Suppression of the DHX9 helicase induces premature senescence in human diploid fibroblasts in a p53-dependent manner.

DHX9 is an ATP-dependent DEXH box helicase with a multitude of cellular functions. Its ability to unwind both DNA and RNA, as well as aberrant, noncanonical polynucleotide structures, has implicated it in transcriptional and translational regulation, DNA replication and repair, and maintenance of genome stability. We report that loss of DHX9 in primary human fibroblasts results in premature senescence, a state of irreversible growth arrest.

Detection of slipped-DNAs at the trinucleotide repeats of the myotonic dystrophy type I disease locus in patient tissues.

Slipped-strand DNAs, formed by out-of-register mispairing of repeat units on complementary strands, were proposed over 55 years ago as transient intermediates in repeat length mutations, hypothesized to cause at least 40 neurodegenerative diseases. While slipped-DNAs have been characterized in vitro, evidence of slipped-DNAs at an endogenous locus in biologically relevant tissues, where instability varies widely, is lacking. Here, using an anti-DNA junction antibody and immunoprecipitation, we identify slipped-DNAs at the unstable trinucleotide repeats (CTG)n•(CAG)n of the myotonic dystrophy disease locus in patient brain, heart, muscle and other tissues, where the largest expansions arise in non-mitotic tissues such as cortex and heart, and are smallest in the cerebellum.

Ku protein levels, localization and association to replication origins in different stages of breast tumor progression.

Human origins of DNA replication are specific sequences within the genome whereby DNA replication is initiated. A select group of proteins, known as the pre-replication (pre-RC) complex, in whose formation the Ku protein (Ku70/Ku86) was shown to play a role, bind to replication origins to initiate DNA replication. In this study, we have examined the involvement of Ku in breast tumorigenesis and tumor progression and found that the Ku protein expression levels in human breast metastatic (MCF10AC1a) cells were higher in the chromatin fraction compared to hyperplastic (MCF10AT) and normal (MCF10A) human breast cells, but remained constant in both the nuclear and cytoplasmic fractions.

Monoallelic chromatin conformation flanking long-range silenced domains in cancer-derived and normal cells.

Epigenetic inactivation of chromatin plays an important role in determining cell phenotype in both normal and cancer cells, but our knowledge is still incomplete with respect to any potential monoallelic nature of the phenomenon. We have genotyped DNA isolated from chromatin of two colorectal cancer-derived lines and a culture of normal human intestinal epithelial cells (HIEC), which was immunoprecipitated with antibodies to acetylated vs. methylated histone H3K9, and presented the data as B allele frequency differences over multiple single-nucleotide polymorphism (SNP) moving window averages.

RNAi screening uncovers Dhx9 as a modifier of ABT-737 resistance in an Eμ-myc/Bcl-2 mouse model.

ABT-737 is a promising chemotherapeutic agent that promotes apoptosis by acting as a selective BH3 mimetic to neutralize Bcl-2-like family members. One shortcoming with its use is that Mcl-1, a member of the Bcl-2 family, is poorly inhibited by ABT-737 and thus is a major cause of resistance. We performed a short hairpin RNA (shRNA)-based drop-out screen to identify novel genes and pathways that could reverse resistance to ABT-737 treatment in Eµ-myc/Bcl-2 lymphoma cells engineered to rely on endogenous Mcl-1 for survival.

Differential chromatin structure encompassing replication origins in transformed and normal cells.

This study examines the chromatin structure encompassing replication origins in transformed and normal cells. Analysis of the global levels of histone H3 acetylated at K9&14 (open chromatin) and histone H3 trimethylated at K9 (closed chromatin) revealed a higher ratio of open to closed chromatin in the transformed cells. Also, the trithorax and polycomb group proteins, Brg-1 and Bmi-1, respectively, were overexpressed and more abundantly bound to chromatin in the transformed cells.

14-3-3 checkpoint regulatory proteins interact specifically with DNA repair protein human exonuclease 1 (hEXO1) via a semi-conserved motif.

Human exonuclease 1 (hEXO1) acts directly in diverse DNA processing events, including replication, mismatch repair (MMR), and double strand break repair (DSBR), and it was also recently described to function as damage sensor and apoptosis inducer following DNA damage. In contrast, 14-3-3 proteins are regulatory phosphorserine/threonine binding proteins involved in the control of diverse cellular events, including cell cycle checkpoint and apoptosis signaling. hEXO1 is regulated by post-translation Ser/Thr phosphorylation in a yet not fully clarified manner, but evidently three phosphorylation sites are specifically induced by replication inhibition leading to protein ubiquitination and degradation.

Cdc6 expression represses E-cadherin transcription and activates adjacent replication origins.

E-cadherin (CDH1) loss occurs frequently in carcinogenesis, contributing to invasion and metastasis. We observed that mouse and human epithelial cell lines overexpressing the replication licensing factor Cdc6 underwent phenotypic changes with mesenchymal features and loss of E-cadherin. Analysis in various types of human cancer revealed a strong correlation between increased Cdc6 expression and reduced E-cadherin levels.

Comparative analysis of pre-replication complex proteins in transformed and normal cells.

This study examines the abundance of the major protein constituents of the pre-replication complex (pre-RC), both genome-wide and in association with specific replication origins, namely the lamin B2, c-myc, 20mer1, and 20mer2 origins. Several pre-RC protein components, namely ORC1-6, Cdc6, Cdt1, MCM4, MCM7, as well as additional replication proteins, such as Ku70/86, 14-3-3, Cdc45, and PCNA, were comparatively and quantitatively analyzed in both transformed and normal cells. The results show that these proteins are overexpressed and more abundantly bound to chromatin in the transformed compared to normal cells.

More is less: inactivation and deletion events and the search for tumor suppressor genes.

Tumor suppressor genes are frequently inactivated in cancer by large-scale deletion events or epigenetic silencing, and experimental demonstration of such inactivation has historically been considered as support for assigning tumor suppressive function to a given gene. However, the discovery of a number of chromosomal domains wherein large deletions naturally occur at frequencies up to 100 times the average for the genome as a whole leads us to reevaluate the significance of sporadic deletions found within genes associated with these hotspots. Similarly, our recent demonstration that epigenetic chromatin silencing frequently spreads in cancer cells from gene-poor into gene-rich regions with apparent indifference to the gene content of the affected domain raises questions about the pertinence of inactivation as a criterion for ascribing tumor suppressor function to a given gene.

Increased origin activity in transformed versus normal cells: identification of novel protein players involved in DNA replication and cellular transformation.

Using libraries of replication origins generated previously, we identified three clones that supported the autonomous replication of their respective plasmids in transformed, but not in normal cells. Assessment of their in vivo replication activity by in situ chromosomal DNA replication assays revealed that the chromosomal loci corresponding to these clones coincided with chromosomal replication origins in all cell lines, which were more active by 2-3-fold in the transformed by comparison to the normal cells. Evaluation of pre-replication complex (pre-RC) protein abundance at these origins in transformed and normal cells by chromatin immunoprecipitation assays, using anti-ORC2, -cdc6 and -cdt1 antibodies, showed that they were bound by these pre-RC proteins in all cell lines, but a 2-3-fold higher abundance was observed in the transformed by comparison to the normal cells.

14-3-3 proteins function in the initiation and elongation steps of DNA replication in Saccharomyces cerevisiae.

14-3-3s are highly conserved abundant eukaryotic proteins essential for viability, at least in lower eukaryotes. We previously showed that they associate with mammalian and yeast replication origins in a cell-cycle-dependent manner, and are involved in the initiation of DNA replication. Here, we present evidence that 14-3-3 proteins are novel regulators of the initiation and elongation steps of DNA replication in Saccharomyces cerevisiae.

Transient dsDNA breaks during pre-replication complex assembly.

Initiation of DNA replication involves the ordered assembly of the multi-protein pre-replicative complex (pre-RC) during G(1) phase. Previously, DNA topoisomerase II (topo II) was shown to associate with the DNA replication origin located in the lamin B2 gene locus in a cell-cycle-modulated manner. Here we report that activation of both the early-firing lamin B2 and the late-firing hOrs8 human replication origins involves DNA topo II-dependent, transient, site-specific dsDNA-break formation.

Ku is involved in cell growth, DNA replication and G1-S transition.

The Ku protein (Ku70-Ku80) is involved in various genome-maintenance processes such as DNA replication and repair, telomere maintenance, and chromosomal stability. We previously found that Ku80 is implicated in the loading of members of the pre-replicative complex (pre-RC) onto replication origins. Here, we report that acute reduction of Ku80 to 10% of its normal levels leads to impaired DNA replication and activation of a replication stress checkpoint.

Fine mapping and functional activity of the adenosine deaminase origin in murine embryonic fibroblasts.

DNA replication initiates at origins within the genome. The late-firing murine adenosine deaminase (mAdA) origin is located within a 2 kb fragment of DNA, making it difficult to examine by realtime technology. In this study, fine mapping of the mAdA region by measuring the abundance of nascent strand DNA identified two origins, mAdA-1 and mAdA-C, located 397 bp apart from each other.

14-3-3 cruciform-binding proteins as regulators of eukaryotic DNA replication.

Cruciforms are secondary DNA structures, serving as recognition signals at or near eukaryotic (yeast and mammalian) origins of DNA replication. The cruciform-binding protein is a member of the 14-3-3 protein family and binds to origins of DNA replication in a cell cycle-dependent manner. Five 14-3-3 protein isoforms (beta, gamma, epsilon, zeta and sigma) have been identified as having cruciform binding activity.

Deletion of the cruciform binding domain in CBP/14-3-3 displays reduced origin binding and initiation of DNA replication in budding yeast.

Background: Initiation of eukaryotic DNA replication involves many protein-protein and protein-DNA interactions. We have previously shown that 14-3-3 proteins bind cruciform DNA and associate with mammalian and yeast replication origins in a cell cycle dependent manner.

Results: By expressing the human 14-3-3epsilon, as the sole member of 14-3-3 proteins family in Saccharomyces cerevisiae, we show that 14-3-3epsilon complements the S.

Identification and functional analysis of a human homologue of the monkey replication origin ors8.

We previously isolated from African green monkey (CV-1) cells a replication origin, ors8, that is active at the onset of S-phase. Here, its homologous sequence (hors8, accession number: DQ230978) was amplified from human cells, using the monkey-ors8-specific primers. Sequence alignment between the monkey and the human fragment revealed a 92% identity.

Differentially active origins of DNA replication in tumor versus normal cells.

Previously, a degenerate 36 bp human consensus sequence was identified as a determinant of autonomous replication in eukaryotic cells. Random mutagenesis analyses further identified an internal 20 bp of the 36 bp consensus sequence as sufficient for acting as a core origin element. Here, we have located six versions of the 20 bp consensus sequence (20mer) on human chromosome 19q13 over a region spanning approximately 211 kb and tested them for ectopic and in situ replication activity by transient episomal replication assays and nascent DNA strand abundance analyses, respectively.

Proliferating human cells hypomorphic for origin recognition complex 2 and pre-replicative complex formation have a defect in p53 activation and Cdk2 kinase activation.

The Origin Recognition Complex (ORC) is a critical component of replication initiation. We have previously reported generation of an Orc2 hypomorph cell line (Delta/-) that expresses very low levels of Orc2 but is viable. We have shown here that Chk2 is phosphorylated, suggesting that DNA damage checkpoint pathways are activated.

Decreased origin usage and initiation of DNA replication in haploinsufficient HCT116 Ku80+/- cells.

One of the functions of the abundant heterodimeric nuclear protein, Ku (Ku70/Ku80), is its involvement in the initiation of DNA replication through its ability to bind to chromosomal replication origins in a sequence-specific and cell cycle dependent manner. Here, using HCT116 Ku80+/- cells, the effect of Ku80 deficiency on cell cycle progression and origin activation was examined. Western blot analyses revealed a 75% and 36% decrease in the nuclear expression of Ku80 and Ku70, respectively.

Ku80 binds to human replication origins prior to the assembly of the ORC complex.

The Ku heterodimer, an abundant nuclear protein, binds DNA replication origins in a sequence-specific manner and promotes initiation. In this study, using HCT116 Ku80+/- haplo-insufficient and Orc2(delta/-) hypomorphic cells, the order of binding of Ku and the human origin recognition complex (HsORC) was determined. The nuclear expression of Ku80 was found to be decreased by 60% in Ku80+/- cells, while its general association with chromatin was decreased by 33%.

The human stress-activated protein kin17 belongs to the multiprotein DNA replication complex and associates in vivo with mammalian replication origins.

The human stress-activated protein kin17 accumulates in the nuclei of proliferating cells with predominant colocalization with sites of active DNA replication. The distribution of kin17 protein is in equilibrium between chromatin-DNA and the nuclear matrix. An increased association with nonchromatin nuclear structure is observed in S-phase cells.

NAP-2 is part of multi-protein complexes in HeLa cells.

We previously reported that a complex of nuclear proteins from HeLa cells, among them histone H1 and casein kinase 2 co-eluted from immobilized nucleosome assembly protein 2 (NAP-2)-Sepharose. Here, using HeLa cell nuclear extracts, we found NAP-2 migrates in a blue-native polyacrylamide gel with an apparent molecular weight of 300 kDa. HeLa cell NAP-2, labeled in vivo with radioactive orthophosphate, co-precipitated with at least two phosphoproteins, with an apparent mass of 100 and 175 kDa, respectively, as determined by SDS-PAGE.

Eucaryotic replication origin binding proteins.

Initiation of eukaryotic DNA replication is a tightly controlled process. Replication initiates at multiple specific sites (replication origins) that have been licensed for replication, following the cell cycle-dependent, multi-step assembly of specific factors. Thus, replication origins occur in two chromatin states: a replication-competent pre-replicative (pre-RC) state, when a number of replication proteins assemble on the origin in a stepwise fashion, and a replication-incompetent post-replicative (post-RC) state, in which the origin (or elements of it) is bound only by the origin recognition complex (ORC) (or subunits of it).