RESUMEN
BACKGROUND: Melasma is a therapeutically challenging hyperpigmented skin condition. Currently, there is a lack of in vivo observation regarding changes in melanin and dendritic melanocytes after laser treatment. OBJECTIVE: To investigate alterations in melanin and melanocytes in melasma before and after laser treatment using optical coherence tomography (OCT). METHODS: Eight female melasma patients were enrolled in Taiwan. Based on the baseline OCT scans, the patients were categorized into either epidermal-type or mixed-type melasma and were assigned different treatment protocols accordingly. Sequential OCT images were collected from melasma lesions and normal skin at baseline, Week 4 and Week 8. RESULTS: After 8 weeks of laser treatment, the mean Melasma Area Severity Index (MASI) score improved from 10.92 to 6.30. Results from OCT showed no significant changes in the normalized density, area, or intensity of melanin in both lesional and normal skin. At baseline, the mean length of dendritic melanocytes in the affected skin was 15% longer than those in normal skin; at Week 8, the mean length of lesional dendritic melanocytes became the same as those in normal skin. Additionally, the mean width of dendritic melanocytes decreased from being 4% wider to only 2% wider than those in normal skin. CONCLUSION: After 8 weeks of treatment, an improvement of MASI score was noted, mainly attributable to a reduction in lesional area. OCT showed no notable change regarding melanin, but a decrease in length and width of dendritic melanocytes was noted in the lesional skin of melasma patients.
RESUMEN
The Mcm2-7 complex is the catalytic core of the eukaryotic replicative helicase. Here, we identify a new role for this complex in maintaining genome integrity. Using both genetic and cytological approaches, we find that a specific mcm allele (mcm2DENQ) causes elevated genome instability that correlates with the appearance of numerous DNA-damage associated foci of γH2AX and Rad52. We further find that the triggering events for this genome instability are elevated levels of RNA:DNA hybrids and an altered DNA topological state, as over-expression of either RNaseH (an enzyme specific for degradation of RNA in RNA:DNA hybrids) or Topoisomerase 1 (an enzyme that relieves DNA supercoiling) can suppress the mcm2DENQ DNA-damage phenotype. Moreover, the observed DNA damage has several additional unusual properties, in that DNA damage foci appear only after S-phase, in G2/M, and are dependent upon progression into metaphase. In addition, we show that the resultant DNA damage is not due to spontaneous S-phase fork collapse. In total, these unusual mcm2DENQ phenotypes are markedly similar to those of a special previously-studied allele of the checkpoint sensor kinase ATR/MEC1, suggesting a possible regulatory interplay between Mcm2-7 and ATR during unchallenged growth. As RNA:DNA hybrids primarily result from transcription perturbations, we suggest that surveillance-mediated modulation of the Mcm2-7 activity plays an important role in preventing catastrophic conflicts between replication forks and transcription complexes. Possible relationships among these effects and the recently discovered role of Mcm2-7 in the DNA replication checkpoint induced by HU treatment are discussed.
Asunto(s)
Daño del ADN/genética , Replicación del ADN/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de Mantenimiento de Minicromosoma/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Saccharomyces cerevisiae/genética , Puntos de Control del Ciclo Celular/genética , ADN Helicasas/genética , Inestabilidad Genómica/genética , Mutación , Fosforilación , ARN/genética , Saccharomyces cerevisiae/genéticaRESUMEN
The DNA replication checkpoint (DRC) monitors and responds to stalled replication forks to prevent genomic instability. How core replication factors integrate into this phosphorylation cascade is incompletely understood. Here, through analysis of a unique mcm allele targeting a specific ATPase active site (mcm2DENQ), we show that the Mcm2-7 replicative helicase has a novel DRC function as part of the signal transduction cascade. This allele exhibits normal downstream mediator (Mrc1) phosphorylation, implying DRC sensor kinase activation. However, the mutant also exhibits defective effector kinase (Rad53) activation and classic DRC phenotypes. Our previous in vitro analysis showed that the mcm2DENQ mutation prevents a specific conformational change in the Mcm2-7 hexamer. We infer that this conformational change is required for its DRC role and propose that it allosterically facilitates Rad53 activation to ensure a replication-specific checkpoint response.
Asunto(s)
Replicación del ADN , ADN de Hongos/genética , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Dominio Catalítico , Proteínas de Ciclo Celular/metabolismo , Quinasa de Punto de Control 2/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Mantenimiento de Minicromosoma/química , Proteínas de Mantenimiento de Minicromosoma/genética , Mutación , Proteínas Nucleares/metabolismo , Multimerización de Proteína , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Transducción de SeñalRESUMEN
DNA damage activates the cell cycle checkpoint to regulate cell cycle progression. The checkpoint clamp (Rad9-Hus1-Rad1 complex) is recruited to damage sites, and is required for checkpoint activation. While functions of the checkpoint clamp in checkpoint activation have been well studied, its functions in DNA repair regulation remain elusive. Here we show that Rad9 is required for efficient homologous recombination (HR), and facilitates DNA-end resection. The role of Rad9 in homologous recombination is independent of its function in checkpoint activation, and this function is important for preventing alternative non-homologous end joining (altNHEJ). These findings reveal novel function of the checkpoint clamp in HR.