Puri, T.; (2006) Characterisation of the human TIP48 and TIP49 AAA+ proteins and their complexes. Doctoral thesis , University of London.

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Abstract

TIP48 and TIP49 are two closely related proteins, which are highly conserved in all eukaryotes. They are crucial components of various nuclear complexes involved in transcription activation, DNA repair, and snoRNP accumulation and trafficking, but the function of TIP48 and TIP49 within these complexes is not understood. They are part of the AAA+ family of ATPases and show homology to the bacterial branch migration motor RuvB. However, reports about their enzymatic activities, such as DNA helicase activities, are contradictory and subject to speculations. In this study the enzymatic activities of recombinant TIP48, TIP49 and catalytically inactive mutants were examined. The proteins displayed very low ATPase activities, which were not stimulated in the presence of DNA, and no DNA helicase activities. Both TIP48 and TIP49 bound adenine nucleotides. However, whilst TIP48 formed oligomers, upon incubation with adenine nucleotides, TIP49 did not. An equimolar TIP48/TIP49 complex was assembled in solution and purified. The ATPase activity of the complex was significantly higher than the activities of the constituent proteins. Within this complex, both proteins bound and hydrolyzed ATP, as shown by using catalytically inactive mutant. The ATPase activity of the complex was not stimulated in the presence of DNA and no DNA helicase or branch migration activities were detected. The TIP48/TIP49 complex consists of two stacked hexameric rings with C6 symmetry as shown by electron microscopy. Structural differences between the top and bottom rings indicate that the complex may consist of two homohexamers. The localizations of TIP48 and TIP49 in cells were examined by immunofluoresence microscopy. Both proteins showed specific localisation to the mitotic spindle, which strongly suggests a novel role in mitosis. Surprisingly, a dramatic accumulation of TIP48 at the midbody was observed in the late stages of mitosis, which differed from that of TIP49. The observations suggest that TIP48 may have a separate function at this stage of the cell cycle.

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