Biomedical subjects

Sahar Sibani

Publications and source records attributed to Sahar Sibani.

7 recordsLinked to original sources

PubMed2006

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. Nascent DNA abundance analysis, involving quantification by real-time PCR, indicated that hors8 is an active replication origin, as the abundance of nascent DNA from a genomic region containing it was 97-fold higher relative to a non-origin region in the same locus. Furthermore, the data showed that the hors8 fragment is capable of supporting the episomal replication of its plasmid, when cloned into pBlueScript (pBS), as assayed by the DpnI resistance assay after transfection of HeLa cells. A quantitative chromatin immunoprecipitation (ChIP) assay, using antibodies against Ku, Orc2, and Cdc6, showed that these DNA replication initiator proteins were associated in vivo with the human ors8 (hors8). Finally, nascent DNA abundance experiments from human cells synchronized at different phases of the cell cycle revealed that hors8 is a late-firing origin of DNA replication, having the highest activity 8 h after release from late G(1).

Animals↗

PubMed2005

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. This was concomitant with a 33% and 40% decrease in chromatin binding of both proteins, respectively. Cell cycle analysis of asynchronous and late G1 synchronized Ku80+/- cells revealed a prolonged G1 phase. Furthermore, these Ku-deficient cells had a 4.5-, 3.4- and 4.3-fold decrease in nascent strand DNA abundance at the lamin B2, beta-globin and c-myc replication origins, respectively. Chromatin immunoprecipitation (ChIP) assays showed that the association of Ku80 with the lamin B2, beta-globin and c-myc origins was decreased by 1.5-, 2.3- and 2.5-fold, respectively, whereas that of Ku70 was similarly decreased (by 2.1-, 1.5- and 1.7-fold, respectively) in Ku80+/- cells. The results indicate that a deficiency of Ku80 resulted in a prolonged G1 phase, as well as decreased Ku binding to and activation of origins of DNA replication.

Animals↗

PubMed2005

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%. Coimmunoprecipitation studies indicated that the Ku heterodimer associates specifically with the human HsOrc-2, -3, -4, and -6 subunits. Chromatin immunoprecipitation (ChIP) experiments, using cells synchronized to late G1, showed that the association of Ku80 with the lamin B2, beta-globin, and c-myc origins in vivo was decreased by 1.5-, 2.3-, and 2.5-fold, respectively, in Ku80+/- cells. The association of HsOrc-3, -4, and -6 was consistently decreased in all three origins examined in Ku80+/- cells, while that of HsOrc-2 showed no significant variation, indicating that the HsOrc-3, -4, and -6 subunits bind to the origins after Ku80. In Orc2(delta/-) cells, the association of HsOrc-2 with the lamin B2, beta-globin, and c-myc origins was decreased by 2.8-, 4.9-, and 2.8-fold, respectively, relative to wild-type HCT116 cells. Furthermore, nascent strand abundance at these three origins was decreased by 4.5-, 2.3-, and 2.6-fold in Orc2(delta/-) relative to HCT116 cells, respectively. Interestingly, the association of Ku80 with these origins was not affected in this hypomorphic cell line, indicating that Ku and HsOrc-2 bind to origins independently of each other.

Antigens, Nuclear↗

PubMed2004

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). This review summarizes the origin binding proteins that have been have been identified to date.

14-3-3 Proteins↗

PubMed2003

Characterization of mutations in severe methylenetetrahydrofolate reductase deficiency reveals an FAD-responsive mutation.

Methylenetetrahydrofolate reductase (MTHFR) synthesizes 5-methyltetrahydrofolate, a major methyl donor for homocysteine remethylation to methionine. Severe MTHFR deficiency results in marked hyperhomocysteinemia and homocystinuria. Patients display developmental delay and a variety of neurological and vascular symptoms. Cloning of the human cDNA and gene has enabled the identification of 29 rare mutations in homocystinuric patients and two common variants [677C>T (A222V) and 1298A>C (E429A)] with mild enzymatic deficiency. Homozygosity for 677C>T or combined heterozygosity for both polymorphisms is associated with mild hyperhomocysteinemia. In this communication, we describe four novel mutations in patients with homocystinuria: two missense mutations (471C>G, I153M; 1025T>C, M338T), a nonsense mutation (1274G>A, W421X), and a 2-bp deletion (1553delAG). We expressed the 1025T>C mutation as well as two previously reported amino acid substitutions [983A>G (N324S) and 1027T>G (W339G)] and observed decreased enzyme activity at 10%, 36%, and 21% of control levels, respectively, with little or no effect on affinity for 5-methyltetrahydrofolate. One of these mutations, 983A>G (N324S), showed flavin adenine dinucleotide (FAD) responsiveness in vitro. Expression of these mutations in cis with the 677C>T polymorphism, as observed in the patients, resulted in an additional 50% decrease in enzyme activity. This report brings the total to 33 severe mutations identified in patients with severe MTHFR deficiency.

Age of Onset↗

PubMed2003

Impact of Dnmt1 deficiency, with and without low folate diets, on tumor numbers and DNA methylation in Min mice.

Although a number of studies have suggested that diets with low intake of folate, an important methyl donor, are associated with increased risks of colon cancer and its precursor the adenomatous polyp, the underlying mechanisms are poorly understood. Dysregulation and instability of DNA methylation and alterations in the levels of the predominant DNA methylating enzyme, DNA (cytosine-5)-methyltransferase 1 (Dnmt1), have also been linked to tumorigenesis. We have used a combination of genetic and dietary manipulation to assess the effects of reduced Dnmt1 expression with and without folate deficiency on tumor induction in the Apc(Min) mouse. Apc(Min) mice with a reduction in Dnmt1 expression (Apc(Min/+)/Dnmt1(C/+)) had significantly lower tumor numbers than Apc(Min) mice with normal Dnmt1 (Apc(Min/+)/Dnmt1(+/+)). Dietary folate deficiency from weaning to 13 weeks of age did not affect tumor number or size in Apc(Min/+)/Dnmt(+/+) mice. However, in Apc(Min/+)/Dnmt1(C/+) mice with high baseline tumor numbers (41 +/- 4), folate deficiency was associated with a decreased absolute number of tumors (27 +/- 3), but a higher proportion of larger tumors as compared with mice on the control diet. In the repeat experiment, Apc(Min/+)/Dnmt1(C/+) mice had low baseline tumor numbers (20 +/- 2) and folate deficiency did not affect tumor number (23 +/- 4) or size as compared with the same mice on the control diet. These results suggest that, in the presence of Dnmt1 deficiency, the effects of folate deficiency on tumor number and size may depend on the stage of adenoma development when folate deficiency is initiated. We also show that folate deficiency with or without reductions in Dnmt1 did not affect overall genomic DNA methylation or the methylation levels of two candidate genes, E-cadherin or p53, in normal or neoplastic intestinal tissue. In conclusion, genetic deficiency in Dnmt1 with or without folate deficiency decreases tumor number in the Apc(Min) mouse model, but this effect may not be mediated by changes in SAM or SAH levels, nor by alterations in global methylation in the pre-neoplastic intestinal tissue.

Animals↗

PubMed2002

Studies of methionine cycle intermediates (SAM, SAH), DNA methylation and the impact of folate deficiency on tumor numbers in Min mice.

Several epidemiological studies have suggested a modulatory effect of dietary folate intake on the risk of colorectal cancer. The molecular basis for this inverse association is not clearly understood, but may involve alterations in DNA methylation. In this study, we examined the levels of methylation intermediates [S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH)] and of global DNA methylation in the pre-neoplastic small intestine of Min (multiple intestinal neoplasia) mice. We also studied the effect of folate/choline deficiency on these parameters and on tumor multiplicity in this animal model. In folate-adequate Min mice, we identified positive linear correlations between SAM or SAH and tumor numbers (R(2) = 0.38, P < 0.005; R(2) = 0.26, P = 0.025, respectively). A positive correlation between global DNA hypomethylation and tumor multiplicity was also observed (R(2) = 0.29, P = 0.014). These three biochemical determinants (SAM, SAH and DNA hypomethylation) may, therefore, serve as early markers of cell transformation. Folate/choline deficiency, however, did not produce a consistent effect on tumor numbers in three separate experiments. As an increase in tumor numbers was observed only in folate- and choline-deficient mice with low levels of SAM and DNA hypomethylation, the modulatory role of folate may be dependent on the transformation state of the cell.

Animals↗