating rapid adaptation to environmental stresses without sexual reproduction.^[14] In organisms like diatoms, interhomolog mitotic recombination occurs at rates of approximately 4.2 events per 100 cell divisions, producing copy-neutral LOH and novel protein variants that enhance survival under conditions such as oxidative or heavy metal stress.^[14] This mechanism relaxes clonal interference constraints, allowing beneficial allele combinations to emerge and fix in asexual lineages.^[14] Beyond repair and evolution, mitotic recombination supports immune function by aiding the repair of DSBs generated during somatic hypermutation (SHM) and class-switch recombination (CSR) in proliferating B cells.^[46] Activation-induced cytidine deaminase (AID) introduces lesions that, if unresolved, could lead to B cell death; HR via mitotic recombination processes these into viable mutations, enhancing antibody affinity and diversity.^[46] In aging, accumulated somatic mosaicism from mitotic recombination contributes to tissue heterogeneity and functional decline, as LOH events in stem cells drive clonal expansions that impair regeneration and increase disease susceptibility.^[44]

Methods of study

Experimental induction

Mitotic recombination can be experimentally induced in model organisms to study its mechanisms and outcomes under controlled conditions. In yeast, such as Saccharomyces cerevisiae, chemical agents that generate DNA lesions or double-strand breaks (DSBs) are widely used. Methyl methanesulfonate (MMS) alkylates purine bases, creating replication-blocking lesions that lead to DSBs and stimulate homologous recombination during S phase.^[47] Camptothecin, a topoisomerase I inhibitor, traps the enzyme on DNA, causing fork collapse and DSBs that trigger recombination repair pathways.^[47] Ultraviolet (UV) light induces cyclobutane pyrimidine dimers, which, upon replication, generate one-ended DSBs or gaps that promote mitotic crossovers and gene conversions.^[47] Site-specific genetic induction is achieved using endonucleases in yeast. The HO endonuclease, expressed from an inducible promoter like GAL1, cleaves at a specific 24-bp recognition sequence engineered into the genome, generating a DSB that initiates recombination with homologous sequences, enabling analysis of repair fidelity and products.^[17] This method has been instrumental in dissecting recombination intermediates and outcomes in haploid and diploid cells.^[48] In mammalian systems, targeted DSBs are induced using CRISPR-Cas9 in cell lines such as HEK293T or human hematopoietic stem/progenitor cells. Cas9 guided by single-guide RNAs creates DSBs at chosen loci, often resulting in copy-neutral loss of heterozygosity (LOH) via mitotic recombination, with events spanning up to 5 Mb from the break site in up to 1.1% of edited clones.^[49] Ionizing radiation protocols, involving X-ray exposure at 2–10 Gy, potently trigger DSBs in mouse embryonic stem cells, elevating recombination-mediated LOH frequencies by 35–60% of total mutants, far exceeding contributions from deletions or conversions.^[50] Experimental setups often incorporate cell cycle synchronization to enrich for G2 phase, where homologous recombination predominates. Nocodazole treatment arrests mammalian cells in G2/M by disrupting microtubules, allowing timed DSB induction and repair monitoring post-release, with synchronization efficiencies exceeding 90% in human pluripotent stem cells.30489-2) In yeast, analogous controls use hydroxyurea to synchronize in early S phase before damage induction.^[17]

Detection techniques

Mitotic recombination events can be detected using genetic marker systems that exploit visible phenotypic changes in heterozygous cells. In Drosophila melanogaster, the twin-spot assay relies on heterozygous markers, such as those affecting eye or wing pigmentation, to identify sectors of homozygous tissue arising from mitotic crossing-over during development.^[51] This method, first described by Stern in 1936, produces adjacent "twin spots" of contrasting colors in adult tissues, allowing quantification of recombination frequency proximal to the markers.^[51] Similarly, in yeast (Saccharomyces cerevisiae), sectoring assays use heterozygous auxotrophic or color markers on homologous chromosomes to detect mitotic recombination as colony sectors with altered growth or pigmentation, often in diploid strains.^[52] Molecular techniques provide precise identification of loss of heterozygosity (LOH), a common outcome of mitotic recombination, by analyzing DNA from cell populations or clones. PCR-based methods amplify polymorphic markers flanking potential recombination sites to detect LOH through loss of one allele's signal, commonly applied in mammalian cell lines and tumor samples.^[53] SNP arrays enable genome-wide scanning for LOH tracts by comparing allele intensities across single nucleotide polymorphisms, revealing breakpoints of mitotic crossovers with resolution down to kilobases in yeast and human cells.^[19] Whole-genome sequencing offers the highest resolution for mapping recombination breakpoints and LOH extents, as demonstrated in budding yeast where it identifies spontaneous crossover events across chromosomes.^[54] Imaging approaches visualize recombination intermediates or products at the chromosomal level. Fluorescence in situ hybridization (FISH), particularly centromere-oriented FISH (Cen-CO-FISH), detects exchanges by probing specific loci on metaphase chromosomes, showing elevated recombination rates at centromeres in mammalian cells compared to telomeres.^[55] Live-cell microscopy with fluorescently tagged proteins, such as RAD51-GFP, monitors homologous recombination foci formation during mitosis, revealing protective roles of RAD51 in resolving under-replicated DNA in human cells.^[56] Recent advances in single-nucleus DNA sequencing have enabled detection of mosaic LOH in tumors, mapping subclonal mitotic recombination events with high sensitivity in heterogeneous tissues like cerebral cavernous malformations.^[57] This post-2020 technique resolves low-frequency variants missed by bulk sequencing, providing insights into tumor evolution driven by interhomolog recombination.^[57]

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Jun 23, 2021 · Mitotic recombination can lead to interstitial (I-LOH) and terminal (T-LOH) LOH events. The I-LOH events are mostly caused by gene conversions ...
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Somatic recombination in adult tissues: What is there to learn?
Mitotic recombination has important consequences on adult tissues, creating genetic mosaicism, promoting tumor formation and having the potential to modify ...
[38]
Mitotic Recombination and Rapid Genome Evolution in the Invasive ...
Mar 12, 2019 · Alien species are often successful invaders in new environments, despite the introduction of a few isolates with a reduced genetic pool.
[39]
Double-Strand DNA Breaks | Learn Science at Scitable - Nature
DSBs are particularly troublesome because they can lead to cell death if not repaired. And, if not repaired correctly, DSBs can cause deletions, translocations ...
[40]
The RAD51 recombinase protects mitotic chromatin in human cells
Sep 10, 2021 · RAD51 is best known as the central catalyst of homologous recombination (HR), which provides error-free repair of double-stranded DNA breaks ( ...
[41]
Polθ is phosphorylated by PLK1 to repair double-strand breaks in ...
Sep 6, 2023 · Here we show that DNA polymerase theta 6 (Polθ) repairs mitotic DSBs and thereby maintains genome integrity.
[42]
Analysis of matched primary and recurrent BRCA1/2 mutation ...
Nov 7, 2022 · Our study reveals multiple potential drivers of recurrent disease in BRCA1/2 mutation-associated cancer, improving our understanding of tumor evolution.
[43]
Multi-omic machine learning predictor of breast cancer therapy ...
Dec 7, 2021 · HLA class I allelic loss confers resistance Loss of heterozygosity (LOH) over the HLA class I locus21 was identified in 29 cases and associated ...
[44]
https://www.cell.com/cell-reports/fulltext/S2211-1247(23)01497-3
[45]
REV7 is required for processing AID initiated DNA lesions ... - Nature
Jun 4, 2020 · Activation-induced cytidine deaminase (AID) initiates both antibody class switch recombination (CSR) and somatic hypermutation (SHM) in ...
[46]
https://www.nature.com/articles/s41467-020-16632-8
[47]
https://doi.org/10.1016/j.fgb.2010.02.007
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CRISPR-Cas9 globin editing can induce megabase-scale copy ...
Aug 13, 2021 · Here we report that genome editing targeting globin genes induces megabase-scale losses of heterozygosity (LOH) from the globin CRISPR-Cas9 cut-site to the ...Results · Cell Culture · Snp Analysis By Sanger...
[49]
Ionizing radiation is a potent inducer of mitotic recombination in ...
The primary mechanism underlying the elevated loss of heterozygosity after irradiation is mitotic recombination, with lesser contributions from deletions and ...
[50]
Genetic odyssey to generate marked clones in Drosophila mosaics
In 1936 Stern (3) first coined the term “twin spots” to refer to the two homozygous daughter cells generated by mitotic recombination (MR) in a heterozygous ...
[51]
A Fine-Structure Map of Spontaneous Mitotic Crossovers in the ...
Although mitotic recombination between homologous chromosomes was first described in 1936 [1], our understanding of the mechanism of spontaneous mitotic ...
[52]
Loss of Heterozygosity Occurs via Mitotic Recombination in Trp53 + ...
Abstract. Loss of heterozygosity (LOH) occurs commonly in cancers causing disruption of tumor suppressor genes and promoting tumor progression.Abstract · INTRODUCTION · MATERIALS AND METHODS · RESULTS
[53]
Genome-Wide Analysis of Mitotic Recombination in Budding Yeast
In this chapter, we describe an approach that utilizes whole genome sequencing (WGS) data to support the analysis of tracts of loss-of-heterozygosity (LOH) ...
[54]
Centromere mitotic recombination in mammalian cells
Jun 9, 2008 · We use a chromosome orientation fluorescence in situ hybridization technique to visualize and quantify recombination events at mouse centromeres ...
[55]
The RAD51 recombinase protects mitotic chromatin in human cells
In this study, we show that RAD51 protects under-replicated DNA in mitotic human cells and, in this way, promotes mitotic DNA synthesis (MiDAS) and successful ...
[56]
Single-nucleus DNA sequencing reveals hidden somatic loss-of ...
Nov 2, 2023 · Here we report a single-nucleus DNA-sequencing approach that leverages the underlying biology of CCMs to identify lesions with somatic loss-of-heterozygosity.Results · Whole Genome Sequencing · Haplotype Bias Within...<|control11|><|separator|>