TY - JOUR
T1 - c-Myc-induced aberrant DNA synthesis and activation of DNA damage response in p300 knockdown cells
AU - Sankar, Natesan
AU - Kadeppagari, Ravi Kumar
AU - Thimmapaya, Bayar
PY - 2009/5/29
Y1 - 2009/5/29
N2 - 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 G1 early and continue to accumulate in S phase but do not progress into G2/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 γ-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 downregulated cells is likely to be because of c-Myc-induced deregulated replication origin activity, which results in replicative stress, activation of aDNAdamage response, and S-phase arrest. Our results point to an important role for p300 in maintaining genomic integrity by negatively regulating c-myc.
AB - 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 G1 early and continue to accumulate in S phase but do not progress into G2/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 γ-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 downregulated cells is likely to be because of c-Myc-induced deregulated replication origin activity, which results in replicative stress, activation of aDNAdamage response, and S-phase arrest. Our results point to an important role for p300 in maintaining genomic integrity by negatively regulating c-myc.
UR - http://www.scopus.com/inward/record.url?scp=67649386533&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=67649386533&partnerID=8YFLogxK
U2 - 10.1074/jbc.M900776200
DO - 10.1074/jbc.M900776200
M3 - Article
C2 - 19332536
AN - SCOPUS:67649386533
VL - 284
SP - 15193
EP - 15205
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 22
ER -