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Sp1 is a ubiquitously expressed transcription factor that is phosphorylated by ataxia telangiectasia mutated kinase (ATM) in response to ionizing radiation and H(2)O(2). Here, we show by indirect immunofluorescence that Sp1 phosphorylated on serine 101 (pSp1) localizes to ionizing radiation-induced foci with phosphorylated histone variant γH2Ax and members of the MRN (Mre11, Rad50, and Nbs1) complex. More precise analysis of occupancy of DNA double-strand breaks (DSBs) by chromatin immunoprecipitation (ChIP) shows that Sp1, like Nbs1, resides within 200 bp of DSBs. Using laser microirradiation of cells, we demonstrate that pSp1 is present at DNA DSBs by 7.5 min after induction of damage and remains at the break site for at least 8 h. Depletion of Sp1 inhibits repair of site-specific DNA breaks, and the N-terminal 182-amino-acid peptide, which contains targets of ATM kinase but lacks the zinc finger DNA binding domain, is phosphorylated, localizes to DSBs, and rescues the repair defect resulting from Sp1 depletion. Together, these data demonstrate that Sp1 is rapidly recruited to the region immediately adjacent to sites of DNA DSBs and is required for DSB repair, through a mechanism independent of its sequence-directed transcriptional effects.

In mammalian somatic cells, the Anaphase Promoting Complex (APC) is inactivated during S phase by active cyclin A-Cyclin dependent kinase (Cdk) 2 complexes promoting accumulation of mitotic regulators, such as cyclin B and Polo like kinase 1 (Plk1). However, mitotic entry does not appear to be perturbed in some human cancer cells or in normal mouse cells following Cdk2 RNA interference (i) or deletion of the Cdk2 gene. These results suggest functional complementation of APC regulation by a compensatory kinase. Using Plk1 protein level as readout of APC activity, we show that APC is inactivated during S phase in human cells by both cyclin A-Cdk2 and cyclin A-Cdk1 complexes. Expression of a dominant negative mutant of Cdk2 or Cdk2 RNAi in early S phase destabilizes Plk1 as it begins to accumulate. However, this effect wanes in late S phase, where destabilization of Plk1 also requires Cdk1 RNAi. Although Cdk2 is the dominant partner of cyclin A in these settings, cyclin A also binds Cdk1. Both complexes bind the APC targeting factor Cdh1, but Cdk1 complexes are inactive in early S phase, accounting for the stronger regulation of APC function by Cdk2. These results provide further evidence that cyclin A-Cdk2 and -Cdk1 complexes display overlapping and partially redundant roles in preparing cells for mitosis, through regulation of the APC.