Uloga ATP-a u funkciji humanog proteina ORC4

Abstract

Humani protein ORC4, podjedinica ORC kompleksa (eng. Origin Recognition Complex), pripada familiji AAA+ adenozin-trifosfat (ATP)-aza povezanih sa različitim funkcijama u ćeliji. Za pripadnike ove familije proteina je karakteristično da je ATP neophodan za njihovu funkciju i da po vezivanju ATP-a prolaze kroz konformacionu promenu ili je indukuju u svojim partnerima. Humani protein ORC4 indukuje strukturne promene u supstratnim DNK, tako što promoviše renaturaciju i formiranje nekanonskih struktura, kao i konverziju jednolančanih oligonukleotida u višelančane strukture. ATP je neophodan za ove funkcije ORC4 proteina, što je pokazano analizom aktivnosti mutanta koji ne može da veže ATP, i koji nije aktivan u ovim reakcijama. Da bismo bliže ispitali ulogu ATP-a u aktivnosti ORC4 napravljen je protein sa mutacijom u domenu za hidrolizu ATP-a (Voker B motiv), koji ima očuvanu ATP-vezivnu aktivnost. Ovaj mutant je bio aktivan u reakcijama restrukturiranja DNK, tako da se može zaključiti da je uloga ATP-a strukturna, kao kofaktora, i da njegova hidroliza nije neophodna za funkciju humanog proteina ORC4.

Human origin recognition complex 4 (ORC4) protein, a subunit of the origin recognition complex, belongs to the AAA+ superfamily of adenosine triphosphate (ATP) ases. Proteins belonging to this family require ATP for their function and interactions with ATP lead to conformational changes in them or in their partners. Human ORC4 protein induces structural changes in DNA substrates, promoting renaturation and formation of non-canonical structures, as well as conversion of single-stranded into multi-stranded oligonucleotide structures. The aim of this study was to further investigate the role of ATP in the function of human ORC4 protein. For this purpose, a mutant in the conserved Walker B motif of ORC4, which is able to bind but not to hydrolyze ATP, was constructed and its activity in DNA restructuring reactions was investigated. The obtained results showed that ATP hydrolysis is not necessary for the function of human ORC4. It is proposed that ATP has a structural role as a cofactor in the function of human ORC4 as a DNA restructuring agent.