RESUMEN
BACKGROUND: The target substrates of DNA mismatch recognising factors MutSalpha (MSH2+MSH6) and MutSbeta (MSH2+MSH3) have already been widely researched. However, the extent of their functional redundancy and clinical substance remains unclear. Mismatch repair (MMR)-deficient tumours are strongly associated with microsatellite instability (MSI) and the degree and type of MSI seem to be dependent on the MMR gene affected, and is linked to its substrate specificities. Deficiency in MSH2 and MSH6 is associated with both mononucleotide and dinucleotide repeat instability. Although no pathogenic MSH3 mutations have been reported, its deficiency is also suggested to cause low dinucleotide repeat instability. METHODS: To assess the substrate specificities and functionality of MutSalpha and MutSbeta we performed an in vitro MMR assay using three substrate constructs, GT mismatch, 1 and 2 nucleotide insertion/deletion loops (IDLs) in three different cell lines. RESULTS: Our results show that though MutSalpha alone seems to be responsible for GT and IDL1 repair, MutSalpha and MutSbeta indeed have functional redundancy in IDL2 repair and in contrast with earlier studies, MutSbeta seems to exceed MutSalpha. CONCLUSION: The finding is clinically relevant because the strong role of MutSbeta in IDL2 repair indicates MSH3 deficiency in tumours with low dinucleotide and no mononucleotide repeat instability.
Asunto(s)
Reparación de la Incompatibilidad de ADN/genética , Proteínas de Unión al ADN/fisiología , Repeticiones de Dinucleótido/genética , Proteína 2 Homóloga a MutS/fisiología , Conformación de Ácido Nucleico , Animales , Células Cultivadas , Proteínas de Unión al ADN/genética , Células HCT116 , Células HeLa , Humanos , Mutación INDEL/genética , Proteína 2 Homóloga a MutS/genética , Proteína 3 Homóloga de MutS , Mutación Missense/fisiología , Spodoptera , Especificidad por SustratoRESUMEN
We describe an enzyme immunoassay for determination of total estriol in urine. Estriol covalently bound to horseradish peroxidase is used as tracer, and free and bound hormone are separated by precipitation with polyethylene glycol. The method can be used with either acid hydrolysis at 100 degrees C for 30 min or enzyme hydrolysis at 50 degrees C for 40 min; results by the former procedure are about 15% lower than results by the latter. Results were practically identical when we compared the enzyme immunoassay with a radioimmunoassay, using the same antiserum and method of hydrolysis. The day-to-day CV for three different concentrations was 10.7-12.0%, the within-series CV 6.6-8.6%. The additional time required for the enzyme reaction is compensated for by the rapid measurement of light absorbance. Thus this method is faster than radioimmunoassay when more than 25 samples are to be assayed.