Double hexamer disruption and biochemical activities of Methanobacterium thermoautotrophicum MCM.
Methanobacterium thermoautotrophicum MCM (mtMCM) is a helicase required for DNA replication. Previous electron microscopy studies have shown mtMCM in several oligomeric forms. However, biochemical studies suggest that mtMCM is a dodecamer, likely a double hexamer (dHex). The crystal structure of the N-terminal fragment of mtMCM reveals a stable dHex architecture. To further confirm that the dHex is not an artifact of crystal packing of two hexamers, we investigated the relevance of the dHex by disrupting the hexamer-hexamer interactions seen in the crystal structure via site-directed mutagenesis and examining various biochemical activities of the mutants in vitro. Using a combination of biochemical and structural assays, we demonstrated that changing arginine to alanine at amino acid position 161 or the insertion of a six-aminoacid peptide at the hexamer-hexamer interface disrupted dHex formation and produced stable single hexamers (sHex). Furthermore, we showed that the sHex mutants retained wild-type level of ATPase and DNA binding activities but had decreased helicase activity when compared with the wild type dHex protein. These biochemical properties of mtMCM are reminiscent of those of SV40 large T antigen, suggesting that the dHex form of mtMCM may be the active helicase for DNA unwinding during the bidirectional DNA replication.
Fletcher
Ryan J
RJ
Molecular and Computational Biology, University of Southern California, Los Angeles, California 90089, USA.
Shen
Jingping
J
Gómez-Llorente
Yacob
Y
Martín
Carmen San
CS
Carazo
José M
JM
Chen
Xiaojiang S
XS
D000255:Adenosine Triphosphate / Q000378:metabolism; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D001483:Base Sequence; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D017931:DNA Primers; D006868:Hydrolysis; D017014:Methanobacterium / Q000378:metabolism*; D008854:Microscopy, Electron; D008958:Models, Molecular; D009154:Mutation
D019843:Archaeal Proteins; D017931:DNA Primers; D000255:Adenosine Triphosphate; D004265:DNA Helicases; C403162:MCM protein, Methanobacterium thermoautotrophicum