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1.
Using semi-quantitative PCR-based approach, we have shown that the breakpoint cluster region of the AML1 gene was associated with the nuclear matrix. We have demonstrated that inhibition of topoisomerase II by etoposide stimulates the appearance of histone gammaH2AX foci, an indicator for the presence of DNA double-strand breaks. Furthermore, the major part of these foci was associated with the nuclear matrix. We also visualized nuclear matrix--associated multiprotein complexes involved in topoisomerase II--induced DNA double-strand break repair. Colocalization studies have demonstrated that these complexes included the principal components of the non-homologous end joining repair system (Ku80, DNA-PKcs and DNA ligase IV). Thus, it is reasonable to suggest that the non-homologous DNA end joining is a possible mechanism of topoisomerase II--induced chromosomal rearrangements.  相似文献   

2.
Molecular mechanisms of exon shuffling: illegitimate recombination   总被引:5,自引:0,他引:5  
van Rijk A  Bloemendal H 《Genetica》2003,118(2-3):245-249
Illegitimate recombination (IR) is a process that takes place far more often than homologous recombination and is characterized by the recombination between non-homologous or short homologous sequences. The consequences of IR frequently emerge after the introduction of DNA in cell lines because it more frequently integrates in non-homologous than in homologous regions of the host genome. As a result, unexpected truncated or elongated products may be found. By not discarding those products as transfection artifacts, but by studying how they are generated, it might elucidate a possible molecular mechanism of IR. Here we review the current literature describing different mechanisms by which non-homologous DNA recombination can be induced.  相似文献   

3.
    
We have developed a plasmid test system to study recombination in vitro and in mammalian cells in vivo, and to analyze the possible role of DNA topoisomerase II. The system is based on a plasmid construct containing an inducible marker gene ccdB (\"killer\" (KIL) gene) whose product is lethal for bacterial cells, flanked by two different potentially recombinogenic elements. The plasmids were subjected to recombinogenic conditions in vitro or in vivo after transient transfection into COS-1 cells, and subsequently transformed into E. coli which was then grown in the presence of the ccdB gene inducer. Hence, all viable colonies contained recombinant plasmids since only recombination between the flanking regions could remove the KIL gene. Thus, it was possible to detect recombination events and to estimate their frequency. We found that the frequency of topoisomerase II-mediated recombination in vivo is significantly higher than in a minimal in vitro system. The presence of VM-26, an inhibitor of the religation step of the topoisomerase II reaction, increased the recombination frequency by 60%. We propose that cleavable complexes of topoisomerase II are either not religated, triggering error-prone repair of the DNA breaks, or are incorrectly religated resulting in strand exchange. We also studied the influence of sequences known to contain preferential breakpoints for recombination in vivo after chemotherapy with topoisomerase II-targeting drugs, but no preferential stimulation of recombination by these sequences was detected in this non-chromosomal context.  相似文献   

4.
Ultraviolet light (UV) can provoke genome instability, partly through its ability to induce homologous recombination (HR). However, the mechanism(s) of UV-induced recombination is poorly understood. Although double-strand breaks (DSBs) have been invoked, there is little evidence for their generation by UV. Alternatively, single-strand DNA lesions that stall replication forks could provoke recombination. Recent findings suggest efficient initiation of UV-induced recombination in G1 through processing of closely spaced single-strand lesions to DSBs. However, other scenarios are possible, since the recombination initiated in G1 can be completed in the following stages of the cell cycle. We developed a system that could address UV-induced recombination events that start and finish in G2 by manipulating the activity of the sister chromatid cohesion complex. Here we show that sister-chromatid cohesion suppresses UV-induced recombination events that are initiated and resolved in G2. By comparing recombination frequencies and survival between UV and ionizing radiation, we conclude that a substantial portion of UV-induced recombination occurs through DSBs. This notion is supported by a direct physical observation of UV-induced DSBs that are dependent on nucleotide excision repair. However, a significant role of nonDSB intermediates in UV-induced recombination cannot be excluded.  相似文献   

5.
Ultraviolet light (UV) can provoke genome instability, partly through its ability to induce homologous recombination (HR). However, the mechanism(s) of UV-induced recombination is poorly understood. Although double-strand breaks (DSBs) have been invoked, there is little evidence for their generation by UV. Alternatively, single-strand DNA lesions that stall replication forks could provoke recombination. Recent findings suggest efficient initiation of UV-induced recombination in G1 through processing of closely spaced single-strand lesions to DSBs. However, other scenarios are possible, since the recombination initiated in G1 can be completed in the following stages of the cell cycle. We developed a system that could address UV-induced recombination events that start and finish in G2 by manipulating the activity of the sister chromatid cohesion complex. Here we show that sister-chromatid cohesion suppresses UV-induced recombination events that are initiated and resolved in G2. By comparing recombination frequencies and survival between UV and ionizing radiation, we conclude that a substantial portion of UV-induced recombination occurs through DSBs. This notion is supported by a direct physical observation of UV-induced DSBs that are dependent on nucleotide excision repair. However, a significant role of nonDSB intermediates in UV-induced recombination cannot be excluded.  相似文献   

6.
Plasmid-based transfection assays provide a rapid means to measure homologous and nonhomologous recombination in mammalian cells. Often it is of interest to examine the stimulation of recombination by DNA damage induced by radiation, genotoxic chemicals, or nucleases. Transfection is frequently performed by using calcium phosphate coprecipitation (CPP), because this method is well suited for handling large sample sets, and it does not require expensive reagents or equipment. Alternative transfection methods include lipofection, microinjection, and electroporation. Since DNA strand breaks are known to stimulate both homologous and nonhomologous recombination, the induction of nonspecific damage during transfection would increase background recombination levels and thereby reduce the sensitivity of assays designed to detect the stimulation of recombination by experimentally induced DNA damage. In this article, we compare the stimulatory effects of nuclease-induced double-strand breaks (DSBs) on homologous and nonhomologous recombination for molecules transfected by CPP and by electroporation. Although electroporation yielded fewer transfectants, both nonhomologous and homologous recombination were stimulated by nuclease-induced DSBs to a greater degree than with CPP. Ionizing radiation is an effective agent for inducing DNA strand breaks, but previous studies using CPP generally showed little or no stimulation of homologous recombination among plasmids damaged with ionizing radiation. By contrast, we found clear dose-dependent enhancement of recombination with irradiated plasmids transfected using electroporation. Thus, electroporation provides a higher signal-to-noise ratio for transfection-based studies of damage-induced recombination, possibly reflecting less nonspecific damage to plasmid DNA during transfection of mammalian cells.  相似文献   

7.
Repetitive DNA is present in the eukaryotic genome in the form of segmental duplications, tandem and interspersed repeats, and satellites. Repetitive sequences can be beneficial by serving specific cellular functions (e.g. centromeric and telomeric DNA) and by providing a rapid means for adaptive evolution. However, such elements are also substrates for deleterious chromosomal rearrangements that affect fitness and promote human disease. Recent studies analyzing the role of nuclear organization in DNA repair and factors that suppress non-allelic homologous recombination (NAHR) have provided insights into how genome stability is maintained in eukaryotes. In this review, we outline the types of repetitive sequences seen in eukaryotic genomes and how recombination mechanisms are regulated at the DNA sequence, cell organization, chromatin structure, and cell cycle control levels to prevent chromosomal rearrangements involving these sequences.  相似文献   

8.
Homologous recombination in DNA repair and DNA damage tolerance   总被引:20,自引:0,他引:20  
Li X  Heyer WD 《Cell research》2008,18(1):99-113
Homologous recombination (HR) comprises a series of interrelated pathways that function in the repair of DNA double-stranded breaks (DSBs) and interstrand crosslinks (ICLs). In addition, recombination provides critical support for DNA replication in the recovery of stalled or broken replication forks, contributing to tolerance of DNA damage. A central core of proteins, most critically the RecA homolog Rad51, catalyzes the key reactions that typify HR: homology search and DNA strand invasion. The diverse functions of recombination are reflected in the need for context-specific factors that perform supplemental functions in conjunction with the core proteins. The inability to properly repair complex DNA damage and resolve DNA replication stress leads to genomic instability and contributes to cancer etiology. Mutations in the BRCA2 recombination gene cause predisposition to breast and ovarian cancer as well as Fanconi anemia, a cancer predisposition syndrome characterized by a defect in the repair of DNA interstrand crosslinks. The cellular functions of recombination are also germane to DNA-based treatment modalities of cancer, which target replicating cells by the direct or indirect induction of DNA lesions that are substrates for recombination pathways. This review focuses on mechanistic aspects of HR relating to DSB and ICL repair as well as replication fork support.  相似文献   

9.
A hypothesis has been proposed that the regions of DNA loop anchorage to the nuclear matrix are the preferential sites (hot spots) of illegitimate recombination mediated or triggered by topoisomerase II of the nuclear matrix. Recombination between the regions of DNA loop anchorage to the nuclear matrix may result in deletion or repositioning of DNA loops or their groups. The proposed hypothesis is confirmed by the results of original experiments and published data obtained by other researchers.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 4, 2005, pp. 633–638.Original Russian Text Copyright © 2005 by Razin, Iarovaia.  相似文献   

10.
The repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is an essential process in maintenance of chromosomal stability. A key player of HR is the strand exchange factor RAD51 whose assembly at sites of DNA damage is tightly regulated. We detected an endogenous complex of RAD51 with the calcium-binding protein S100A11, which is localized at sites of DNA repair in HaCaT cells as well as in normal human epidermal keratinocytes (NHEK) synchronized in S phase. In biochemical assays, we revealed that S100A11 enhanced the RAD51 strand exchange activity. When cells expressing a S100A11 mutant lacking the ability to bind Ca2+, a prolonged persistence of RAD51 in repair sites and nuclear γH2AX foci was observed suggesting an incomplete DNA repair. The same phenotype became apparent when S100A11 was depleted by RNA interference. Furthermore, down-regulation of S100A11 resulted in both reduced sister chromatid exchange confirming the restriction of the recombination capacity of the cells, and in an increase of chromosomal aberrations reflecting the functional requirement of S100A11 for the maintenance of genomic stability. Our data indicate that S100A11 is involved in homologous recombination by regulating the appearance of RAD51 in DSB repair sites. This function requires the calcium-binding activity of S100A11.  相似文献   

11.
As a member of imitation switch (ISWI) family in ATP-dependent chromatin remodeling factors, RSF complex consists of SNF2h ATPase and Rsf-1. Although it has been reported that SNF2h ATPase is recruited to DNA damage sites (DSBs) in a poly(ADP-ribosyl) polymerase 1 (PARP1)-dependent manner in DNA damage response (DDR), the function of Rsf-1 is still elusive. Here we show that Rsf-1 is recruited to DSBs confirmed by various cellular analyses. Moreover, the initial recruitment of Rsf-1 and SNF2h to DSBs shows faster kinetics than that of γH2AX after micro-irradiation. Signals of Rsf-1 and SNF2h are retained over 30 min after micro-irradiation, whereas γH2AX signals are gradually reduced at 10 min. In addition, Rsf-1 is accumulated at DSBs in ATM-dependent manner, and the putative pSQ motifs of Rsf-1 by ATM are required for its accumulation at DSBs. Furtheremore, depletion of Rsf-1 attenuates the activation of DNA damage checkpoint signals and cell survival upon DNA damage. Finally, we demonstrate that Rsf-1 promotes homologous recombination repair (HRR) by recruiting resection factors RPA32 and Rad51. Thus, these findings reveal a new function of chromatin remodeler Rsf-1 as a guard in DNA damage checkpoints and homologous recombination repair.  相似文献   

12.
    
《Molecular cell》2023,83(14):2434-2448.e7
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13.
14.
    
Programmed and unprogrammed double-strand breaks (DSBs) often arise from such physiological requirements as meiotic recombination, and exogenous insults, such as ionizing radiation (IR). Due to deleterious impacts on genome stability, DSBs must be appropriately processed and repaired in a regulatory manner. Recent investigations have indicated that ubiquitination is a critical factor in DNA damage response and meiotic recombination repair. This review summarizes the effects of proteins and complexes associa...  相似文献   

15.
    
Chromosomal rearrangements frequently occur at specific places (“hot spots”) in the genome. These recombination hot spots are usually separated by 50–100 kb regions of DNA that are rarely involved in rearrangements. It is quite likely that there is a correlation between the above‐mentioned distances and the average size of DNA loops fixed at the nuclear matrix. Recent studies have demonstrated that DNA loop anchorage regions can be fairly long and can harbor DNA recombination hot spots. We previously proposed that chromosomal DNA loops may constitute the basic units of genome organization in higher eukaryotes. In this review, we consider recombination between DNA loop anchorage regions as a possible source of genome evolution.  相似文献   

16.
    
DNA double-strand breaks (DSBs) induced by genotoxic agents can cause cell death or contribute to chromosomal instability, a major driving force of cancer. By contrast, Spo11-dependent DSBs formed during meiosis are aimed at generating genetic diversity. In eukaryotes, CtIP and the Mre11 nuclease complex are essential for accurate processing and repair of both unscheduled and programmed DSBs by homologous recombination (HR). Here, we applied bioinformatics and genetic analysis to identify Paramecium tetraurelia CtIP (PtCtIP), the smallest known Sae2/Ctp1/CtIP ortholog, as a key factor for the completion of meiosis and the recovery of viable sexual progeny. Using in vitro assays, we find that purified recombinant PtCtIP preferentially binds to double-stranded DNA substrates but does not contain intrinsic nuclease activity. Moreover, mutation of the evolutionarily conserved C-terminal 'RHR' motif abrogates DNA binding of PtCtIP but not its ability to functionally interact with Mre11. Translating our findings into mammalian cells, we provide evidence that disruption of the 'RHR' motif abrogates accumulation of human CtIP at sites of DSBs. Consequently, cells expressing the DNA binding mutant CtIPR837A/R839A are defective in DSB resection and HR. Collectively, our work highlights minimal structural requirements for CtIP protein family members to facilitate the processing of DSBs, thereby maintaining genome stability as well as enabling sexual reproduction.  相似文献   

17.
Two L5178Y (LY) murine lymphoma cell sublines, LY-R, resistant, and LY-S, sensitive, to X-irradiation display inverse cross-sensitivity to camptothecin (CPT): LY-R cells were more susceptible to this specific topoisomerase I inhibitor than LY-S cells. After 1 h incubation with CPT, the doses that inhibited growth by 50 per cent (ID50) after 48 h of incubation were 0·54μM for LY-R cells and 1·25 μM for LY-S cells. Initial numbers of DNA–protein crosslinks (DPCs) measured at this level of growth inhibition were two-fold higher in LY-R (5·6 Gray-equivalents) than in LY-S cells (3·1 Gray-equivalents), which corresponds well with the greater in vitro sensitivity of Topo I from LY-R cells to CPT.1,2 Conversely, the initial levels of single-strand DNA breaks (SSBs) and double-strand DNA breaks (DSBs) were lower in LY-R cells (4·2 Gray-equivalent SSBs and 5·8 Gray equivalent DSBs) than in LY-S cells (8·0 Gray-equivalent SSBs and 12·0 Gray-equivalent DSBs). Dissimilarity in the replication-dependent DNA damage observed after 1 h of treatment with CPT was not due to a difference in the rate of DNA synthesis between the two cell lines, but may have arisen from a substantially slower repair of DNA breaks in LY-S cells.3 Release from G2 block by caffeine co-treatment significantly increased cell killing in the LY-S subline, and only slightly inhibited growth of LY-R cells. These results show that after CPT treatment cells arrest in G2, allowing them time to repair the long-lived DSBs. As LY-S cells are slower in repairing the DSBs, they were more susceptible to CPT in the presence of caffeine.  相似文献   

18.
DNA topoisomerase (topo) II catalyses topological genomic changes essential for many DNA metabolic processes. It is also regarded as a structural component of the nuclear matrix in interphase and the mitotic chromosome scaffold. Mammals have two isoforms (alpha and beta) with similar properties in vitro. Here, we investigated their properties in living and proliferating cells, stably expressing biofluorescent chimera of the human isozymes. Topo IIalpha and IIbeta behaved similarly in interphase but differently in mitosis, where only topo IIalpha was chromosome associated to a major part. During interphase, both isozymes joined in nucleolar reassembly and accumulated in nucleoli, which seemed not to involve catalytic DNA turnover because treatment with teniposide (stabilizing covalent catalytic DNA intermediates of topo II) relocated the bulk of the enzymes from the nucleoli to nucleoplasmic granules. Photobleaching revealed that the entire complement of both isozymes was completely mobile and free to exchange between nuclear subcompartments in interphase. In chromosomes, topo IIalpha was also completely mobile and had a uniform distribution. However, hypotonic cell lysis triggered an axial pattern. These observations suggest that topo II is not an immobile, structural component of the chromosomal scaffold or the interphase karyoskeleton, but rather a dynamic interaction partner of such structures.  相似文献   

19.
It has been recently demonstrated that yeast cells are able to partially regress chromosome segregation in telophase as a response to DNA double-strand breaks (DSBs), likely to find a donor sequence for homology-directed repair (HDR). This regression challenges the traditional concept that establishes anaphase events as irreversible, hence opening a new field of research in cell biology. Here, the nature of this new behavior in yeast is summarized and the underlying mechanisms are speculated about. It is also discussed whether it can be reproduced in other eukaryotes. Overall, this work brings forwards the need of understanding how cells attempt to repair DSBs when transiting the latest stages of mitosis, i.e., anaphase and telophase.  相似文献   

20.
  总被引:2,自引:0,他引:2  
The nonchromatin structure or nuclear matrix in developing spermatogenic cells of the rat was studied using a biochemical fractionation in concert with resinless section electron microscopy. Observations demonstrated that the nuclear matrix of spermatogenic cells consisted of a three-dimensional network of filaments of variable thicknesses. In spermatogonia and spermatocytes the nuclear matrix consisted of relatively thin filaments, while that of round spermatids consisted of a thicker interconnecting network of filament. In elongating spermatids, the interior of the nuclear matrix consisted of a network of dense filaments bounded by a peripheral lamina. The protein composition of the nuclear matrix in spermatogenic cells was examined by high-resolution two-dimensional gel electrophoresis and correlated with morphological changes characteristic of each stage. The results showed that the proteins of nuclear matrix changed in a cell stage-specific manner. These stage-specific changes corresponded to the major transitions of chromatin structure and function during spermatogenesis. Furthermore, immunocytochemical and immunoblotting analysis of DNA topoisomerase II (topo II) revealed that this enzyme exhibited stage-specific variations and was associated with the nuclear matrix. These results suggest that the nuclear matrix in spermatogenic cells may be involved in mediating DNA modifications and maintaining nuclear organization during spermatogenesis. Mol. Reprod. Dev. 59:314-321, 2001.  相似文献   

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