Base excision repair (BER) is the major pathway for processing of simple lesions in DNA, including single-strand breaks, base damage, and base loss. The scaffold protein XRCC1, DNA polymerase beta, and DNA ligase IIIalpha play pivotal roles in BER. Although all these enzymes are essential for development, their cellular levels must be tightly regulated because increased amounts of BER enzymes lead to elevated mutagenesis and genetic instability and are frequently found in cancer cells. Here we report that BER enzyme levels are linked to and controlled by the level of DNA lesions. We demonstrate that stability of BER enzymes increases after formation of a repair complex on damaged DNA and that proteins not involved in a repair complex are ubiquitylated by the E3 ubiquitin ligase CHIP and subsequently rapidly degraded. These data identify a molecular mechanism controlling cellular levels of BER enzymes and correspondingly the efficiency and capacity of BER.

SummaryThe deubiquitylation enzyme USP7/HAUSP plays a major role in regulating genome stability and cancer prevention by controlling the key proteins involved in the DNA damage response. Despite this important role in controlling other proteins, USP7 itself has not been recognized as a target for regulation. Here, we report that USP7 regulation plays a central role in DNA damage signal transmission. We find that stabilization of Mdm2, and correspondingly p53 downregulation in unstressed cells, is accomplished by a specific isoform of USP7 (USP7S), which is phosphorylated at serine 18 by the protein kinase CK2. Phosphorylation stabilizes USP7S and thus contributes to Mdm2 stabilization and downregulation of p53. After ionizing radiation, dephosphorylation of USP7S by the ATM-dependent protein phosphatase PPM1G leads to USP7S downregulation, followed by Mdm2 downregulation and accumulation of p53. Our findings provide a quantitative transmission mechanism of the DNA damage signal to coordinate a p53-dependent DNA damage response.

Base excision repair (BER) is the major cellular pathway involved in removal of endogenous/spontaneous DNA lesions. Here, we study the mechanism that controls the steady-state levels of BER enzymes in human cells. By fractionating human cell extract, we purified the E3 ubiquitin ligase Mule (ARF-BP1/HectH9) as an enzyme that can ubiquitylate DNA polymerase b (Pol b), the major BER DNA polymerase. We identified lysines 41, 61 and 81 as the major sites of modification and show that replacement of these lysines to arginines leads to increased protein stability. We further show that the cellular levels of Pol b and its ubiquitylated derivative are modulated by Mule and ARF and siRNA knockdown of Mule leads to accumulation of Pol b and increased DNA repair. Our findings provide a novel mechanism regulating steady-state levels of BER proteins.