Characterization of Schizosaccharomyces pombe Hus1: a PCNA-Related Protein That Associates with Rad1 and Rad9.

Hus1 is one of six checkpoint Rad proteins required for all Schizosaccharomyces pombe DNA integrity checkpoints. MYC-tagged Hus1 reveals four discrete forms. The main form, Hus1-B, participates in a protein complex with Rad9 and Rad1, consistent with reports that Rad1-Hus1 immunoprecipitation is dependent on the rad9+ locus. A small proportion of Hus1-B is intrinsically phosphorylated in undamaged cells and more becomes phosphorylated after irradiation. Hus1-B phosphorylation is not increased in cells blocked in early S phase with hydroxyurea unless exposure is prolonged. The Rad1-Rad9-Hus1-B complex is readily detectable, but upon cofractionation of soluble extracts, the majority of each protein is not present in this complex. Indirect immunofluorescence demonstrates that Hus1 is nuclear and that this localization depends on Rad17. We show that Rad17 defines a distinct protein complex in soluble extracts that is separate from Rad1, Rad9, and Hus1. However, two-hybrid interaction, in vitro association and in vivo overexpression experiments suggest a transient interaction between Rad1 and Rad17

Clinical characterization and the mutation spectrum in Swedish adenomatous polyposis families

<p>Abstract</p> <p>Background</p> <p>The dominantly inherited condition familial adenomatous polyposis (FAP) is caused by germline mutations in the <it>APC </it>gene. Finding the causative mutations has great implications for the families. Correlating the genotypes to the phenotypes could help to improve the diagnosis and follow-up of patients.</p> <p>Methods</p> <p>Mutation screening of <it>APC </it>and the clinical characterization of 96 unrelated FAP patients from the Swedish Polyposis Registry was performed. In addition to generally used mutation screening methods, analyses of splicing-affecting mutations and investigations of the presence of low-frequency mutation alleles, indicating mosaics, have been performed, as well as quantitative real-time polymerase chain reaction to detect lowered expression of <it>APC</it>.</p> <p>Results</p> <p>Sixty-one different <it>APC </it>mutations in 81 of the 96 families were identified and 27 of those are novel. We have previously shown that 6 of the 96 patients carried biallelic <it>MUTYH </it>mutations. The 9 mutation-negative cases all display an attenuated or atypical phenotype. Probands with a genotype (codon 1250–1464) predicting a severe phenotype had a median age at diagnosis of 21.8 (range, 11–49) years compared with 34.4 (range, 14–57) years among those with mutations outside this region (<it>P </it>< 0.017). Dense polyposis (> 1000) occurred in 75% of the probands with a severe phenotype compared with 30% in those with mutations outside this region. The morbidity in colorectal cancer among probands was 25% at a mean age of 37.5 years and 29% at a mean age of 46.6 years.</p> <p>Conclusion</p> <p>Using a variety of mutation-detection techniques, we have achieved a 100% detection frequency in classical FAP. Probands with <it>APC </it>mutations outside codon 1250–1464, although exhibiting a less-severe phenotype, are at high risk of having a colorectal cancer at diagnosis indicating that age at diagnosis is as important as the severity of the disease for colorectal cancer morbidity.</p