Substrate Requirements for Duplex DNA Translocation by the Eukaryal and Archaeal Minichromosome Maintenance Helicases

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 streptavidinbiotin 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.

Biochemical Characterization of the Methanothermobacter thermautotrophicus Minichromosome Maintenance (MCM) Helicase N-terminal Domains

“…6B). It was hypothesized that dsDNA translocation by the archaeal, eukaryal, and bacterial helicases may play a regulatory role during the process of initiation of DNA replication (15,29,31,32).…”

“…Additional evidence of the impaired ability of the mutant proteins in efficient DNA translocation comes from the observation that the mutant proteins cannot displace streptavidin from biotinylated oligonucleotides, whereas the fulllength enzyme can (Ref. 15; data not shown).…”

“…It was recently demonstrated that the M. thermautotrophicus and eukaryotic MCM complexes are capable of translocating along duplex DNA (15,29). Thus, the mutant proteins that show some level of helicase activity (Fig.…”

“…4 and 5), it was anticipated that most of the mutants would have very low, if any, helicase activity. Therefore, the helicase and dsDNA translocation experiments were performed using a higher protein concentration (10 -50-fold) than that required to detect helicase activity with the full-length enzyme (14,15).…”

The DNA-unwinding mechanism of the ring helicase of bacteriophage T7

“…During DNA unwinding, T7 helicase ring surrounds the 5Ј strand and excludes the 3Ј strand from its central channel (31,32). No DNA unwinding is observed when the ring helicase surrounds both strands of the DNA (6,33,34).In this paper, we study the DNA-unwinding reaction catalyzed by T7 helicase at different dTTP concentrations. We measure the single-turnover kinetics of DNA unwinding and propose a modified stepping model that allows global fitting of the unwinding kinetics to obtain the helicase's kinetic step size, stepping rate, and processivity.…”