Studies On The Nuclear Exporting Mechanism Of DNA Helicase RecQL4, And A Novel Method For Isolation And Purification Of Mitochondria

RecQL4, one of the five human RecQ helicases, is crucial for genomic stability and RecQL4when mutated leads to premature aging phenotypes in humans. Mutations in RecQL4result in Rothmund-Thomson syndrome (RTS), RAPADILINO and Baller-Gerold (BGS) syndromes. RecQL4structurally differs from other human RecQ helicases by having nuclear localization and retention domains at the N-terminus as well as a replication-associated domain homologous to yeast Sld2which functions in replisome assembly. Unlike other human RecQ helicases, RecQL4is localized to both the nucleus and the cytoplasm. While the nuclear localization signal (NLS) and the retention domain at the N-terminus are responsible for the nuclear localization of RecQL4, the signal for its cytoplasmic localization is essentially unknown. We have studied the potential mechanisms responsible for the nuclear exporting of RecQL4protein including ubiquitination-and nuclear exporting signal-mediated signalings.To facilitate the research in the regulation of mitochondrial function, we have established a convenient yet powerful approach for isolating high purity mitochondria component, whereas the similar approach is not commercially available at present. We have used Flag-tagged TOM complex, the translocase of the outer mitochondrial membrane to establish the stable cell lines expressing Flag-TOM20and Flag-TOM22, respectively. The mitochondrial component can be pulled down and purified by Flag antibody-conjugated beads from cell lysates.