RESUMO
In Schizosaccharomyces pombe, rad18 is an essential gene involved in the repair of DNA damage produced by ionizing radiation and in tolerance of UV-induced DNA damage. The Rad18 protein is a member of the SMC (structural maintenance of chromosomes) superfamily, and we show that, like the other SMC proteins in condensin and cohesin, Rad18 is a component of a high-molecular-weight complex. This complex contains at least six other proteins, the largest of which is Spr18, a novel SMC family member closely related to Rad18, and likely to be its heterodimeric partner. SMC proteins have ATP-binding domains at the N- and C-termini, and two extended coiled-coil domains separated by a hinge in the middle. We show that the N-terminal ATP-binding domain of Rad18 is essential for all functions, and overexpression of an N-terminal mutant has a dominant-negative effect. We have identified an important mutation (S1045A) near the C-terminus of Rad18 that separates its repair and essential roles. Potential models for the role of the Rad18-Spr18 complex during DNA repair are discussed.
Assuntos
Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Proteínas Cromossômicas não Histona/genética , Reparo do DNA , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Teste de Complementação Genética , Substâncias Macromoleculares , Dados de Sequência Molecular , Peso Molecular , Mutagênese Sítio-Dirigida , Fenótipo , Schizosaccharomyces/genética , Homologia de Sequência de AminoácidosRESUMO
Nucleotide excision repair (NER) removes damage from DNA in a tightly regulated multiprotein process. Defects in NER result in three different human disorders, xeroderma pigmentosum (XP), trichothiodystrophy (TTD) and Cockayne syndrome (CS). Two cases with the combined features of XP and CS have been assigned to the XP-D complementation group. Despite their extreme UV sensitivity, these cells appeared to incise their DNA as efficiently as normal cells in response to UV damage. These incisions were, however, uncoupled from the rest of the repair process. Using cell-free extracts, we were unable to detect any incision activity in the neighbourhood of the damage. When irradiated plasmids were introduced into unirradiated XP-D/CS cells, the ectopically introduced damage triggered the induction of breaks in the undamaged genomic DNA. XP-D/CS cells thus have a unique response to sensing UV damage, which results in the introduction of breaks into the DNA at sites distant from the damage. We propose that it is these spurious breaks that are responsible for the extreme UV sensitivity of these cells.
