UbcH7 levels control the entry and exit from S phase. (A) Cells were treated with siRNA to deplete UbcH7 (U7) or a non silencing siRNA (NS) for 72 h. The cell cycle profile was determined by propidium iodide staining. (B) HeLa cells were treated for 48 h with UbcH7 specific siRNA (U7) or a non silencing siRNA (NS) as indicated. Cells were then synchronized at the G1/S boundary by treatment with 2 mM hydroxyurea for 18 h. Cells were allowed to enter cycle after removal of hydroxyurea and culture in drug-free medium. The cell cycle profile at each time point after drug removal was determined. (C) COS cells were transiently transfected with plasmids containing UbcH7 or an empty vector. After 48 h of expression, the cell cycle profile was determined as above.

Possible mechanisms of UbcH7 regulation of Chk1 levels sand S phase. Conditions under normal or high levels of UbcH7 depicted on the left; after UbcH7 depletion, on the right. Decreased protein levels or decreased signaling pathways are noted by gray or lighter shading. (A) UbcH7 directly targets Chk1 for ubiquitination and degradation. If UbcH7 is directly involved in Chk1 ubiquitination, depletion of UbcH7 (right side) would result in an increase in Chk1. (B) UbcH7 increases Chk1 through a PTEN/Akt pathway. Depletion of UbcH7 (right) leads to decreased P-PTEN. Decreased activity through PTEN would increase Akt activity leading to increased P280-Chk1. The effect of UbcH7 on PTEN could be through inhibition of a phosphatase which affects the phosphorylation state of PTEN or through the inhibition of the E3 that targets PTEN for degradation. (C) UbcH7 activates Chk1 via alteration of Mcm7 levels. Decreased ubiquitination of Mcm7 through a UbcH7/E6-AP pathway (right) would lead to the imbalance of proteins in the Mcm2-7 complex. This in turn could lead to the activation of the S phase checkpoint and an increase in Chk1 levels. (D) UbcH7 depletion activates Chk1 through an increase in BRCA1/BARD1 function. The release of inhibition caused by UbcH7 (right) would lead to increased BRCA1/BARD1 ubiquitination and activation of the S phase checkpoint.