Studies On Regulatory Function Of Orc1/Cdc6 Of Sulfolobus Solfataricus During DNA Replication Initiation

DNA replication is the most essential contents on the life and a complex molecular process involving the combined efforts of dozens of proteins that ensures the precise and timely duplication of the genetic information.Archaea is the third domain of life that is different from the eubacteria and eukaryotes.Although recent studies reveal that archaeal DNA replication appears to be similar to that of eukaryotes,it is a simpler version of it. Thus,the study of archaeal DNA replication could provide important clues for elucidating the complex replication mechanism of higher eukaryotes.Cdc6 and MCM are two such proteins involved in the initiation of eukaryotic DNA replication.It has been reported that the circular chromosome of hyperthermophilic crenarchaeon Sulfolobus solfataricus contains three active origins of replication and its genome encodes three eukaryote-like Cdc6/Orc1 homologues(SsoCdc6-1,-2 and-3) and one MCM-like helicases(SsoMCM).Although S.solfataricus has the huge potential to become a model system to understand the central mechanism of eukaryotic DNA replication,it is unclear now how to the replication initiation proteins intervene the molecular details such as firing of duplex DNA and the forming of replication initiation complex in archaea and eukaryotes.This thesis designed three groups of specific DNA substrates with forked and blunt structure based on the structure characteristic of S. solfataricus oriC1 and oriC2.Using the standard electrophoretic mobility shift assay (EMSA),bacterial two-hybrid assays and Pull-down/western blot,we systematically studied the physical and functional interactions between three Orc1/Cdc6 proteins of S. solfataricus,between three Orc1/Cdc6 proteins and MCM,polB1,SSB of S.solfataricus. The idiographic research results have four aspects and are as followed:1.SsoCdc6 proteins showed a significant difference on the binding to DNA molecules from the origin.Both SsoCdc6-1 and SsoCdc6-3 bind the DNA molecular from origin,not only in structure-specific but also in sequence-specific way.SsoCdc6-1 had the higher DNA-binding activity to the DNA from oriC1,with the higher DNA-binding activity to the blunt DNA than the forked one.In contrast,SsoCdc6-3 had the higher DNA-binding activity to the DNA substrates from oriC2,with the lower DNA-binding activity on the blunt DNA than the forked one at the lower concentration and no obvious difference on the blunt and forked DNA at the higher concentration.SsoCdc6-2 could bind to the origin DNA without obvious sequence-specific on three groups of DNA substrates,with the higher DNA-binding activity to the forked DNA than the blunt one.In addition,their C-terminal WH domain deleted proteins showed the DNA-binding activities in vitro using EMSA although obviously much weaker than the full-length proteins.We speculated that not only the C-terminal WH domain of SsoCdc6 protein,but their N-terminal domain and the DNA sequence have been suggested to be responsible for their DNA-binding ability.2.N-terminal catalytic region of three SsoCdc6 proteins was involved in their physical and functional interaction that could regulate their origin DNA-binding activities: Using bacterial two-hybrid assays and Pull-down/western blot,we found that three SsoCdc6 proteins could physically interact by their N-terminal domain.By using EMSA, we found that SsoCdc6-1 stimulated DNA-binding activities of SsoCdc6-3,and simultaneously formed the big complex of SsoCdc6-1/DNA/SsoCdc6-3;In contrast,both SsoCdc6-3 and SsoCdc6-1 inhibited DNA-binding activities of SsoCdc6-2;Furthermore, SsoCdc6-1 can form the big complex of SsoCdc6-1/DNA/SsoCdc6-2 with SsoCdc6-2. These results suggest that these proteins may functionally interact and regulate their assembly to the origin,collectively contribute to DNA replication initiation as the part of Pre-RC(pre-replication complex).3.Three SsoCdc6 proteins could physically interact with SsoMCM by different domain and these physical interactions play important roles in regulating the binding of SsoMCM to origin DNA:SsoCdc6-2 physically interacted with SsoMCM by its C-terminal domain,and stimulated the loading of SsoMCM on origin DNA.On the contrary,SsoCdc6-1 and SsoCdc6-3 physical interacted with SsoMCM protein by their N-terminal portion and inhibited the loading of SsoMCM on the origin DNA.Although three SsoCdc6 proteins could interact with SsoMCM,SsoCdc6-2 exhibited tighter binding compared to SsoCdc6-1 or-3,which suggesting a role for SsoCdc6-2 in helicase loading.4.We detected the physical interaction between S.solfataricus Cdc6,polB1,MCM and SSB using bacterial two-hybrid assay,and the physical interaction were found between SsoCdc6-1â–³C and SsopolB1,SsoCdc6-3 and SsopolB1,SsoCdc6-2 and SsoSSB,SsoCdc6-3 and SsoSSB,SsoSSB and SsoMCM,SsopolB1 and SsoMCM, SsopolB1 and SsoSSB. Summarizing these results of this thesis,we propose that although there might be separate roles for three S.solfataricu Cdc6 proteins during the early events of DNA replication initiation,they might collectively contribute to positively and negatively regulation of DNA replication initiation in the archaeon species.The genes encode SsoCdc6-1 and-3 lies the upstream of origin,thus,SsoCdc6-1 and-3 might be responsible for the specific recognition to origin and are the homologues of eukaryotic ORC and bacterial DnaA.The gene encodes SsoCdc6-2 lies the site near the gene encodes SsoMCM and faraway any of three origins,thus,SsoCdc6-2 might be responsible for the loading of SsoMCM helicase on the origin and is the homologues of eukaryotic Cdc6 and bacterial DnaC.The mentioned important conclusions have provided the important clues for the cognition of DNA replication initiation mechanism in Eukarya and Archaea.