RESUMEN
Women having BRCA1 germ-line mutations develop cancer in breast and ovary, estrogen-regulated tissues, with high penetrance. Binding of estrogens to the estrogen receptor (ER) transiently induces DNA double-strand breaks (DSBs) by topoisomerase II (TOP2) and controls gene transcription. TOP2 resolves catenated DNA by transiently generating DSBs, TOP2-cleavage complexes (TOP2ccs), where TOP2 covalently binds to 5' ends of DSBs. TOP2 frequently fails to complete its catalysis, leading to formation of pathological TOP2ccs. We have previously shown that the endonucleolytic activity of MRE11 plays a key role in removing 5' TOP2 adducts in G1 phase. We show here that BRCA1 promotes MRE11-mediated removal of TOP2 adducts in G1 phase. We disrupted the BRCA1 gene in 53BP1-deficient ER-positive breast cancer and B cells. The loss of BRCA1 caused marked increases of pathological TOP2ccs in G1 phase following exposure to etoposide, which generates pathological TOP2ccs. We conclude that BRCA1 promotes the removal of TOP2 adducts from DSB ends for subsequent nonhomologous end joining. BRCA1-deficient cells showed a decrease in etoposide-induced MRE11 foci in G1 phase, suggesting that BRCA1 repairs pathological TOP2ccs by promoting the recruitment of MRE11 to TOP2cc sites. BRCA1 depletion also leads to the increase of unrepaired DSBs upon estrogen treatment both in vitro in G1-arrested breast cancer cells and in vivo in epithelial cells of mouse mammary glands. BRCA1 thus plays a critical role in removing pathological TOP2ccs induced by estrogens as well as etoposide. We propose that BRCA1 suppresses tumorigenesis by removing estrogen-induced pathological TOP2ccs throughout the cell cycle.
Asunto(s)
Proteína BRCA1/fisiología , Neoplasias de la Mama/genética , Carcinogénesis/genética , ADN-Topoisomerasas de Tipo II/metabolismo , Inestabilidad Genómica/genética , Animales , Proteína BRCA1/genética , ADN/metabolismo , Daño del ADN , Reparación del ADN , Estrógenos/fisiología , Femenino , Fase G1 , Histonas/metabolismo , Humanos , Células MCF-7 , Glándulas Mamarias Animales/metabolismo , Ratones , Regiones Promotoras Genéticas , Receptores de Estrógenos/metabolismoRESUMEN
ALC1/CHD1L is a member of the SNF2 superfamily of ATPases carrying a macrodomain that binds poly(ADP-ribose). Poly(ADP-ribose) polymerase (PARP) 1 and 2 synthesize poly(ADP-ribose) at DNA-strand cleavage sites, promoting base excision repair (BER). Although depletion of ALC1 causes increased sensitivity to various DNA-damaging agents (H2O2, UV, and phleomycin), the role played by ALC1 in BER has not yet been established. To explore this role, as well as the role of ALC1's ATPase activity in BER, we disrupted the ALC1 gene and inserted the ATPase-dead (E165Q) mutation into the ALC1 gene in chicken DT40 cells, which do not express PARP2. The resulting ALC1-/- and ALC1-/E165Q cells displayed an indistinguishable hypersensitivity to methylmethane sulfonate (MMS), an alkylating agent, and to H2O2, indicating that ATPase plays an essential role in the DNA-damage response. PARP1-/- and ALC1-/-/PARP1-/- cells exhibited a very similar sensitivity to MMS, suggesting that ALC1 and PARP1 collaborate in BER. Following pulse-exposure to H2O2, PARP1-/- and ALC1-/-/PARP1-/- cells showed similarly delayed kinetics in the repair of single-strand breaks, which arise as BER intermediates. To ascertain ALC1's role in BER in mammalian cells, we disrupted the ALC1 gene in human TK6 cells. Following exposure to MMS and to H2O2, the ALC1-/- TK6 cell line showed a delay in single-strand-break repair. We therefore conclude that ALC1 plays a role in BER. Following exposure to H2O2, ALC1-/- cells showed compromised chromatin relaxation. We thus propose that ALC1 is a unique BER factor that functions in a chromatin context, most likely as a chromatin-remodeling enzyme.
