The regulatory function of N-terminal AAA+ ATPase domain of eukaryote-like archaeal Orc1/Cdc6 protein during DNA replication initiation.
Archaeal replication machinery represents a core version of this in eukaryotes. The crenarchaeon Sulfolobus solfataricus has the potential to be a powerful model system to understand the central mechanism of eukaryotic DNA replication because it contains three active origins of replication and three eukaryote-like Orc1/Cdc6 proteins (SsoCdc6-1, SsoCdc6-2, and SsoCdc6-3). In this study, we investigate the DNA-binding activities of the N-terminal AAA+ ATPase domains of these Orc1/Cdc6 proteins, including their functional interactions with the other SsoCdc6 proteins, on duplex DNA substrates derived from the origins of S. solfataricus. We showed that the ATPase domain of SsoCdc6-2 retained to a great extent the origin DNA-binding activity, and likewise maintained its stimulating effect on SsoCdc6-3. Second, the ATPase domain of SsoCdc6-1, which also stimulated the DNA-binding ability of SsoCdc6-3, demonstrated a significantly improved DNA-binding activity at the forked substrate, but only showed a very weak ability towards the blunt DNA. Third, the ATPase domain of SsoCdc6-3, although having lost much of its DNA-binding activity from the origin, inhibited both SsoCdc6-1 and SsoCdc6-2. These imply that the N-terminal AAA+ ATPase domain of archaeal Orc1/Cdc6 protein could be differentially involved in origin recognition during DNA replication initiation even if lacking conventional C-terminal winged helix DNA-binding elements. Our findings further propose that conserved AAA+ ATPase domains of Orc1/Cdc6 proteins determine their defined and coordinated functions not only in the archaeon species but also in eukaryotes during the early events of DNA replication.
He
Zheng-Guo
ZG
National Key Laboratory of Agricultural Microbiology, Center for Proteomics Research, College of Life Science and Technology, Huazhong Agricultural University, 1 Sizisan Street, Wuhan 430070, China. hezhengguo@mail.hzau.edu.cn
Feng
Ying
Y
Wang
Jun
J
Jiang
Pei-Xia
PX
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D004261:DNA Replication / Q000235:genetics / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D005057:Eukaryotic Cells / Q000737:chemistry; D008969:Molecular Sequence Data; D051738:Origin Recognition Complex / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018741:Replication Origin / Q000235:genetics / Q000502:physiology; D048229:Sulfolobus solfataricus / Q000201:enzymology* / Q000235:genetics
D019843:Archaeal Proteins; D004268:DNA-Binding Proteins; D051738:Origin Recognition Complex; D000251:Adenosine Triphosphatases