DNA helicases are enzymes capable of unwinding double-stranded DNA (dsDNA) to provide the single-stranded DNA (ssDNA) template required in many biological events, such as replication, recombination and repair. In that process they hydrolyse ATP to translocate along one strand of the double helix displacing the other. How exactly is this translocation coupled to unwinding is a much debated issue. In particular two models confront each other. A passive model whereby the translocation of the helicase simply traps unwinding fluctuations of the upstream double-stranded DNA (dsDNA) and an active mechanism whereby the interaction of the enzyme with the dsDNA is sufficient to destabilise it and eliminate its possible action as a "road block" on the progression of the enzyme. How exactly active or passive an enzyme is has rested on problematic estimations of the free energy of interaction of the enzyme with the DNA fork. We  propose to cut this Gordian knot by defining a passive enzyme as one whose rate is very sensitive to the DNA sequence and increases significantly by a force acting to unzip the two DNA strands. By contrast the unwinding rate of an active enzyme will be insensitive to the DNA sequence and unchanged (or even possibly decreased) by a force acting to separate the two strands. We have studied how these features characterize the different behaviors of various helicases: UvrD and RecQ which are exemples of active helicases and gp41 (the T4 replication helicase), which is a passive one.

To monitor in real time the activity of these helicases we have used two types of substrates, see Fig.1.

1) A nicked double stranded DNA unwound by a helicase at a tension larger than about 25pN to prevent rehybridization of the separated strands in the wake of the enzyme.

2) A single stranded DNA forming a hairpin under a tension smaller than about 12pN (at which force the two strands are unzipped by the stretching force).

Fig.1: Schematics of the two configurations used to study the action of helicases with a magnetic tweezers set-up. a) a hairpin DNA under a tension (F<12 pN) ; b) a  nicked DNA under a tension larger than 25pN to prevent rehybridization of the separated strands in the wake of the enzyme.

The typical extension signals that we observe are shown in Fig.2. They present an ATP dependent increase in the molecule's extension arising from the unwinding of the dsDNA followed by a rapid (ATP independent) decrease in extension resulting from the rehybridization of the two strands upon the enzyme falling off.  Sometimes one also observes a slow ATP dependent decrease in extension resulting from the rehybridization of the two strands in the wake of an enzyme advancing on ssDNA (either through strand switching or in the case of a hairpin DNA, if the enzyme progresses beyond the hairpin apex). 


Fig.2: Typical signals observed upon unwinding of a hairpin by the T4 helicase (gp41). a) as the enzyme unwinds the hairpin the molecule's extension increases. As the enzymes falls off the two strands rehybridize and the extension returns to its initial value. b) If the enzyme progresses on the hairpin pass its apex, the two strands can rehybridize it its wake, reforming the hairpin.

 The rate of unwinding is given by the slope of the rising part of the signal, the velocity of the enzyme on ssDNA is given by the slope of the ATP-dependent decreasing part of the signal. The processivity of the enzyme is given by the change in extension of the molecule, its on-time by the time span of the signal.


Coupling helicase unwinding activity and primase priming activity

DNA replication is a fundamental process of all living organisms.  It consists of duplicating the information stored in each strand of the genetic DNA in order to transfer it to the next generation. DNA replication is achieved by separating and duplicating the two complementary DNA strands to generate two identical copies. This process is carried out by a multiprotein complex called the replisome. First, enzymes known as helicases, unwind the DNA duplex generating two replication forks, one on each extreme of the denaturation bubble as the two DNA strands separate. DNA replication proceeds through the replisome, a complex formed by helicase, primase and two polymerases, among others. In each replication fork, DNA polymerases synthesize new DNA strands using the original DNA as a template. DNA polymerases can only synthesize nascent DNA in a given polarity (5′ to 3′ direction), starting from either a RNA or DNA primer. Therefore DNA synthesis occurs continuously in one strand (the so called leading strand), whereas the polymerase synthesizes DNA in short, approximately 1 kbp segments known as Okazaki fragments in the other strand (the so called lagging strand). Repetitive Okazaki fragments synthesis in the lagging strand is initiated by the synthesis of RNA primers by the primase.

Replisomes from different organisms can vary in size and complexity. For example, only four proteins are needed to assembly the T7 bacteriophage replisome, whereas in humans tens of proteins are required. Despite these differences, many components of the replisome are functionally and structurally conserved from organism to organism. Studies in prokaryotic and viral systems have strongly contributed to our present understanding of DNA replication.  Here, we investigate the replisome of the T4 bacteriophage, which is a model system to study DNA replication and is formed by eight different proteins that work in a very coordinated fashion to efficiently copy the genomic DNA.  

The coordination between the continuous leading strand DNA synthesis and the discontinuous lagging strand counterpart is regulated by a subassembly of the replisome called the primosome.  In the T4 system the primosome is formed by a hexameric helicase (gp41) that unwinds dsDNA and an oligomeric primase (gp61) that synthesizes RNA primers to initiate repetitive Okazaki fragment synthesis.  Three possible models have been suggested to explain how helicase is able to unwind dsDNA translocating 5′ to 3′ on the lagging strand while primase travels in the opposite direction (3′ to 5′) in order to synthesize an RNA primer. In the first model (pausing), the helicase temporarily pauses or stops translocating to allow for primer synthesis and then resumes unwinding the DNA; helicase pausing would necessitate the pausing of the entire replisome while a primer is being synthesized. In the second model (disassembly), one or more primase subunits dissociate from the helicase and remain behind to synthesize a primer while the helicase and any remaining primase subunits continue to translocate along the lagging strand. In this model, leading strand synthesis would continue uninterrupted; however, new primase subunits might need to be recruited for each cycle of Okazaki fragment synthesis. In the third model (DNA looping), the primosome remains intact and the DNA that is continuously unwound by the helicase during primer synthesis forms a loop which is released once the primer is transferred to the lagging strand polymerase.

In this work, the unwinding and priming activities of the bacteriophage T4 primosome have been investigated on DNA hairpins manipulated by a magnetic trap. We find that the T4 primosome continuously unwinds the DNA duplex while allowing for primer synthesis through a primosome disassembly mechanism or a DNA looping mechanism. Other proteins within the replisome control the partitioning of these two mechanisms disfavoring primosome disassembly thereby increasing primase processivity. In contrast priming in bacteriophage T7 may involve either discrete pausing [1] of the primosome or DNA looping [2] , and in Escherichia coli appears to be associated primarily with dissociation of the primase from the helicase. Thus nature appears to use several strategies to couple the disparate helicase and primase activities within primosomes.

Replisome assembly

Fig.3:  Schematic of replication process


Other references: 

[1] Lee J.B. et al. DNA primase acts as a molecular brake in DNA replication. Nature 439, 621-624 (2006).

[2] Pandey M. et al. Coordinating DNA replication by means of priming loop and differential synthesis rate. Nature 462, 940-943 (2009).