DNA synthesis provides the driving force to accelerate DNA unwinding by a helicase

Department of Biochemistry, UMDNJ-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854, USA.
Nature (Impact Factor: 41.46). 06/2005; 435(7040):370-3. DOI: 10.1038/nature03615
Source: PubMed


Helicases are molecular motors that use the energy of nucleoside 5'-triphosphate (NTP) hydrolysis to translocate along a nucleic acid strand and catalyse reactions such as DNA unwinding. The ring-shaped helicase of bacteriophage T7 translocates along single-stranded (ss)DNA at a speed of 130 bases per second; however, T7 helicase slows down nearly tenfold when unwinding the strands of duplex DNA. Here, we report that T7 DNA polymerase, which is unable to catalyse strand displacement DNA synthesis by itself, can increase the unwinding rate to 114 base pairs per second, bringing the helicase up to similar speeds compared to its translocation along ssDNA. The helicase rate of stimulation depends upon the DNA synthesis rate and does not rely on specific interactions between T7 DNA polymerase and the carboxy-terminal residues of T7 helicase. Efficient duplex DNA synthesis is achieved only by the combined action of the helicase and polymerase. The strand displacement DNA synthesis by the DNA polymerase depends on the unwinding activity of the helicase, which provides ssDNA template. The rapid trapping of the ssDNA bases by the DNA synthesis activity of the polymerase in turn drives the helicase to move forward through duplex DNA at speeds similar to those observed along ssDNA.

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    • "The unwinding k cat (with SSB) is 160 bp/s and decreases minimally to 140 bp/s in the presence of T7 helicase, whereas the dNTPs K m decreases by 24-fold from ∼120 μM (with SSB) to 5 μM in the presence of T7 helicase (Figure 3G). The 5 μM dNTPs K m is slightly lower than the DNAP's dNTP K m on ssDNA template (∼10–20 μM) (Patel et al., 1991; Stano et al., 2005). Thus, T7 helicase stimulates T7 DNAP by promoting dNTP binding, and in the presence of helicase, T7 DNAP behaves like a motor translocating on ssDNA template. "
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    ABSTRACT: Leading strand DNA synthesis requires functional coupling between replicative helicase and DNA polymerase (DNAP) enzymes, but the structural and mechanistic basis of coupling is poorly understood. This study defines the precise positions of T7 helicase and T7 DNAP at the replication fork junction with single-base resolution to create a structural model that explains the mutual stimulation of activities. Our 2-aminopurine studies show that helicase and polymerase both participate in DNA melting, but each enzyme melts the junction base-pair partially. When combined, the junction base-pair is melted cooperatively provided the helicase is located one nucleotide ahead of the primer-end. The synergistic shift in equilibrium of junction base-pair melting by combined enzymes explains the cooperativity, wherein helicase stimulates the polymerase by promoting dNTP binding (decreasing dNTP Km), polymerase stimulates the helicase by increasing the unwinding rate-constant (kcat), consequently the combined enzymes unwind DNA with kinetic parameters resembling enzymes translocating on single-stranded DNA.
    eLife Sciences 05/2015; 4. DOI:10.7554/eLife.06562 · 9.32 Impact Factor
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    • "Even in situations where an isolated replicative helicase cannot efficiently unwind protein-bound duplex DNA, it must be borne in mind that such helicases function within the context of large macromolecular machines that have both a thermodynamic and a kinetic effect on helicase activity [17] [21]. These interactions amongst the helicase, polymerases, ssDNA binding proteins and clamp loaders accelerate DNA unwinding and enhance processivity in bacteria, bacteriophages and mitochondria [26] [27] [28] [29]. As an example, a single lac repressor–operator complex inhibits the E. coli replicative helicase DnaB in the absence of other components of the replisome [23] but this high-affinity protein–DNA complex is much less effective at inhibiting translocation of the E. coli replisome [30]. "
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    ABSTRACT: Complete, accurate duplication of the genetic material is a prerequisite for successful cell division. Achieving this accuracy is challenging since there are many barriers to replication forks that may cause failure to complete genome duplication or result in possibly catastrophic corruption of the genetic code. One of the most important types of replicative barriers are proteins bound to the template DNA, especially transcription complexes. Removal of these barriers demands energy input to not only separate the DNA strands but also to disrupt the multiple bonds between the protein and DNA. Replicative helicases that unwind the template DNA for polymerases at the fork can displace proteins bound to the template. However, even occasional failures in protein displacement by the replicative helicase could spell disaster. In such circumstances, failure to restart replication could result in incomplete genome duplication. Avoiding incomplete genome duplication via the repair and restart of blocked replication forks also challenges viability since the involvement of recombination enzymes is associated with the risk of genome rearrangements. Organisms have therefore evolved accessory replicative helicases that aid replication fork movement along protein-bound DNA. These helicases reduce the dangers associated with replication blockage by protein-DNA complexes, aiding clearance of blocks and resumption of replication by the same replisome thus circumventing the need for replication repair and restart. This review summarises recent work in bacteria and eukaryotes that has begun to delineate features of accessory replicative helicases and their importance in genome stability.
    Journal of Molecular Biology 10/2014; 426(24). DOI:10.1016/j.jmb.2014.10.001 · 4.33 Impact Factor
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    • "Unlike RNA polymerases, however, most replicative polymerases lack a mechanism to prevent reannealing of the unwound bases, a role fulfilled by the associated helicase that traps the complementary bases by translocating on the lagging strand. We propose that replisomes of bacteria, mitochondria, and phages where the helicase and polymerase are bound to opposite strands of the replication fork use this general mechanism to couple DNA unwinding to DNA synthesis (Dong et al., 1996; Korhonen et al., 2004; Manosas et al., 2012b; Patel et al., 2011b; Stano et al., 2005). However, eukaryotic replicative helicases such as MCM (mini chromosome maintenance) that unwind DNA in the 3 0 –5 0 direction are expected to bind to the same strand as the polymerase and work in a different manner. "
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    ABSTRACT: By simultaneously measuring DNA synthesis and dNTP hydrolysis, we show that T7 DNA polymerase and T7 gp4 helicase move in sync during leading-strand synthesis, taking one-nucleotide steps and hydrolyzing one dNTP per base-pair unwound/copied. The cooperative catalysis enables the helicase and polymerase to move at a uniformly fast rate without guanine:cytosine (GC) dependency or idling with futile NTP hydrolysis. We show that the helicase and polymerase are located close to the replication fork junction. This architecture enables the polymerase to use its strand-displacement synthesis to increase the unwinding rate, whereas the helicase aids this process by translocating along single-stranded DNA and trapping the unwound bases. Thus, in contrast to the helicase-only unwinding model, our results suggest a model in which the helicase and polymerase are moving in one-nucleotide steps, DNA synthesis drives fork unwinding, and a role of the helicase is to trap the unwound bases and prevent DNA reannealing.
    Cell Reports 03/2014; 6(6). DOI:10.1016/j.celrep.2014.02.025 · 8.36 Impact Factor
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