The Interaction Between DNA Replication Initiators And DNA Polymerase In The Hyperthermophilic Archaeon And The Construction Of Archaeal ORFeome Cloning Library

Sulfolobus solfataricus P2 (Sso) and Sulfolobus tokodaii strain7 (Sto) are typical representatives of hyperthermophilic archaeon. They contain a deal of eukaryote-like genes involved in DNA replication and repair. They also have multiple replication origins in their chromosomes. Archaeal DNA replication machinery represents a core version of that found in eukaryotes. Thus it has become a powerful tool for addressing related mechanistic issues in eukaryotic cell-cycle control of replication.In this study, we found for the first time that three replication initiators (SsoCdc6-1, SsoCdc6-2 and SsoCdc6-3) physically and functionally interact with DNA polymerase B1 (SsoPolB1) in the Crenarchaeon S. solfataricus. The major results are as follows: 1) Using bacterial 2-hybrid technique and Pull-down/Western blotting assays, we showed that SsoCdc6-2 had stronger interaction with SsoPolB1 than SsoCdc6-1 and SsoCdc6-3. 2) SsoCdc6s were found to inhibit the polymerase activity of SsoPolB1, of which SsoCdc6-1 has the strongest inhibition. Both SsoCdc6-1 and SsoCdc6-2 significantly inhibited the nuclease activity of SsoPolB1, but SsoCdc6-3 didn’t have such activity. 3) SsoCdc6 proteins mutually regulated their interactions with SsoPolB1. SsoCdc6-1 could enhance the inhibition of SsoCdc6-2 on the activity of SsoPolB1. SsoCdc6-3 enhanced the inhibition of SsoCdc6-2 on the polymerase activity of SsoPolB1, but reduced that on the nuclease activity. On the contrary, SsoCdc6-3 reduced the inhibition of SsoCdc6-1 on both of polymerase and nuclease activities of SsoPolB1. 4) SsoCdc6s were found to regulate the DNA binding activities of SsoPolB1c467 and to enhance its polymerase activities.On the other hand, we have also constructed an ORFeome cloning library for S. tokodaii strain7. Using the bacterial 2-hybrid technique, we have studied the interactions between its DNA replication proteins. Based on a preliminary screening, we have confirmed some reported interactions (such as ST0469 and ST0942) . Meanwhile, we have also found some new interactions.In this study, we presented the first evidence on the interaction between Orc1/Cdc6 and DNA polymerase SsoPolB1 in an archaeon. Our present results, together with previous findings, indicated that archaeal Orc1/Cdc6 proteins could potentially play critical roles in the coordination of origin selection with cell-cycle control of replication. In addition, the archaeal ORPeome cloning library will lay the foundation for the construction of an archaeal replication regulation network.