RESUMEN
Phosphorylation of replication protein A (RPA) by Cdk2 and the checkpoint kinase ATR (ATM and Rad3 related) during replication fork stalling stabilizes the replisome, but how these modifications safeguard the fork is not understood. To address this question, we used single-molecule fiber analysis in cells expressing a phosphorylation-defective RPA2 subunit or lacking phosphatase activity toward RPA2. Deregulation of RPA phosphorylation reduced synthesis at forks both during replication stress and recovery from stress. The ability of phosphorylated RPA to stimulate fork recovery is mediated through the PALB2 tumor suppressor protein. RPA phosphorylation increased localization of PALB2 and BRCA2 to RPA-bound nuclear foci in cells experiencing replication stress. Phosphorylated RPA also stimulated recruitment of PALB2 to single-strand deoxyribonucleic acid (DNA) in a cell-free system. Expression of mutant RPA2 or loss of PALB2 expression led to significant DNA damage after replication stress, a defect accentuated by poly-ADP (adenosine diphosphate) ribose polymerase inhibitors. These data demonstrate that phosphorylated RPA recruits repair factors to stalled forks, thereby enhancing fork integrity during replication stress.
Asunto(s)
Daño del ADN/genética , Reparación del ADN/genética , Replicación del ADN/genética , Proteínas Nucleares/genética , Proteína de Replicación A/genética , Proteína de Replicación A/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas de la Ataxia Telangiectasia Mutada , Proteína BRCA2/genética , Camptotecina/farmacología , Línea Celular Tumoral , Cromatina/genética , Quinasa 2 Dependiente de la Ciclina , Replicación del ADN/efectos de los fármacos , ADN de Cadena Simple/genética , Proteínas de Unión al ADN/metabolismo , Proteína del Grupo de Complementación N de la Anemia de Fanconi , Humanos , Hidroxiurea/farmacología , Complejos Multiproteicos/genética , Proteínas Nucleares/biosíntesis , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Fosfoproteínas Fosfatasas/genética , Fosforilación/efectos de los fármacos , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Interferencia de ARN , ARN Interferente Pequeño , Inhibidores de Topoisomerasa I/farmacología , Proteínas Supresoras de Tumor/biosíntesisRESUMEN
Cyclobutane pyrimidine dimer (CPD) photolyases are structure specific DNA-repair enzymes that specialize in the repair of CPDs, the major photoproducts that are formed upon irradiation of DNA with ultraviolet light. The purified enzyme binds a flavin adenine dinucleotide (FAD), which is in the neutral radical semiquinone (FADH(*)) form. The CPDs are repaired by a light-driven, electron transfer from the anionic hydroquinone (FADH(-)) singlet excited state to the CPD, which is followed by reductive cleavage of the cyclobutane ring and subsequent monomerization of the pyrimidine bases. CPDs formed between two adjacent thymidine bases (T< >T) are repaired with greater efficiency than those formed between two adjacent cytidine bases (C< >C). In this paper, we investigate the changes in Escherichia coli photolyase that are induced upon binding to DNA containing C< >C lesions using resonance Raman, UV-vis absorption, and transient absorption spectroscopies, spectroelectrochemistry, and computational chemistry. The binding of photolyase to a C< >C lesion modifies the energy levels of FADH(*), the rate of charge recombination between FADH(-) and Trp(306)(*), and protein-FADH(*) interactions differently than binding to a T< >T lesion. However, the reduction potential of the FADH(-)/FADH(*) couple is modified in the same way with both substrates. Our calculations show that the permanent electric dipole moment of C< >C is stronger (12.1 D) and oriented differently than that of T< >T (8.7 D). The possible role of the electric dipole moment of the CPD in modifying the physicochemical properties of photolyase as well as in affecting CPD repair will be discussed.
