Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P16104 (H2AX)
 3,930 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human checkpoint kinase 1 (Chk1) is an essential kinase required to preserve genome stability. Here, we show that Chk1 inhibition by two distinct drugs, UCN-01 and CEP-3891, or by Chk1 small interfering RNA (siRNA) leads to phosphorylation of ATR targets. Chk1-inhibition triggered rapid, pan-nuclear phosphorylation of histone H2AX, p53, Smc1, replication protein A, and Chk1 itself in human S-phase cells. These phosphorylations were inhibited by ATR siRNA and caffeine, but they occurred independently of ATM. Chk1 inhibition also caused an increased initiation of DNA replication, which was accompanied by increased amounts of nonextractable RPA protein, formation of single-stranded DNA, and induction of DNA strand breaks. Moreover, these responses were prevented by siRNA-mediated downregulation of Cdk2 or the replication initiation protein Cdc45, or by addition of the CDK inhibitor roscovitine. We propose that Chk1 is required during normal S phase to avoid aberrantly increased initiation of DNA replication, thereby protecting against DNA breakage. These results may help explain why Chk1 is an essential kinase and should be taken into account when drugs to inhibit this kinase are considered for use in cancer treatment.
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PMID:Inhibition of human Chk1 causes increased initiation of DNA replication, phosphorylation of ATR targets, and DNA breakage. 1583 61

We previously showed that in quiescent cells, p300/CBP (CREB-binding protein)family coactivators repress c-myc and prevent premature induction of DNA synthesis. p300/CBP-depleted cells exit G(1) early and continue to accumulate in S phase but do not progress into G(2)/M, and eventually they die of apoptosis. Here, we show that the S-phase arrest in these cells is because of an intra-S-phase block. The inappropriate DNA synthesis that occurs as a result of forced expression of c-myc leads to the activation of the DNA damage response as evidenced by the phosphorylation of several checkpoint related proteins and the formation of foci containing gamma-H2AX. The activation of checkpoint response is related to the induction of c-myc, as the phosphorylation of checkpoint proteins can be reversed when cells are treated with a c-Myc inhibitor or when Myc synthesis is blocked by short hairpin RNA. Using the DNA fiber assay, we show that in p300-depleted cells initiation of replication occurs from multiple replication origins. Chromatin loading of the Cdc45 protein also indicates increased origin activity in p300 knockdown cells. Immunofluorescence experiments indicate that c-Myc colocalizes with replication foci, consistent with the recently reported direct role of c-Myc in the initiation of DNA synthesis. Thus, the inappropriate S-phase entry of p300 down-regulated cells is likely to be because of c-Myc-induced deregulated replication origin activity, which results in replicative stress, activation of a DNA damage response, and S-phase arrest. Our results point to an important role for p300 in maintaining genomic integrity by negatively regulating c-myc.
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PMID:c-Myc-induced aberrant DNA synthesis and activation of DNA damage response in p300 knockdown cells. 1933 36

H2AX phosphorylation at serine 139 (gammaH2AX) is a sensitive indicator of both DNA damage and DNA replication stress. Here we show that gammaH2AX formation is greatly enhanced in response to replication inhibitors but not ionizing radiation in HCT116 or SW480 cells depleted of Chk1. Although H2AX phosphorylation precedes the induction of apoptosis in such cells, our results suggest that cells containing gammaH2AX are not committed to death. gammaH2AX foci in these cells largely colocalize with RPA foci and their formation is dependent upon the essential replication helicase cofactor Cdc45, suggesting that H2AX phosphorylation occurs at sites of stalled forks. However Chk1-depleted cells released from replication inhibitors retain gammaH2AX foci and do not appear to resume replicative DNA synthesis. BrdU incorporation only occurs in a minority of Chk1-depleted cells containing gammaH2AX foci after release from thymidine arrest and, in cells incorporating BrdU, DNA synthesis does not occur at sites of gammaH2AX foci. Furthermore activated ATM and Chk2 persist in these cells. We propose that the gammaH2AX foci in Chk1-depleted cells may represent sites of persistent replication fork damage or abandonment that are unable to resume DNA synthesis but do not play a direct role in the Chk1 suppressed death pathway.
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PMID:Enhanced H2AX phosphorylation, DNA replication fork arrest, and cell death in the absence of Chk1. 2005 81

Cdc45 is an essential protein that together with Mcm2-7 and GINS forms the eukaryotic replicative helicase CMG. Cdc45 seems to be rate limiting for the initial unwinding or firing of replication origins. In line with this view, Cdc45-overexpressing cells fired at least twice as many origins as control cells. However, these cells displayed an about 2-fold diminished fork elongation rate, a pronounced asymmetry of replication fork extension, and an early S phase arrest. This was accompanied by H2AX-phosphorylation and subsequent apoptosis. Unexpectedly, we did not observe increased ATR/Chk1 signaling but rather a mild ATM/Chk2 response. In addition, we detected accumulation of long stretches of single-stranded DNA, a hallmark of replication catastrophe. We conclude that increased origin firing by upregulated Cdc45 caused exhaustion of the single-strand binding protein RPA, which in consequence diminished the ATR/Chk1 response; the subsequently occurring fork breaks led to an ATM/Chk2 mediated phosphorylation of H2AX and eventually to apoptosis.
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PMID:Cdc45 is limiting for replication initiation in humans. 2715 31