RESUMEN
Base excision repair (BER) is a cellular process that removes damaged bases arising from exogenous and endogenous sources including reactive oxygen species, alkylation agents, and ionizing radiation. BER is mediated by the actions of multiple proteins which work in a highly concerted manner to resolve DNA damage efficiently to prevent toxic repair intermediates. During the initiation of BER, the damaged base is removed by one of 11 mammalian DNA glycosylases, resulting in abasic sites. Many DNA glycosylases are product-inhibited by binding to the abasic site more avidly than the damaged base. Traditionally, apurinic/apyrimidinic endonuclease 1, APE1, was believed to help turn over the glycosylases to undergo multiple rounds of damaged base removal. However, in a series of papers from our laboratory, we have demonstrated that UV-damaged DNA binding protein (UV-DDB) stimulates the glycosylase activities of human 8-oxoguanine glycosylase (OGG1), MUTY DNA glycosylase (MUTYH), alkyladenine glycosylase/N-methylpurine DNA glycosylase (AAG/MPG), and single-strand selective monofunctional glycosylase (SMUG1), between three- and five-fold. Moreover, we have shown that UV-DDB can assist chromatin decompaction, facilitating access of OGG1 to 8-oxoguanine damage in telomeres. This review summarizes the biochemistry, single-molecule, and cell biology approaches that our group used to directly demonstrate the essential role of UV-DDB in BER.
Asunto(s)
Cromatina , ADN Glicosilasas , Humanos , Cromatina/genética , Reparación del ADN , Daño del ADN , ADN Glicosilasas/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismoRESUMEN
UV-damaged DNA-binding protein (UV-DDB) is a heterodimeric protein, consisting of DDB1 and DDB2 subunits, that works to recognize DNA lesions induced by UV damage during global genome nucleotide excision repair (GG-NER). Our laboratory previously discovered a non-canonical role for UV-DDB in the processing of 8-oxoG, by stimulating 8-oxoG glycosylase, OGG1, activity 3-fold, MUTYH activity 4-5-fold, and APE1 (apurinic/apyrimidinic endonuclease 1) activity 8-fold. 5-hydroxymethyl-deoxyuridine (5-hmdU) is an important oxidation product of thymidine which is removed by single-strand selective monofunctional DNA glycosylase (SMUG1). Biochemical experiments with purified proteins indicated that UV-DDB stimulates the excision activity of SMUG1 on several substrates by 4-5-fold. Electrophoretic mobility shift assays indicated that UV-DDB displaced SMUG1 from abasic site products. Single-molecule analysis revealed that UV-DDB decreases the half-life of SMUG1 on DNA by â¼8-fold. Immunofluorescence experiments demonstrated that cellular treatment with 5-hmdU (5 µM for 15 min), which is incorporated into DNA during replication, produces discrete foci of DDB2-mCherry, which co-localize with SMUG1-GFP. Proximity ligation assays supported a transient interaction between SMUG1 and DDB2 in cells. Poly(ADP)-ribose accumulated after 5-hmdU treatment, which was abrogated with SMUG1 and DDB2 knockdown. These data support a novel role for UV-DDB in the processing of the oxidized base, 5-hmdU.
Asunto(s)
Daño del ADN , Proteínas de Unión al ADN , Proteínas de Unión al ADN/metabolismo , Reparación del ADN , ADN/química , Timidina , Rayos UltravioletaRESUMEN
Recent data from our laboratory has shown that the nucleotide excision repair (NER) proteins UV-damaged DNA-binding protein (UV-DDB), xeroderma pigmentosum group C (XPC), and xeroderma pigmentosum group A (XPA) play important roles in the processing of 8-oxoG. This review first discusses biochemical studies demonstrating how UV-DDB stimulates human 8-oxoG glycosylase (OGG1), MUTYH, and apurinic/apyrimidinic (AP) endonuclease (APE1) to increase their turnover at damage sites. We further discuss our single-molecule studies showing that UV-DDB associates with these proteins at abasic moieties on DNA damage arrays. Data from cell experiments are then described showing that UV-DDB interacts with OGG1 at sites of 8-oxoG. Finally, since many glycosylases are inhibited from working on damage in the context of chromatin, we present a working model of how UV-DDB may be the first responder to alter the structure of damage containing-nucleosomes to allow access by base excision repair (BER) enzymes.
Asunto(s)
Xerodermia Pigmentosa , Humanos , Daño del ADN , Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Guanina/metabolismo , Xerodermia Pigmentosa/metabolismo , Proteínas de Unión al ADN/metabolismoRESUMEN
Single-strand selective monofunctional uracil DNA glycosylase 1 (SMUG1) works to remove uracil and certain oxidized bases from DNA during base excision repair (BER). This review provides a historical characterization of SMUG1 and 5-hydroxymethyl-2'-deoxyuridine (5-hmdU) one important substrate of this enzyme. Biochemical and structural analyses provide remarkable insight into the mechanism of this glycosylase: SMUG1 has a unique helical wedge that influences damage recognition during repair. Rodent studies suggest that, while SMUG1 shares substrate specificity with another uracil glycosylase UNG2, loss of SMUG1 can have unique cellular phenotypes. This review highlights the multiple roles SMUG1 may play in preserving genome stability, and how the loss of SMUG1 activity may promote cancer. Finally, we discuss recent studies indicating SMUG1 has moonlighting functions beyond BER, playing a critical role in RNA processing including the RNA component of telomerase.
Asunto(s)
Genoma , Neoplasias/enzimología , Neoplasias/genética , Uracil-ADN Glicosidasa/metabolismo , Animales , Citoprotección , Humanos , Nucleosomas/metabolismo , Especificidad por SustratoRESUMEN
The six major mammalian DNA repair pathways were discovered as independent processes, each dedicated to remove specific types of lesions, but the past two decades have brought into focus the significant interplay between these pathways. In particular, several studies have demonstrated that certain proteins of the nucleotide excision repair (NER) and base excision repair (BER) pathways work in a cooperative manner in the removal of oxidative lesions. This review focuses on recent data showing how the NER proteins, XPA, XPC, XPG, CSA, CSB and UV-DDB, work to stimulate known glycosylases involved in the removal of certain forms of base damage resulting from oxidative processes, and also discusses how some oxidative lesions are probably directly repaired through NER. Finally, since many glycosylases are inhibited from working on damage in the context of chromatin, we detail how we believe UV-DDB may be the first responder in altering the structure of damage containing-nucleosomes, allowing access to BER enzymes.