RESUMEN
Early detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been proven crucial during the efforts to mitigate the effects of the COVID-19 pandemic. Several diagnostic methods have emerged in the past few months, each with different shortcomings and limitations. The current gold standard, RT-qPCR using fluorescent probes, relies on demanding equipment requirements plus the high costs of the probes and specific reaction mixes. To broaden the possibilities of reagents and thermocyclers that could be allocated towards this task, we have optimized an alternative strategy for RT-qPCR diagnosis. This is based on a widely used DNA-intercalating dye and can be implemented with several different qPCR reagents and instruments. Remarkably, the proposed qPCR method performs similarly to the broadly used TaqMan-based detection, in terms of specificity and sensitivity, thus representing a reliable tool. We think that, through enabling the use of vast range of thermocycler models and laboratory facilities for SARS-CoV-2 diagnosis, the alternative proposed here can increase dramatically the testing capability, especially in countries with limited access to costly technology and reagents.
Asunto(s)
Benzotiazoles/química , Prueba de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Diaminas/química , Sustancias Intercalantes/química , Quinolinas/química , ARN Viral/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , SARS-CoV-2/genética , COVID-19/virología , Prueba de Ácido Nucleico para COVID-19/normas , ADN/análisis , ADN/biosíntesis , Cartilla de ADN/química , Cartilla de ADN/metabolismo , Humanos , Nasofaringe/virología , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Sensibilidad y EspecificidadRESUMEN
Transcription-coupled DNA repair (TCR) removes bulky DNA lesions impeding RNA polymerase II (RNAPII) transcription. Recent studies have outlined the stepwise assembly of TCR factors CSB, CSA, UVSSA, and TFIIH around lesion-stalled RNAPII. However, the mechanism and factors required for the transition to downstream repair steps, including RNAPII removal to provide repair proteins access to the DNA lesion, remain unclear. Here, we identify STK19 as a new TCR factor facilitating this transition. Loss of STK19 does not impact initial TCR complex assembly or RNAPII ubiquitylation but delays lesion-stalled RNAPII clearance, thereby interfering with the downstream repair reaction. Cryo-EM and mutational analysis reveal that STK19 associates with the TCR complex, positioning itself between RNAPII, UVSSA, and CSA. The structural insights and molecular modeling suggest that STK19 positions the ATPase subunits of TFIIH onto DNA in front of RNAPII. Together, these findings provide new insights into the factors and mechanisms required for TCR.