JMJD2 promotes acquired cisplatin resistance in non-small cell lung carcinoma cells.

Platinum-based drugs such as cisplatin (CP) are the first-line chemotherapy for non-small-cell lung carcinoma (NSCLC). Unfortunately, NSCLC has a low response rate to CP and acquired resistance always occurs. Histone methylation regulates chromatin structure and is implicated in DNA repair.

MASTL overexpression promotes chromosome instability and metastasis in breast cancer.

MASTL kinase is essential for correct progression through mitosis, with loss of MASTL causing chromosome segregation errors, mitotic collapse and failure of cytokinesis. However, in cancer MASTL is most commonly amplified and overexpressed. This correlates with increased chromosome instability in breast cancer and poor patient survival in breast, ovarian and lung cancer.

Single molecule mtDNA fiber FISH for analyzing numtogenesis.

Somatic human cells contain thousands of copies of mitochondrial DNA (mtDNA). In eukaryotes, natural transfer of mtDNA into the nucleus generates nuclear mitochondrial DNA (NUMT) copies. We name this phenomenon as "numtogenesis".

Effective intra-S checkpoint responses to UVC in primary human melanocytes and melanoma cell lines.

The objective of this study was to assess potential functional attenuation or inactivation of the intra-S checkpoint during melanoma development. Proliferating cultures of skin melanocytes, fibroblasts, and melanoma cell lines were exposed to increasing fluences of UVC and intra-S checkpoint responses were quantified. Melanocytes displayed stereotypic intra-S checkpoint responses to UVC qualitatively and quantitatively equivalent to those previously demonstrated in skin fibroblasts.

SWI/SNF complexes are required for full activation of the DNA-damage response.

SWI/SNF complexes utilize BRG1 (also known as SMARCA4) or BRM (also known as SMARCA2) as alternative catalytic subunits with ATPase activity to remodel chromatin. These chromatin-remodeling complexes are required for mammalian development and are mutated in ~20% of all human primary tumors. Yet our knowledge of their tumor-suppressor mechanism is limited.

DNA damage checkpoint responses in the S phase of synchronized diploid human fibroblasts.

We investigated the hypothesis that the strength of the activation of the intra-S DNA damage checkpoint varies within the S phase. Synchronized diploid human fibroblasts were exposed to either 0 or 2.5 J m(-2) UVC in early, mid- and late-S phase.

Human CST promotes telomere duplex replication and general replication restart after fork stalling.

Mammalian CST (CTC1-STN1-TEN1) associates with telomeres and depletion of CTC1 or STN1 causes telomere defects. However, the function of mammalian CST remains poorly understood. We show here that depletion of CST subunits leads to both telomeric and non-telomeric phenotypes associated with DNA replication defects.

Titanium dioxide nanoparticles activate the ATM-Chk2 DNA damage response in human dermal fibroblasts.

The use of nanoparticles in consumer products increases their prevalence in the environment and the potential risk to human health. Although recent studies have shown in vivo and in vitro toxicity of titanium dioxide nanoparticles (nano-TiO2), a more detailed view of the underlying mechanisms of this response needs to be established. Here, the effects of nano-TiO2 on the DNA damage response and DNA replication dynamics were investigated in human dermal fibroblasts.

Temporal and functional analysis of DNA replicated in early S phase.

In summary, recently developed technologies have begun to draw back the curtain of mystery that obscures some of the basic mechanisms of DNA replication at multiple levels. Studies using extended DNA and chromatin fiber techniques have proven valuable for identifying the location of origins of replication at specific genomic sites and determining their temporal order of replication, for identifying and quantifying sites of DNA damage and localizing chromatin proteins in relation to sites of DNA replication. The future potential of these methods include further discoveries in functional genomics and contributions to the elucidation of the histone code.

Accumulation of true single strand breaks and AP sites in base excision repair deficient cells.

Single strand breaks (SSBs) are one of the most frequent DNA lesions caused by endogenous and exogenous agents. The most utilized alkaline-based assays for SSB detection frequently give false positive results due to the presence of alkali-labile sites that are converted to SSBs. Methoxyamine, an acidic O-hydroxylamine, has been utilized to measure DNA damage in cells.

BRG1 co-localizes with DNA replication factors and is required for efficient replication fork progression.

For DNA replication to occur, chromatin must be remodeled. Yet, we know very little about which proteins alter nucleosome occupancy at origins and replication forks and for what aspects of replication they are required. Here, we demonstrate that the BRG1 catalytic subunit of mammalian SWI/SNF-related complexes co-localizes with origin recognition complexes, GINS complexes, and proliferating cell nuclear antigen at sites of DNA replication on extended chromatin fibers.

Abasic sites preferentially form at regions undergoing DNA replication.

We investigated whether apurinic/apyrimidinic (AP/abasic) sites were more frequent in regions of DNA replication in cells and whether their number increased during oxidative stress. DNA fiber spreading and fluorescent immunostaining were used to detect areas of DNA replication and sites of AP lesions in extended DNA fibers. The distribution of AP sites was determined in DNA fibers from vertebrate cells maintained under normal culture conditions or stressed with exogenous H(2)O(2).

FEN1 functions in long patch base excision repair under conditions of oxidative stress in vertebrate cells.

From in vitro studies, flap endonuclease 1 (FEN1) has been proposed to play a role in the long patch (LP) base excision repair (BER) subpathway. Yet the role of FEN1 in BER in the context of the living vertebrate cell has not been thoroughly explored. In the present study, we cloned a DT40 chicken cell line with a deletion in the FEN1 gene and found that these FEN1-deficient cells exhibited hypersensitivity to H(2)O(2).

DNA replication and the GINS complex: localization on extended chromatin fibers.

Background: The GINS complex is thought to be essential for the processes of initiation and elongation of DNA replication. This complex contains four subunits, one of which (Psf1) is proposed to bind to both chromatin and DNA replication-associated proteins. To date there have been no microscopic analyses to evaluate the chromatin distribution of this complex.

Analysis of re-replication from deregulated origin licensing by DNA fiber spreading.

A major challenge each human cell-division cycle is to ensure that DNA replication origins do not initiate more than once, a phenomenon known as re-replication. Acute deregulation of replication control ultimately causes extensive DNA damage, cell-cycle checkpoint activation and cell death whereas moderate deregulation promotes genome instability and tumorigenesis. In the absence of detectable increases in cellular DNA content however, it has been difficult to directly demonstrate re-replication or to determine if the ability to re-replicate is restricted to a particular cell-cycle phase.

Human immunodeficiency virus type 1 Vpr-binding protein VprBP, a WD40 protein associated with the DDB1-CUL4 E3 ubiquitin ligase, is essential for DNA replication and embryonic development.

Damaged DNA binding protein 1, DDB1, bridges an estimated 90 or more WD40 repeats (DDB1-binding WD40, or DWD proteins) to the CUL4-ROC1 catalytic core to constitute a potentially large number of E3 ligase complexes. Among these DWD proteins is the human immunodeficiency virus type 1 (HIV-1) Vpr-binding protein VprBP, whose cellular function has yet to be characterized but has recently been found to mediate Vpr-induced G(2) cell cycle arrest. We demonstrate here that VprBP binds stoichiometrically with DDB1 through its WD40 domain and through DDB1 to CUL4A, subunits of the COP9/signalsome, and DDA1.

DNA replication in early S phase pauses near newly activated origins.

During the S phase of the cell cycle, the entire genome is replicated. There is a high level of orderliness to this process through the temporally and topologically coordinated activation of many replication origins situated along chromosomes. We investigated the program of replication from origins initiating in early S phase by labeling synchronized normal human fibroblasts (NHF1) with nucleotide analogs for various pulse times and measuring labeled tracks in combed DNA fibers.

The human Tim/Tipin complex coordinates an Intra-S checkpoint response to UV that slows replication fork displacement.

UV-induced DNA damage stalls DNA replication forks and activates the intra-S checkpoint to inhibit replicon initiation. In response to stalled replication forks, ATR phosphorylates and activates the transducer kinase Chk1 through interactions with the mediator proteins TopBP1, Claspin, and Timeless (Tim). Murine Tim recently was shown to form a complex with Tim-interacting protein (Tipin), and a similar complex was shown to exist in human cells.

Mapping of an origin of DNA replication in the promoter of fragile X gene FMR1.

An origin of bidirectional DNA replication was mapped to the promoter of the FMR1 gene in human chromosome Xq27.3, which has been linked to the fragile X syndrome. This origin is adjacent to a CpG island and overlaps the site of expansion of the triplet repeat (CGG) at the fragile X instability site, FRAXA.

Architecture of the bacteriophage T4 replication complex revealed with nanoscale biopointers.

Our previous electron microscopy of DNA replicated by the bacteriophage T4 proteins showed a single complex at the fork, thought to contain the leading and lagging strand proteins, as well as the protein-covered single-stranded DNA on the lagging strand folded into a compact structure. "Trombone" loops formed from nascent lagging strand fragments were present on a majority of the replicating molecules (Chastain, P., Makhov, A.

Nonrandom AP site distribution in highly proliferative cells.

Reactive oxygen species (ROS) and the oxidative DNA damage they produce [e.g., 8-oxo-guanine and apurinic/apyrimidinic (AP) sites] have been linked to the pathogenesis of several age-related and chronic diseases.

Checkpoint regulation of replication dynamics in UV-irradiated human cells.

At any moment during S phase, regions of genomic DNA are in various stages of replication (i.e., initiation, chain elongation, and termination).

A late origin of DNA replication in the trinucleotide repeat region of the human FMR2 gene.

We confirmed that the replication of the fragile-X E site (FRAXE) in human chromosomal band Xq28 occurs at six hours into the eight-hour S phase of normal human fibroblasts. In this late-replicating region, we mapped an origin of DNA replication within the promoter of FMR2. This origin is coincident with CpG islands, the trinucleotide repeat, and exon 1 of the FMR2 gene.