RESUMO
Bloom syndrome (BS) is rare autosomal recessive disorder associated with chromosomal instability. The gene responsible for BS, BLM, encodes a protein belonging to the RecQ helicase family. Disruptions of the SGS1 gene of Saccharomyces cerevisiae, which encodes the RecQ helicase homologue in the budding yeast, causes accelerated aging, and this phenotype is enhanced by the disruption of MGS1, the budding yeast homologue for WRNIP1. To examine the functional relationship between RecQ and WRNIP1 in vertebrate cells, we generated and characterized wrnip1/blm cells derived from the chicken B-lymphocyte line DT40. wrnip1/blm cells showed an additive elevation of sister chromatid exchange (SCE), suggesting that both genes independently contribute to the suppression of excess SCE formation. The double mutants were more sensitive to DNA damage from camptothecin (CPT), but not to damage from methyl methanesulfonate, than either single mutant. This result suggests that WRNIP1 and BLM function independently to repair DNA or induce tolerance to the lesions induced by CPT.
Assuntos
Adenosina Trifosfatases/fisiologia , DNA Helicases/fisiologia , Proteínas de Ligação a DNA/fisiologia , Instabilidade Genômica , Adenosina Trifosfatases/genética , Animais , Síndrome de Bloom/genética , Células Cultivadas , Galinhas , Dano ao DNA , DNA Helicases/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Humanos , Mutação , RecQ Helicases , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Troca de Cromátide IrmãRESUMO
Werner was originally identified as a protein that interacts with the product of the Werner syndrome (WS) gene, WRN. To examine the function of the WRNIP1/WRN complex in cells, we generated knock-out cell lines that were deficient in either WRN (WRN(-/-)), WRNIP1 (WRNIP10(-/-/-)), or both (WRNIP1(-/-/-)/WRN(-/-)), using a chicken B lymphocyte cell line, DT40. WRNIP1(-/-/-)/WRN(-/-) DT40 cells grew at a similar rate as wild-type cells, but the rate of spontaneous sister-chromatid exchange was augmented compared to that of either of the single mutant cell lines. Moreover, while WRNIP1(-/-/-) and WRN(-/-) cells were moderately sensitive to camptothecin (CPT), double mutant cells showed a synergistic increase in CPT sensitivity. This suggested that WRNIP1 and WRN do not always function cooperatively to repair DNA lesions. The lack of a discernable functional interaction between WRNIP1 and WRN prompted us to reevaluate the nature of the physical interaction between these proteins. We found that MBP-tagged WRNIP1 interacted directly with WRN, and that the interaction was enhanced by the addition of ATP. Mutations in the Walker A motifs of the two proteins revealed that WRNIP1, but not WRN, must bind ATP before an efficient interaction can occur.
Assuntos
Proteínas de Transporte/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Proteínas de Transporte/genética , Linhagem Celular , Proliferação de Células , Galinhas , DNA Helicases/deficiência , DNA Helicases/genética , Primers do DNA/genética , Reparo do DNA , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Exodesoxirribonucleases , Humanos , Técnicas In Vitro , Camundongos , RecQ Helicases , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Troca de Cromátide Irmã , Técnicas do Sistema de Duplo-Híbrido , Síndrome de Werner/genética , Síndrome de Werner/metabolismo , Helicase da Síndrome de WernerRESUMO
The WRNIP1 protein interacts with WRN, the product of the causative gene for Werner syndrome. Mutation of the Saccharomyces cerevisiae gene MGS1, the yeast counterpart of WRNIP1, confers synthetic lethality with mutation of RAD18. To examine the functional relationship between WRNIP1 and Rad18 in higher eukaryotic cells, we generated WRNIP1-/-/-/RAD18-/- lines from chicken DT40 cells and compared them with single mutant cell lines. Unlike the corresponding yeast mutant, WRNIP1-/-/-/RAD18-/- cells are viable but grow more slowly than single mutants and wild type cells, and they show an additive or synergistic elevation in the frequency of sister chromatid exchanges. As reported, WRNIP1-/-/- cells and RAD18-/- cells are moderately and severely sensitive to camptothecin (CPT), respectively. Unexpectedly, the severe CPT sensitivity of RAD18-/- cells is slightly suppressed by disruption of the WRNIP1 gene.