Expansion of chromosome territories with chromatin decompaction in BAF53-depleted interphase cells

Chromosomes are compartmentalized into discrete chromosome territories during interphase in mammalian cells. A chromosome territory is generated by the tendency of chromatin to occupy the smallest shell volume, which is determined by the polymeric properties and interactions of the internal meshwork of the chromatin fiber. Here, we show that BAF53 knockdown by small interfering RNA interference led to the expansion of chromosome territories. This was accompanied by a reduction in chromatin compaction, an increase in the micrococcal nuclease sensitivity of the chromatin, and an alteration in H3-K9 and H3-K79 dimethylation. Interestingly, the BAF53 knockdown cells suffer a cell cycle defect. Despite the significant irregularity and decompaction of the polynucleosomes isolated from the BAF53 knockdown cells, the chromatin loading of H1 and core histones remained unaltered, as did the nucleosome spacing. The histone hyperacetylation and down-regulation of BRG-1, mBrm, and Tip49, the catalytic components of the SWI/SNF complex and the TIP60 complex, respectively, did not expand chromosome territories. These results indicate that BAF53 contributes to the polymeric properties and/or the internal meshwork interactions of the chromatin fiber probably via a novel mechanism.     

Suppression of HPV E6 and E7 expression by BAF53 depletion in cervical cancer cells

Deregulation of the expression of human papillomavirus (HPV) oncogenes E6 and E7 plays a pivotal role in cervical carcinogenesis because the E6 and E7 proteins neutralize p53 and Rb tumor suppressor pathways, respectively. In approximately 90% of all cervical carcinomas, HPVs are found to be integrated into the host genome. Following integration, the core-enhancer element and P105 promoter that control expression of E6 and E7 adopt a chromatin structure that is different from that of episomal HPV, and this has been proposed to contribute to activation of E6 and E7 expression. However, the molecular basis underlying this chromatin structural change remains unknown. Previously, BAF53 has been shown to be essential for the integrity of higher-order chromatin structure and interchromosomal interactions. Here, we examined whether BAF53 is required for activated expression of E6 and E7 genes. We found that BAF53 knockdown led to suppression of expression of E6 and E7 genes from HPV integrants in cervical carcinoma cell lines HeLa and SiHa. Conversely, expression of transiently transfected HPV18-LCR-Luciferase was not suppressed by BAF53 knockdown. The level of the active histone marks H3K9Ac and H4K12Ac on the P105 promoter of integrated HPV 18 was decreased in BAF53 knockdown cells. BAF53 knockdown restored the p53-dependent signaling pathway in HeLa and SiHa cells. These results suggest that activated expression of the E6 and E7 genes of integrated HPV is dependent on BAF53-dependent higher-order chromatin structure or nuclear motor activity.     

53BP1 nuclear bodies form around DNA lesions generated by mitotic transmission of chromosomes under replication stress

Completion of genome duplication is challenged by structural and topological barriers that impede progression of replication forks. Although this can seriously undermine genome integrity, the fate of DNA with unresolved replication intermediates is not known. Here, we show that mild replication stress increases the frequency of chromosomal lesions that are transmitted to daughter cells. Throughout G1, these lesions are sequestered in nuclear compartments marked by p53-binding protein 1 (53BP1) and other chromatin-associated genome caretakers. We show that the number of such 53BP1 nuclear bodies increases after genetic ablation of BLM, a DNA helicase associated with dissolution of entangled DNA. Conversely, 53BP1 nuclear bodies are partially suppressed by knocking down SMC2, a condensin subunit required for mechanical stability of mitotic chromosomes. Finally, we provide evidence that 53BP1 nuclear bodies shield chromosomal fragile sites sequestered in these compartments against erosion. Together, these data indicate that restoration of DNA or chromatin integrity at loci prone to replication problems requires mitotic transmission to the next cell generations.     

Association of BAF53 with mitotic chromosomes

The conversion of mitotic chromosome into interphase chromatin consists of at least two separate processes, the decondensation of the mitotic chromosome and the formation of the higher-order structure of interphase chromatin. Previously, we showed that depletion of BAF53 led to the expansion of chromosome territories and decompaction of the chromatin, suggesting that BAF53 plays an essential role in the formation of higher-order chromatin structure. We report here that BAF53 is associated with mitotic chromosomes during mitosis. Immunostaining with two different anti-BAF53 antibodies gave strong signals around the DNA of mitotic preparations of NIH3T3 cells and mouse embryo fibroblasts (MEFs). The immunofluorescent signals were located on the surface of mitotic chromosomes prepared by metaphase spread. BAF53 was also found in the mitotic chromosome fraction of sucrose gradients. Association of BAF53 with mitotic chromosomes would allow its rapid activation on the chromatin upon exit from mitosis.     

DNA damage responses by human ELG1 in S phase are important to maintain genomic integrity

Genomic integrity depends on DNA replication, recombination, and repair, particularly in S phase. We demonstrate that a human homologue of yeast Elg1 plays an important role in S phase to preserve genomic stability. The level of ELG1 is induced during recovery from a variety of DNA damage. In response to DNA damage, ELG1 forms distinct foci at stalled DNA replication forks that are different from DNA double strand break foci. Targeted gene knockdown of ELG1 resulted in spontaneous foci formation of γ-H2AX, 53BP1, and phosphorylated-ATM that mark chromosomal breaks. Abnormal chromosomes including fusions, inversions and hypersensitivity to DNA damaging agents were also observed in cells expressing low level of ELG1 by targeted gene knockdown. Knockdown of ELG1 by siRNA reduced homologous recombination frequency in the I-SceI induced double strand break-dependent assay. In contrast, spontaneous homologous recombination frequency and sister chromatin exchange rate were up-regulated when ELG1 was silenced by shRNA. Taken together, we propose that ELG1 would be a new member of proteins involved in maintenance of genomic integrity.     

Yeast chromosomal DNA molecules have strands which are cross-linked at their termini

The microbial eukaryote Saccharomyces cerevisiae has 18 chromosomes, each consisting of a DNA molecule of 1 to 15×10⁸ daltons (150 to 2,300 kilobase pairs). Interstand cross-links have now been found in molecules of all sizes by examining the ability of high molecular weight DNA to snap back, i.e., to rapidly renature after denaturation. Experiments in which snap back was assessed for molecules broken by shearing indicate that there are probably two cross-links in each chromosome. Evidence that the cross-links occur at specific sites in the genome was obtained by treating total chromosomal DNA with the endonuclease EcoRI which cleaves the yeast genome into approximately 2,000 discrete fragments. Cross-link containing fragments were separated from fragments without cross-links. This purification resulted in enrichment for about 18 specific fragments. To determine whether the cross-links are terminal or at internal sites in chromosomal DNA, large shear-produced fragments were examined by electron microscopy. With complete denaturation few fragments exhibited the X-shaped single strand configuration expected for internal cross-links. When partially denatured fragments were examined some ends had single strand loops as expected for (AT-rich) cross-linked termini. The percentage of looped ends was sufficient to account for all the cross-links in the population of chromosomal molecules. The data suggest that yeast chromosomal DNA molecules have cross-linked termini. We propose that a duplex chromosomal DNA molecule in this eukaryote consists of a continuous, single, self-complementary strand of DNA. This structure has implications for the mechanism of chromosome replication and may be the basis of telomere behavior.     

Control of eukaryotic DNA replication at the chromosomal level.

A hypothesis for the control of eukaryotic DNA replication at the chromosomal level is proposed. The specific regulatory problem arises from the subdivision of the genome into thousands of individually replicating units, each of which must be duplicated a single time during S-phase. The hypothesis is based on the finding of direct repeats at replication origins. Such repeats can adopt, beyond the full-length double helical structure, another configuration exposing two single-stranded loops that provide suitable templates for the initiation of DNA replication. Any further initiation at the same origin is excluded as the single strandedness is eliminated by the replication process. Restoration of the initiable loop structure is proposed to occur by DNA-protein rearrangements involved in chromosome condensation and duplication of the chromosomal protein backbone during mitosis. A possible role of the maturation promoting factor (MPF) is suggested.     

Reorganization of the DNA-nuclear matrix interactions in a 210 kb genomic region centered on c-myc after DNA replication in vivo

In the interphase nucleus of metazoan cells DNA is organized in supercoiled loops anchored to a nuclear matrix (NM). DNA loops are operationally classified in structural and facultative. Varied evidence indicates that DNA replication occurs in replication foci organized upon the NM and that structural DNA loops may correspond to the replicons in vivo. In normal rat liver the hepatocytes are arrested in G0 but synchronously re-enter the cell cycle after partial-hepatectomy leading to liver regeneration. Using this model we have previously determined that the DNA loops corresponding to a gene-rich genomic region move in a sequential fashion towards the NM during replication and then return to their original configuration in newly quiescent cells, once liver regeneration has been achieved. In the present work we determined the organization into structural DNA loops of a gene-poor region centered on c-myc and tracked-down its movement at the peak of S phase and after the return to cellular quiescence during and after liver regeneration. The results confirmed that looped DNA moves towards the NM during replication but in this case the configuration of the gene-poor region into DNA loops becomes reorganized and after replication only the loop containing c-myc resembles the original in the control G0 hepatocytes. Our results suggest that the local chromatin configuration around potentially active genes constraints the formation of specific structural DNA loops after DNA replication, while in non-coding regions the structural DNA loops are only loosely determined after DNA replication by structural constraints that modulate the DNA-NM interactions.     

Bidirectional chromosome replication in Bacillus subtilis

We have examined autoradiographically the pattern of DNA replication following the germination of Bacillus subtilis spores in [³H]thymidine. Thymine-requiring spores were germinated in low specific activity medium and diluted into higher specific activity medium soon after the first round of replication was expected to start. After a further short period, replication was stopped and the chromosomal structures examined by autoradiography. From the pattern of labelling within individual replicating loops it is clear that the majority (≥75%) expand by growth at two positions that are opposite, i.e. expand bidirectionally. The loops continue to expand at approximately equal rates in both directions until at least 20% of the chromosome has been replicated.From a consideration of the other structural forms that become visible, it seems likely that most chromosomes replicate bidirectionally.     

Essential roles of BCCIP in mouse embryonic development and structural stability of chromosomes

BCCIP is a BRCA2- and CDKN1A(p21)-interacting protein that has been implicated in the maintenance of genomic integrity. To understand the in vivo functions of BCCIP, we generated a conditional BCCIP knockdown transgenic mouse model using Cre-LoxP mediated RNA interference. The BCCIP knockdown embryos displayed impaired cellular proliferation and apoptosis at day E7.5. Consistent with these results, the in vitro proliferation of blastocysts and mouse embryonic fibroblasts (MEFs) of BCCIP knockdown mice were impaired considerably. The BCCIP deficient mouse embryos die before E11.5 day. Deletion of the p53 gene could not rescue the embryonic lethality due to BCCIP deficiency, but partially rescues the growth delay of mouse embryonic fibroblasts in vitro. To further understand the cause of development and proliferation defects in BCCIP-deficient mice, MEFs were subjected to chromosome stability analysis. The BCCIP-deficient MEFs displayed significant spontaneous chromosome structural alterations associated with replication stress, including a 3.5-fold induction of chromatid breaks. Remarkably, the BCCIP-deficient MEFs had a ～20-fold increase in sister chromatid union (SCU), yet the induction of sister chromatid exchanges (SCE) was modestly at 1.5 fold. SCU is a unique type of chromatid aberration that may give rise to chromatin bridges between daughter nuclei in anaphase. In addition, the BCCIP-deficient MEFs have reduced repair of irradiation-induced DNA damage and reductions of Rad51 protein and nuclear foci. Our data suggest a unique function of BCCIP, not only in repair of DNA damage, but also in resolving stalled replication forks and prevention of replication stress. In addition, BCCIP deficiency causes excessive spontaneous chromatin bridges via the formation of SCU, which can subsequently impair chromosome segregations in mitosis and cell division.     

The yeast S phase checkpoint enables replicating chromosomes to bi-orient and restrain spindle extension during S phase distress

The budding yeast S phase checkpoint responds to hydroxyurea-induced nucleotide depletion by preventing replication fork collapse and the segregation of unreplicated chromosomes. Although the block to chromosome segregation has been thought to occur by inhibiting anaphase, we show checkpoint-defective rad53 mutants undergo cycles of spindle extension and collapse after hydroxyurea treatment that are distinct from anaphase cells. Furthermore, chromatid cohesion, whose dissolution triggers anaphase, is dispensable for S phase checkpoint arrest. Kinetochore-spindle attachments are required to prevent spindle extension during replication blocks, and chromosomes with two centromeres or an origin of replication juxtaposed to a centromere rescue the rad53 checkpoint defect. These observations suggest that checkpoint signaling is required to generate an inward force involved in maintaining preanaphase spindle integrity during DNA replication distress. We propose that by promoting replication fork integrity under these conditions Rad53 ensures centromere duplication. Replicating chromosomes can then bi-orient in a cohesin-independent manner to restrain untimely spindle extension.     

Chromosomal association of the Smc5/6 complex reveals that it functions in differently regulated pathways

The SMC protein complexes safeguard genomic integrity through their functions in chromosome segregation and repair. The chromosomal localization of the budding yeast Smc5/6 complex determined here reveals that the complex works specifically on the duplicated genome in differently regulated pathways. The first controls the association to centromeres and chromosome arms in unchallenged cells, the second regulates the association to DNA breaks, and the third directs the complex to the chromosome arm that harbors the ribosomal DNA arrays. The chromosomal interaction pattern predicts a function that becomes more important with increasing chromosome length and that the complex's role in unchallenged cells is independent of DNA damage. Additionally, localization of Smc6 to collapsed replication forks indicates an involvement in their rescue. Altogether this shows that the complex maintains genomic integrity in multiple ways, and evidence is presented that the Smc5/6 complex is needed during replication to prevent the accumulation of branched chromosome structures.     

TopBP1 is required at mitosis to reduce transmission of DNA damage to G1 daughter cells

Genome integrity is critically dependent on timely DNA replication and accurate chromosome segregation. Replication stress delays replication into G2/M, which in turn impairs proper chromosome segregation and inflicts DNA damage on the daughter cells. Here we show that TopBP1 forms foci upon mitotic entry. In early mitosis, TopBP1 marks sites of and promotes unscheduled DNA synthesis. Moreover, TopBP1 is required for focus formation of the structure-selective nuclease and scaffold protein SLX4 in mitosis. Persistent TopBP1 foci transition into 53BP1 nuclear bodies (NBs) in G1 and precise temporal depletion of TopBP1 just before mitotic entry induced formation of 53BP1 NBs in the next cell cycle, showing that TopBP1 acts to reduce transmission of DNA damage to G1 daughter cells. Based on these results, we propose that TopBP1 maintains genome integrity in mitosis by controlling chromatin recruitment of SLX4 and by facilitating unscheduled DNA synthesis.     

High-resolution analysis of DNA replication domain organization across an R/G-band boundary.

Establishing how mammalian chromosome replication is regulated and how groups of replication origins are organized into replication bands will significantly increase our understanding of chromosome organization. Replication time bands in mammalian chromosomes show overall congruency with structural R- and G-banding patterns as revealed by different chromosome banding techniques. Thus, chromosome bands reflect variations in the longitudinal structure and function of the chromosome, but little is known about the structural basis of the metaphase chromosome banding pattern. At the microscopic level, both structural R and G bands and replication bands occupy discrete domains along chromosomes, suggesting separation by distinct boundaries. The purpose of this study was to determine replication timing differences encompassing a boundary between differentially replicating chromosomal bands. Using competitive PCR on replicated DNA from flow-sorted cell cycle fractions, we have analyzed the replication timing of markers spanning roughly 5 Mb of human chromosome 13q14.3/q21.1. This is only the second report of high-resolution analysis of replication timing differences across an R/G-band boundary. In contrast to previous work, however, we find that band boundaries are defined by a gradient in replication timing rather than by a sharp boundary separating R and G bands into functionally distinct chromatin compartments. These findings indicate that topographical band boundaries are not defined by specific sequences or structures.     

Local and global functions of Timeless and Tipin in replication fork protection

The eukaryotic cell replicates its chromosomal DNA with almost absolute fidelity in the course of every cell cycle. This accomplishment is remarkable considering that the conditions for DNA replication are rarely ideal. The replication machinery encounters a variety of obstacles on the chromosome, including damaged template DNA. In addition, a number of chromosome regions are considered to be difficult to replicate owing to DNA secondary structures and DNA binding proteins required for various transactions on the chromosome. Under these conditions, replication forks stall or break, posing grave threats to genomic integrity. How does the cell combat such stressful conditions during DNA replication? The replication fork protection complex (FPC) may help answer this question. Recent studies have demonstrated that the FPC is required for the smooth passage of replication forks at difficult-to-replicate genomic regions and plays a critical role in coordinating multiple genome maintenance processes at the replication fork.     

Local and global functions of Timeless and Tipin in replication fork protection

The eukaryotic cell replicates its chromosomal DNA with almost absolute fidelity in the course of every cell cycle. This accomplishment is remarkable considering that the conditions for DNA replication are rarely ideal. The replication machinery encounters a variety of obstacles on the chromosome, including damaged template DNA. In addition, a number of chromosome regions are considered to be difficult to replicate owing to DNA secondary structures and DNA binding proteins required for various transactions on the chromosome. Under these conditions, replication forks stall or break, posing grave threats to genomic integrity. How does the cell combat such stressful conditions during DNA replication? The replication fork protection complex (FPC) may help answer this question. Recent studies have demonstrated that the FPC is required for the smooth passage of replication forks at difficult-to-replicate genomic regions and plays a critical role in coordinating multiple genome maintenance processes at the replication fork.     

The breast cancer susceptibility gene BRCA2 is required for the maintenance of telomere homeostasis

Inactivating mutations in the breast cancer susceptibility gene BRCA2 cause gross chromosomal rearrangements. Chromosome structural instability in the absence of BRCA2 is thought to result from defective homology-directed DNA repair. Here, we show that BRCA2 links the fidelity of telomere maintenance with genetic integrity. Absence of BRCA2 resulted in signs of dysfunctional telomeres, such as telomere shortening, erosions, and end fusions in proliferating mouse fibroblasts. BRCA2 localized to the telomeres in S phase in an ATR-dependent manner, and its absence resulted in the accumulation of common fragile sites, particularly at the G-rich lagging strand, and increased the telomere sister chromatid exchange in unchallenged cells. The incidence of common fragile sites and telomere sister chromatid exchange increased markedly after treatment with replication inhibitors. Congruently, telomere-induced foci were frequently observed in the absence of Brca2, denoting activation of the DNA damage response and abnormal chromosome end joining. These telomere end fusions constituted a significant portion of chromosome aberrations in Brca2-deficient cells. Our results suggest that BRCA2 is required for telomere homeostasis and may be particularly important for the replication of G-rich telomeric lagging strands.     

真核细胞染色体DNA复制起始及复制叉稳定性维持的机制

在真核生物中,DNA复制在染色体上特定的多位点起始．当细胞处在晚M及G1期,多个复制起始蛋白依次结合到DNA复制源,组装形成复制前复合体．pre．RC在Gl-S的转折期得到激活,随后,多个直接参与DNA复制又形成的蛋白结合到DNA复制源,启动DNA的复制,形成两个双向的DNA复制又．在染色体上,移动的DNA复制又经常会碰到复制障碍（二级DNA结构、一些蛋白的结合位点、损伤的碱基等）而暂停下来,此时,需要细胞周期检验点的调控来稳定复制叉,否则,会导致复制又垮塌及基因组不稳定．本文就真核细胞染色体DNA复制起始的机制,以及复制又稳定性的维持机制进行简要综述．