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.
Highlights
Replicative helicases and DNA polymerases (DNAPs) are not efficient at unwinding the duplex DNA when they are working independently
We prepared a set of replication forks (Supplementary file 1-Table 1) where the sequence of the 40-base pair duplex was engineered to contain 20–65% GC content to study the effect of increasing resistance to movement on DNA-unwinding rates
The fluorescence intensity of fluorescein-labeled lagging strand is quenched by BHQ1 when the DNA is duplexed, but when the lagging strand is unwound by T7 DNAP + E. coli single-stranded DNA-binding protein (SSB) the fluorescence intensity increases (Figure 1B)
Summary
Replicative helicases and DNA polymerases (DNAPs) are not efficient at unwinding the duplex DNA when they are working independently. The unwinding rates are slower than their translocation rates on single-stranded (ss) DNA and slower than the rates of DNA replication (Kim et al, 1996; Delagoutte and von Hippel, 2001; Galletto et al, 2004; Stano et al, 2005; Lionnet et al, 2007; Donmez and Patel, 2008). In the presence of an actively synthesizing replicative DNAP, the unwinding rates of the helicase become fast and GC independent (Kim et al, 1996; Delagoutte and von Hippel, 2001; Stano et al, 2005; Manosas et al, 2012b; Pandey and Patel, 2014). Identified in prokaryotic systems (T7, T4 bacteriophage, Escherichia coli), this functional coupling between the helicase and DNAP is found in eukaryotic replication systems (Kang et al, 2012)
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