CHIP-Mediated Degradation and DNA Damage-Dependent Stabilization Regulate Base Excision Repair Proteins

Abstract

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.

Regulation of oxidative DNA damage repair by DNA polymerase λ and MutYH by cross-talk of phosphorylation and ubiquitination

“…In order to shed more light on the regulation of Pol λ in vivo, we set out to identify the E3 ubiquitin ligase responsible for the ubiquitination of Pol λ. To this end, we tested fractions originating from an assay established by the Dianov lab (9), where Pol λ ubiquitination activity was purified from HeLa whole cell extracts in a series of biochemical fractionations via chromatographic columns combined with an in vitro ubiquitination assay (Fig. S1A).…”

“…CHIP plays an important role in the heat shock response (15,16) and has been shown to be involved in regulating cellular levels of proteins like p53 (17). CHIP-mediated ubiquitination of Pol β leads to its degradation by the proteasome under normal circumstances (9,18). However, upon DNA damage, those BER proteins are stabilized and recruited to chromatin to fulfill their roles in the maintenance of genomic integrity in vivo.…”

Comparison of CID versus ETD based MS/MS fragmentation for the analysis of protein ubiquitination

“…The N-terminal domain (10 kDa) of DNA polymerase ␤ was previously shown to be ubiquitinated by the E3 ubiquitin ligase CHIP in vitro and in living cells [22]. In vitro ubiquitinated recombinant DNA Pol ␤ was separated by SDS-PAGE and the corresponding gel band digested with trypsin.…”

“…Formic acid was used to acidify the solution and the samples were kept at Ϫ20°C until analysis. Preparation of DNA polymerase ␤-1 was performed as described [22], and gel bands corresponding to ubiquitinated material were in-gel digested with trypsin as reported previously [23], with the exception that chloroacetamide instead of iodoacetamide was used for the alkylation step.…”

Chaperone-assisted degradation: multiple paths to destruction

“…Another interesting recent study demonstrates that CHIP induces the degradation of the cell growth and proliferation regulator Raf-1 in a manner facilitated by inhibitor of apoptosis proteins (Dogan et al, 2008). Furthermore, Parsons and coworkers showed that CHIP participates in the proteasomal degradation of base excision repair proteins, that are frequently overexpressed in cancer cells (Parsons et al, 2008). Taken together, these studies highlight that CAP acts at multiple stages to control oncogenic transformation, making the chaperone-assisted degradation pathway a promising target for therapeutic intervention in cancer.…”