FIG. 2.

Chk2 depletion or Chk2 inhibition by DBH alters OriP replication timing and function. (A) D98/HR1 cells were transfected with siControl or siChk2 RNA and assayed by Western blotting to verify that Chk2 was depleted at 72 h posttransfection. (B and C) siControl- or siChk2-transfected D98/HR1 cells were pulse-labeled with BrdU, stained with PI, and then fractionated into different cell cycle stages by using FACS. BrdU-labeled DNA specific for the DS (B) or cellular lamin B2 (C) was quantified by anti-BrdU immunoprecipitation and PCR. Cell cycle fractions are indicated. (D and E) A replication timing assay was performed with D98/HR1 cells treated with DBH or control DMSO for 48 h prior to BrdU pulse-labeling, FACS, and IP-PCR analysis. BrdU incorporation was assayed at the EBV DS (D) or at the cellular lamin B2 origin (E). (F) EBV genome copy numbers were assayed in D98/HR1 cells treated with DBH or the DMSO control after 6 days of treatment. The copy number was measured by real-time PCR analysis of the EBV DS relative to cellular actin DNA.