A Study On The Interactions Between Eukaryote-like Orc1/Cdc6 Proteins And Origins Of DNA Replication In The Hyperthermophilic Archaeon

The hyperthermophilic archaeon Sulfolobus solfataricus contains three active origins of replication and three eukaryote-like Orc1/Cdc6 proteins called SsoCdc6-1,SsoCdc6-2 and SsoCdc6-3 proteins.Therefore it has the potential to become a powerful model system to understand the central mechanisms of eukaryotic DNA replication.However,it is not known if these SsoCdc6 proteins can functionally interact and collectively contribute to DNA replication initiation.Using purified SsoCdc6 proteins and their C-terminal deleted forms,we have comparatively studied the interactions between these proteins and two origins DNA in vitro.Specially,we have first systematically analyzed the mutual regulation on their activities of binding to origin DNA.In the current work,we have obtained these results as follows:1.Escherichia coli strains were constructed to express proteins SsoCdc6-2 and its C-terminal deleted form SsoCdc6-2 A C,respectively.Then,three SsoCdc6 proteins and their C-terminal deleted forms SsoCdc6 A C were purified using Ni-NTA agarose after heat treatment,and these purified proteins were greater than 99%pure.According to the structural characters of two origins of the archaeon Sulfolobus solfataricus,we have designed three groups of origin-specific DNA substrates,and each contains both the forked and the blunt DNA substrates.These DNA substrates contain overlapped binding sites of SsoCdc6 proteins.2.Using these origin-specific DNA substrates,SsoCdc6-1 was demonstrated to stimulate DNA-binding activities of both SsoCdc6-2 and SsoCdc6-3.In contrast, SsoCdc6-3 inhibited that of SsoCdc6-1.Moreover,these regulatory functions were differentially affected by their C-terminal domains of the SsoCdc6 proteins.Thus,three SsoCdc6 proteins positively or negatively modulated their DNA-binding activities each other on the duplex DNA substrates.3.The following physical interactions were detected by pull-down/western blot assays:SsoCdc6-1/SsoCdc6-3,SsoCdc6-1â–³C/SsoCdc6-3,SsoCdc6-1/SsoCdc6-2, SsoCdc6-1â–³C/SsoCdc6-2,SsoCdc6-2/SsoCdc6-3,SsoCdc6-2/SsoCdc6-3â–³C.In addition,the interactions between SsoMCM and SsoCdc6 were confirmed by bacterial two-hybrid.4.Escherichia coli strain was constructed to express SsoCdc6-2 Walker A mutant (SsoCdc6-2KA protein).After optimizing the expression condition,we obtained the recombinant protein.In the EMSA,SsoCdc6-2KA protein was also found to bind DNA substrates but it does not autophosphorylate.DNA replication is very complicated and needs the highly harmonious interactions between protein-protein,and protein-DNA.It is a current hotspot to study on DNA replication of hyperthermophilic Archaeon Sulfolobus solfataricus.We have first systematically illuminated the mechanisms on the functional interactions between the multiple archaeal DNA replication initiator Orc1/Cdc6.These results not only lay a basis for understanding the mechanism on DNA replication of the antiquity microorganism,but also give a clue for elucidating the complex DNA replication of Eukaryon.