RESUMO
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.
Assuntos
Dano ao DNA , DNA Ligases/metabolismo , DNA Polimerase beta/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Animais , Cromatina/metabolismo , DNA Ligase Dependente de ATP , DNA Ligases/genética , DNA Polimerase beta/genética , Proteínas de Ligação a DNA/genética , Células HeLa , Humanos , Peróxido de Hidrogênio/metabolismo , Substâncias Macromoleculares/metabolismo , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Oxidantes/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose , Processamento de Proteína Pós-Traducional , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteína 1 Complementadora Cruzada de Reparo de Raio-X , Proteínas de XenopusRESUMO
APE1 (Ref-1) is an essential human protein involved in DNA damage repair and regulation of transcription. Although the cellular functions and biochemical properties of APE1 are well characterized, the mechanism involved in regulation of the cellular levels of this important DNA repair/transcriptional regulation enzyme, remains poorly understood. Using an in vitro ubiquitylation assay, we have now purified the human E3 ubiquitin ligase UBR3 as a major activity that polyubiquitylates APE1 at multiple lysine residues clustered on the N-terminal tail. We further show that a knockout of the Ubr3 gene in mouse embryonic fibroblasts leads to an up-regulation of the cellular levels of APE1 protein and subsequent genomic instability. These data propose an important role for UBR3 in the control of the steady state levels of APE1 and consequently error free DNA repair.
Assuntos
DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Instabilidade Genômica , Ubiquitina-Proteína Ligases/metabolismo , Animais , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , Técnicas de Inativação de Genes , Células HeLa , Humanos , Lisina/metabolismo , Camundongos , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
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 beta (Pol beta), 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 beta and its ubiquitylated derivative are modulated by Mule and ARF and siRNA knockdown of Mule leads to accumulation of Pol beta and increased DNA repair. Our findings provide a novel mechanism regulating steady-state levels of BER proteins.
Assuntos
Reparo do DNA/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Western Blotting , Ensaio Cometa , DNA Polimerase beta/metabolismo , Reparo do DNA/genética , Eletroforese em Gel de Poliacrilamida , Células HeLa , Humanos , Ligação Proteica , Interferência de RNA , Proteínas Supressoras de Tumor , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
XRCC1 is a scaffold protein that interacts with several DNA repair proteins and plays a critical role in DNA base excision repair (BER). XRCC1 protein is in a tight complex with DNA ligase IIIalpha (Lig III) and this complex is involved in the ligation step of both BER and repair of DNA single strand breaks. The majority of XRCC1 has previously been demonstrated to exist in a phosphorylated form and cells containing mutant XRCC1, that is unable to be phosphorylated, display a reduced rate of single strand break repair. Here, in an unbiased assay, we demonstrate that the cytoplasmic form of the casein kinase 2 (CK2) protein is the major protein kinase activity involved in phosphorylation of XRCC1 in human cell extracts and that XRCC1 phosphorylation is required for XRCC1-Lig III complex stability. We demonstrate that XRCC1-Lig III complex containing mutant XRCC1, in which CK2 phosphorylation sites have been mutated, is unstable. We also find that a knockdown of CK2 by siRNA results in both reduced XRCC1 phosphorylation and stability, which also leads to a reduced amount of Lig III and accumulation of DNA strand breaks. We therefore propose that CK2 plays an important role in DNA repair by contributing to the stability of XRCC1-Lig III complex.