Abstract
Replicative DNA helicases are ring-shaped hexamers that play an essential role in DNA synthesis by separating the two strands of chromosomal DNA to provide the single-stranded (ss) substrate for replicative polymerases. Biochemical and structural studies suggest that these helicases translocate along one strand of the duplex, which passes through and interacts with the central channel of these ring-shaped hexamers, and displace the complementary strand. A number of these helicases were shown to also encircle both strands simultaneously and then translocate along double-stranded (ds)DNA. In this report it is shown that the Schizosaccharomyces pombe Mcm4,6,7 complex and archaeal minichromosome maintenance (MCM) helicase from Methanothermobacter thermautotrophicus move along duplex DNA. These two helicases, however, differ in the substrate required to support dsDNA translocation. Although the S. pombe Mcm4,6,7 complex required a 3'-overhang ssDNA region to initiate its association with the duplex, the archaeal protein initiated its transit along dsDNA in the absence of a 3'-overhang region, as well. Furthermore, DNA substrates containing a streptavidin-biotin steric block inhibited the movement of the eukaryotic helicase along ss and dsDNAs but not of the archaeal enzyme. The M. thermautotrophicus MCM helicase, however, was shown to displace a streptavidin-biotin complex from ss, as well as dsDNAs. The possible roles of dsDNA translocation by the MCM proteins during the initiation and elongation phases of chromosomal replication are discussed.
Highlights
Biochemical studies of replicative helicases of bacteria, Archaea, Eukarya, viruses, and bacteriophages revealed that they all form ring-shaped hexamers that encircle and translocate along one strand of the duplex and displace the other strand, utilizing the energy derived from NTP hydrolysis to drive the reaction [1]
The minichromosome maintenance (MCM) complex is believed to function as the replicative helicase of Eukarya and Archaea, suggesting that it is the functional homologue of the bacterial DnaB protein [12,13,14]
It was shown that the MCM proteins preferentially bind to unreplicated DNA [27, 28] and that the complex is distributed over large regions of DNA surrounding origins [29]
Summary
Biochemical studies of replicative helicases of bacteria, Archaea, Eukarya, viruses, and bacteriophages revealed that they all form ring-shaped hexamers that encircle and translocate along one strand of the duplex and displace the other strand, utilizing the energy derived from NTP hydrolysis to drive the reaction [1]. Translocation, all replicative helicases bind ssDNA with high affinity, and their NTPase activity is dramatically stimulated in the presence of ssDNA [2,3,4,5] These proteins, bind dsDNA but with much lower affinity [1, 2, 5, 6]. High affinity dsDNA binding has been observed only for the viral encoded helicases, simian virus 40 large-T antigen [7] and the E1 protein from papilloma viruses [8]. Biochemical studies with the MCM proteins from Methanothermobacter thermautotrophicus (mt) [4, 23,24,25] and Sulfolobus solfataricus [26] revealed that these enzymes possess biochemical properties similar to those of the eukaryotic Mcm complex including 3Ј 3 5Ј helicase, ssDNA binding, and DNA-dependent ATPase activities.
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