Damage-resistant DNA synthesis in Fanconi anemia cells treated with a DNA cross-linking agent

Fanconi anemia (FA) is a recessive disorder associated with diverse congenital anomalies, progressive bone marrow failure, and a marked predisposition to develop cancer. At the cellular level, FA is characterized by a prolonged G(2) phase in proliferating cells and a marked hypersensitivity to both the cytotoxic and the clastogenic effects of agents which produce DNA interstrand cross-links. Treatment with these agents leads to even further prolongation of the G(2) phase in FA cells. We now show that FA cells, from four different complementation groups, fail to decrease their rates of replicative DNA synthesis, as do normal cells, following treatment with a DNA cross-linking agent. This may be responsible for the prolongation of the G2 phase seen in these cells, and suggests that the fundamental defect in response of FA cells to DNA cross-linking agents may be in the S phase, rather than the G(2) phase, of the cell cycle.     

Arrest of S-phase progression is impaired in Fanconi anemia cells

Fanconi anemia (FA) is an inherited cancer-susceptibility disorder, characterized by genomic instability, hypersensitivity to DNA cross-linking agents, and a prolonged G2 phase of the cell cycle. We observed a marked dose-dependent accumulation of FA cells in the G2 compartment after treatment with 4,5',8-trimethylpsoralen (Me(3)Pso) in combination with 365 nm irradiation. Using bivariate DNA distribution methodology, we determined the proportion of replicating and arresting S-phase cells and observed that, whereas normal cells arrested DNA replication in the presence of Me(3)Pso cross-links and monoadducts, FA lymphoblasts failed to arrest DNA synthesis. Taken together, the above data suggest that, in response to damage induced by DNA cross-linking agents, the S-phase checkpoint is inefficient in FA cells. This would lead to accumulation of secondary lesions, such as single- and double-strand breaks and gaps. The prolonged time in G2 phase seen in FA cells therefore exists in order to allow the cells to remove lesions which accumulated during the preceding abnormal S phase.     

Intermediate DNA repair activity associated with the 322delG allele of the fanconi anemia complementation group C gene

Fanconi anemia (FA) is an autosomal recessive disorder associated with pancytopenia and cancer susceptibility. The disorder is heterogeneous, with at least nine complementation groups having been identified. Several recent studies have suggested that defective plasmid DNA end-joining is a consistent feature of FA cells. It was therefore surprising to discover a strain of fibroblasts from an FA patient that possessed wild-type plasmid DNA end-joining activity. Unlike other FA strains, these fibroblasts have wild-type levels of homologous DNA recombination activity and are relatively insensitive to restriction endonuclease-induced death. Interestingly, while end-joining in a number of FA fibroblast strains belonging to complementation groups A, C, and D2 was approximately 70% precise, end-joining in this latter strain of fibroblasts was more than 95% imprecise. Analysis revealed that these latter cells harbored an allele of the FA C gene, referred to as 322delG, that encodes an amino-terminal truncated protein. The relative rarity of this allele precluded the analysis of other FA fibroblast strains; however, studies revealed that overexpression of this allele in normal cells recapitulated the DNA end-joining phenotype seen in the 322delG FA fibroblast strain. These results indicate that DNA end-joining in fibroblasts expressing the 322delG allele of the FA-C gene in fibroblasts is highly imprecise; however, the DNA repair efficiency of these cells is more normal than that commonly associated with FA fibroblasts. This conclusion is intriguing, since a number of reports have suggested that patients harboring this allele exhibit a milder clinical course than do individuals with other alleles of the FA-C gene.     

The histone-fold complex MHF is remodeled by FANCM to recognize branched DNA and protect genome stability

Histone-fold proteins typically assemble in multiprotein complexes to bind duplex DNA. However, one histone-fold complex, MHF, associates with Fanconi anemia (FA) protein FANCM to form a branched DNA remodeling complex that senses and repairs stalled replication forks and activates FA DNA damage response network. How the FANCM-MHF complex recognizes branched DNA is unclear. Here, we solved the crystal structure of MHF and its complex with the MHF-interaction domain (referred to as MID) of FANCM, and performed structure-guided mutagenesis. We found that the MID-MHF complex consists of one histone H3-H4-like MHF heterotetramer wrapped by a single polypeptide of MID. We identified a zinc atom-liganding structure at the central interface between MID and MHF that is critical for stabilization of the complex. Notably, the DNA-binding surface of MHF was altered by MID in both electrostatic charges and allosteric conformation. This leads to a switch in the DNA-binding preference — from duplex DNA by MHF alone, to branched DNA by the MID-MHF complex. Mutations that disrupt either the composite DNA-binding surface or the protein-protein interface of the MID-MHF complex impaired activation of the FA network and genome stability. Our data provide the structural basis of how FANCM and MHF work together to recognize branched DNA, and suggest a novel mechanism by which histone-fold complexes can be remodeled by their partners to bind special DNA structures generated during DNA metabolism.     

Loss of Ezh2 function remodels the DNA replication initiation landscape

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Differential activation of intra-S-phase checkpoint in response to tripchlorolide and its effects on DNA replication

DNA replication is tightly regulated during the S phase of the cell cycle, and the activation of the intra-S-phase checkpoint due to DNA damage usually results in arrest of DNA synthesis. However, the molecular details about the correlation between the checkpoint and regulation of DNA replication are still unclear. To investigate the connections between DNA replication and DNA damage checkpoint, a DNA-damage reagent, tripchlorolide, was applied to CHO (Chinese ovary hamster) cells at early- or middle-stages of the S phase. The early-S-phase treatment with TC signifi-cantly delayed the progression of the S phase and caused the phosphorylation of the Chk 1 checkpoint protein, whereas the middle-S-phase treatment only slightly slowed down the progression of the S phase. Furthermore, the analysis of DNA replication patterns revealed that replication pattern II was greatly prolonged in the cells treated with the drug during the early-S phase, whereas the late-replication patterns of these cells were hardly detected, suggesting that the activation of the intra-S-phase checkpoint inhibits the late-origin firing of DNA replication. We conclude that cells at different stages of the S phase are differentially sensitive to the DNA-damage reagent, and the activation of the intra-S-phase checkpoint blocks the DNA replication progression in the late stage of S phase.     

RAD51 is involved in repair of damage associated with DNA replication in mammalian cells

The RAD51 protein, a eukaryotic homologue of the Escherichia coli RecA protein, plays an important role in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) in mammalian cells. Recent findings suggest that HR may be important in repair following replication arrest in mammalian cells. Here, we have investigated the role of RAD51 in the repair of different types of damage induced during DNA replication with etoposide, hydroxyurea or thymidine. We show that etoposide induces DSBs at newly replicated DNA more frequently than gamma-rays, and that these DSBs are different from those induced by hydroxyurea. No DSB was found following treatment with thymidine. Although these compounds appear to induce different DNA lesions during DNA replication, we show that a cell line overexpressing RAD51 is resistant to all of them, indicating that RAD51 is involved in repair of a wide range of DNA lesions during DNA replication. We observe fewer etoposide-induced DSBs in RAD51-overexpressing cells and that HR repair of etoposide-induced DSBs is faster. Finally, we show that induced long-tract HR in the hprt gene is suppressed in RAD51-overexpressing cells, although global HR appears not to be suppressed. This suggests that overexpression of RAD51 prevents long-tract HR occurring during DNA replication. We discuss our results in light of recent models suggested for HR at stalled replication forks.     

14-3-3sigma is a cruciform DNA binding protein and associates in vivo with origins of DNA replication

A human cruciform binding protein (CBP) was previously shown to bind to cruciform DNA in a structure-specific manner and be a member of the 14-3-3 protein family. CBP had been found to contain the 14-3-3 isoforms beta, gamma, epsilon, and zeta. Here, we show by Western blot analysis that the CBP-cruciform DNA complex eluted from band-shift polyacrylamide gels also contains the 14-3-3sigma isoform, which is present in HeLa cell nuclear extracts. An antibody specific for the 14-3-3sigma isoform was able to interfere with the formation of the CBP-cruciform DNA complex. The effect of the same anti-14-3-3sigma antibody in the in vitro replication of p186, a plasmid containing the minimal replication origin of the monkey origin ors8, was also analyzed. Pre-incubation of total HeLa cell extracts with this antibody decreased p186 in vitro replication to approximately 30% of control levels, while non-specific antibodies had no effect. 14-3-3sigma was found to associate in vivo with the monkey origins of DNA replication ors8 and ors12 in a cell cycle-dependent manner, as assayed by a chromatin immunoprecipitation (ChIP) assay that involved formaldehyde cross-linking, followed by immunoprecipitation with anti-14-3-3sigma antibody and quantitative PCR. The association of 14-3-3sigma with the replication origins was maximal at the G(1)/S phase. The results indicate that 14-3-3sigma is an origin binding protein involved in the regulation of DNA replication via cruciform DNA binding.     

RecQL4 is required for the association of Mcm10 and Ctf4 with replication origins in human cells

Though RecQL4 was shown to be essential for the initiation of DNA replication in mammalian cells, its role in initiation is poorly understood. Here, we show that RecQL4 is required for the origin binding of Mcm10 and Ctf4, and their physical interactions and association with replication origins are controlled by the concerted action of both CDK and DDK activities. Although RecQL4-dependent binding of Mcm10 and Ctf4 to chromatin can occur in the absence of pre-replicative complex, their association with replication origins requires the presence of the pre-replicative complex and CDK and DDK activities. Their association with replication origins and physical interactions are also targets of the DNA damage checkpoint pathways which prevent initiation of DNA replication at replication origins. Taken together, the RecQL4-dependent association of Mcm10 and Ctf4 with replication origins appears to be the first important step controlled by S phase promoting kinases and checkpoint pathways for the initiation of DNA replication in human cells.     

The sequence-directed bent DNA detected in the replication origin of Chlamydomonas reinhardtii chloroplast DNA is important for the replication function

Summary We demonstrated that the 1055 by restriction fragment containing OriA, a chloroplast DNA replication origin of Chlamydomonas reinhardtii, has electrophoretic anomalies characteristic of bent DNA. A tandem dimer of the region was constructed. Quantitative measurement of the relative gel mobility of a set of permuted fragments was used to extrapolate the approximate position of the bent DNA segment. By analyzing the gel mobility of short, sequenced fragments of the bent DNA region, the putative bending locus was identified. Two A₄ tracts and two A5 tracts were located in the bending locus. Oligonucleotide-directed mutagenesis was then used to disrupt the A tract or the spacing between A tracts and the effect of site-specific mutation on electrophoretic mobility was analyzed. To assess the functional role of the bent DNA region, subclones containing the bending locus, mutated bending locus, and regions flanking the bending locus were constructed. Each subclone was used as template in an in vitro DNA replication system which preferentially initiated DNA replication at OriA. A 224 by subclone with the bending locus positioned in the middle displayed the highest replication function and was sufficient to initiate DNA replication in vitro. Site-specific mutations or alterations of the A tracts resulted in decreased DNA bending and decreased DNA replication activity.     

Oligomerization of DNA replication regulatory protein RADX is essential to maintain replication fork stability

Genome integrity requires complete and accurate DNA replication once per cell division cycle. Replication stress poses obstacles to this process that must be overcome to prevent replication fork collapse. An important regulator of replication fork stability is the RAD51 protein, which promotes replication fork reversal and protects nascent DNA strands from nuclease-mediated degradation. Many regulatory proteins control these RAD51 activities, including RADX, which binds both ssDNA and RAD51 at replication forks to ensure that fork reversal is confined to stalled forks. Many ssDNA-binding proteins function as hetero- or homo-oligomers. In this study, we addressed whether this is also the case for RADX. Using biochemical and genetic approaches, we found that RADX acts as a homo-oligomer to control replication fork stability. RADX oligomerizes using at least two different interaction surfaces, including one mapped to a C-terminal region. We demonstrate that mutations in this region prevent oligomerization and prevent RADX function in cells, and that addition of a heterologous dimerization domain to the oligomerization mutants restored their ability to regulate replication. Taken together, our results demonstrate that like many ssDNA-binding proteins, oligomerization is essential for RADX-mediated regulation of genome stability.     

Nek1 interacts with Ku80 to assist chromatin loading of replication factors and S-phase progression

NIMA-related kinases (Neks) play divergent roles in mammalian cells. While several Neks regulate mitosis, Nek1 was reported to regulate DNA damage response, centrosome duplication and primary cilium formation. Whether Nek1 participates in cell cycle regulation is not known. Here we report that loss of Nek1 results in severe proliferation defect due to a delay in S-phase of the cell cycle. Nek1-deficient cells show replication stress and checkpoint activation under normal growth conditions. Nek1 accumulates on the chromatin during normal DNA replication. In response to replication stress, Nek1 is further activated for chromatin localization. Nek1 interacts with Ku80 and, in Nek1-deficient cells chromatin localization of Ku80 and several other DNA replication factors is significantly reduced. Thus, Nek1 may facilitate S-phase progression by interacting with Ku80 and regulating chromatin loading of replication factors.     

Distinct pools of proliferating cell nuclear antigen associated to DNA replication sites interact with the p125 subunit of DNA polymerase delta or DNA ligase I

Proliferating cell nuclear antigen (PCNA) plays an essential role in DNA replication, repair, and cell cycle control. PCNA is a homotrimeric ring that, when encircling DNA, is not easily extractable. Consequently, the dynamics of protein-protein interactions established by PCNA at DNA replication sites is not well understood. We have used DNase I to release DNA-bound PCNA together with replication proteins including the p125-catalytic subunit of DNA polymerase delta (p125-pol delta), DNA ligase I, cyclin A, and cyclin-dependent kinase 2 (CDK2). Interaction with these proteins was investigated by immunoprecipitation with antibodies binding near the interdomain connector loop or to the C-terminal domain of PCNA, respectively, or with antibodies to p125-pol delta or DNA ligase I. PCNA interaction with p125-pol delta or DNA ligase I was detected only by the latter antibodies, and found to be mutually exclusive. In contrast, antibodies to PCNA co-immunoprecipitated only CDK2. A GST-p21(waf1/cip1) C-terminal peptide displaced p125-pol delta and DNA ligase I, but not CDK2, from PCNA. These results suggest that PCNA trimers bound to DNA during the S phase are organized as distinct pools able to bind selectively different partners. Among them, p125-pol delta and DNA ligase I interact with PCNA in a mutually exclusive manner.     

MGMT hypomethylation is associated with DNA damage in workers exposed to low-dose benzene

Objective: This study aims to assess the effects of low-dose benzene on DNA damage and O⁶-methylguanine-DNA methyltransferase (MGMT) methylation in occupational workers.

Materials and methods: We recruited 96 nonsmoking male petrochemical industry workers exposed to low-dose benzene and 100 matched control workers. Urinary S-phenylmercapturic acid (SPMA) and S-benzylmercapturic acid (SBMA) were measured for indicating internal exposure of benzene and toluene. The degree of DNA damage was determined by the Comet assay. The levels of MGMT methylation were detected quantitatively by bisulphite-PCR pyrosequencing assay.

Results: The benzene-exposed workers had significantly higher levels of urinary SPMA, degree of DNA damage but decreased MGMT methylation than the controls (all p?<?0.05). In contrast, the level of urinary SBMA does not differ between benzene-exposed workers and the controls. In all participants, MGMT methylation was negatively associated with the urinary SPMA and the degree of DNA damage, indicating that epigenetic regulation might be involved in response to low-dose benzene exposure-induced genetic damage.

Discussion and conclusion: MGMT methylation could be a potent biomarker associated with low-dose benzene exposure and benzene-induced DNA damage.     

MRE11–RAD50–NBS1 is a critical regulator of FANCD2 stability and function during DNA double‐strand break repair

Monoubiquitination of the Fanconi anaemia protein FANCD2 is a key event leading to repair of interstrand cross‐links. It was reported earlier that FANCD2 co‐localizes with NBS1. However, the functional connection between FANCD2 and MRE11 is poorly understood. In this study, we show that inhibition of MRE11, NBS1 or RAD50 leads to a destabilization of FANCD2. FANCD2 accumulated from mid‐S to G2 phase within sites containing single‐stranded DNA (ssDNA) intermediates, or at sites of DNA damage, such as those created by restriction endonucleases and laser irradiation. Purified FANCD2, a ring‐like particle by electron microscopy, preferentially bound ssDNA over various DNA substrates. Inhibition of MRE11 nuclease activity by Mirin decreased the number of FANCD2 foci formed in vivo. We propose that FANCD2 binds to ssDNA arising from MRE11‐processed DNA double‐strand breaks. Our data establish MRN as a crucial regulator of FANCD2 stability and function in the DNA damage response.     

A unique epigenetic signature is associated with active DNA replication loci in human embryonic stem cells

The cellular epigenetic landscape changes as pluripotent stem cells differentiate to somatic cells or when differentiated cells transform to a cancerous state. These epigenetic changes are commonly correlated with differences in gene expression. Whether active DNA replication is also associated with distinct chromatin environments in these developmentally and phenotypically diverse cell types has not been known. Here, we used BrdU-seq to map active DNA replication loci in human embryonic stem cells (hESCs), normal primary fibroblasts and a cancer cell line, and correlated these maps to the epigenome. In all cell lines, the majority of BrdU peaks were enriched in euchromatin and at DNA repetitive elements, especially at microsatellite repeats, and coincided with previously determined replication origins. The most prominent BrdU peaks were shared between all cells but a sizable fraction of the peaks were specific to each cell type and associated with cell type-specific genes. Surprisingly, the BrdU peaks that were common to all cell lines were associated with H3K18ac, H3K56ac, and H4K20me1 histone marks only in hESCs but not in normal fibroblasts or cancer cells. Depletion of the histone acetyltransferases for H3K18 and H3K56 dramatically decreased the number and intensity of BrdU peaks in hESCs. Our data reveal a unique epigenetic signature that distinguishes active replication loci in hESCs from normal somatic or malignant cells.     

A unique epigenetic signature is associated with active DNA replication loci in human embryonic stem cells

The cellular epigenetic landscape changes as pluripotent stem cells differentiate to somatic cells or when differentiated cells transform to a cancerous state. These epigenetic changes are commonly correlated with differences in gene expression. Whether active DNA replication is also associated with distinct chromatin environments in these developmentally and phenotypically diverse cell types has not been known. Here, we used BrdU-seq to map active DNA replication loci in human embryonic stem cells (hESCs), normal primary fibroblasts and a cancer cell line, and correlated these maps to the epigenome. In all cell lines, the majority of BrdU peaks were enriched in euchromatin and at DNA repetitive elements, especially at microsatellite repeats, and coincided with previously determined replication origins. The most prominent BrdU peaks were shared between all cells but a sizable fraction of the peaks were specific to each cell type and associated with cell type-specific genes. Surprisingly, the BrdU peaks that were common to all cell lines were associated with H3K18ac, H3K56ac, and H4K20me1 histone marks only in hESCs but not in normal fibroblasts or cancer cells. Depletion of the histone acetyltransferases for H3K18 and H3K56 dramatically decreased the number and intensity of BrdU peaks in hESCs. Our data reveal a unique epigenetic signature that distinguishes active replication loci in hESCs from normal somatic or malignant cells.     

Genetic controls of DNA damage avoidance in response to acetaldehyde in fission yeast

Acetaldehyde, a primary metabolite of alcohol, forms DNA adducts and disrupts the DNA replication process, causing genomic instability, a hallmark of cancer. Indeed, chronic alcohol consumption accounts for approximately 3.6% of all cancers worldwide. However, how the adducts are prevented and repaired after acetaldehyde exposure is not well understood. In this report, we used the fission yeast Schizosaccharomyces pombe as a model organism to comprehensively understand the genetic controls of DNA damage avoidance in response to acetaldehyde. We demonstrate that Atd1 functions as a major acetaldehyde detoxification enzyme that prevents accumulation of Rad52-DNA repair foci, while Atd2 and Atd3 have minor roles in acetaldehyde detoxification. We found that acetaldehyde causes DNA damage at the replication fork and activates the cell cycle checkpoint to coordinate cell cycle arrest with DNA repair. Our investigation suggests that acetaldehyde-mediated DNA adducts include interstrand-crosslinks and DNA-protein crosslinks. We also demonstrate that acetaldehyde activates multiple DNA repair pathways. Nucleotide excision repair and homologous recombination, which are both epistatically linked to the Fanconi anemia pathway, have major roles in acetaldehyde tolerance, while base excision repair and translesion synthesis also contribute to the prevention of acetaldehyde-dependent genomic instability. We also show the involvement of Wss1-related metalloproteases, Wss1 and Wss2, in acetaldehyde tolerance. These results indicate that acetaldehyde causes cellular stresses that require cells to coordinate multiple cellular processes in order to prevent genomic instability. Considering that acetaldehyde is a human carcinogen, our genetic studies serve as a guiding investigation into the mechanisms of acetaldehyde-dependent genomic instability and carcinogenesis.