Emmanuelle Delagoutte

A General Model for Nucleic Acid Helicases and Their “Coupling” within Macromolecular Machines

In this article we present a general framework that can highly processive, efficient, specific for the type (DNA be used to describe the molecular mechanisms whereby or RNA) of ssNA substrate (lattice) to which they bind, ATP-driven helicases separate and rearrange the com- and directional (59!39 or 39!59) in their movements plementary strands of double helical nucleic acids. This along the target ssNA lattice (for a general review of framework also permits us to consider how these heli- helicase mechanisms see Lohman and Bjornson, 1996). cases might be functionally coupled to other compo- The processivity of a helicase at a given lattice (e.g., nents within the macromolecular machines that carry template) position is defined as the probability that the out physiological processes. We then proceed to define helicase at that position will continue to translocate forparameters that can be used to quantify helicase func- ward by one step along the NA substrate, divided by tion and derive simple thermodynamic equations that the probability that the helicase will dissociate from the describe helicase reactions in isolation and in coupled substrate lattice at that position. The processivity of a systems. helicase is often regulated by additional protein compoAspects of the molecular mechanisms of helicase nents or “coupling” factors, which may interact with the function are then developed using known systems of helicase either directly, or indirectly via the nucleic acid increasing complexity. We begin by considering simple components of the system (see Table 1 and below). DNA “melting proteins” that can—under some condi- Such processivity coupling factors can be operationally tions—open DNA without binding or hydrolyzing ATP. defined as components that interact functionally with We then discuss the cargo-carrying molecular motors the helicase to “trap” intermediate ssNA reaction prodthat, like helicases, use the chemical free energy of ATP ucts of the dsNA opening reaction and facilitate their hydrolysis to translocate directionally along specific cy- subsequent use (e.g., as an ssNA template) by the macromolecular machine within which the helicase operates toplasmic “tracks” but do not, of course, “open” the

Single-stranded DNA-binding Protein in Vitro Eliminates the Orientation-dependent Impediment to Polymerase Passage on CAG/CTG Repeats

Small insertions and deletions of trinucleotide repeats (TNRs) can occur by polymerase slippage and hairpin formation on either template or newly synthesized strands during replication. Although not predicted by a slippage model, deletions occur preferentially when 5′-CTG is in the lagging strand template and are highly favored over insertion events in rapidly replicating cells. The mechanism for the deletion bias and the orientation dependence of TNR instability is poorly understood. We report here that there is an orientation-dependent impediment to polymerase progression on 5′-CAG and 5′-CTG repeats that can be relieved by the binding of single-stranded DNA-binding protein. The block depends on the primary sequence of the TNR but does not correlate with the thermodynamic stability of hairpins. The orientation-dependent block of polymerase passage is the strongest when 5′-CAG is the template. We propose a “template-push” model in which the slow speed of DNA polymerase across the 5′-CAG leading strand template creates a threat to helicase-polymerase coupling. To prevent uncoupling, the TNR template is pushed out and by-passed. Hairpins do not cause the block, but appear to occur as a consequence of polymerase pass-over.

5'CAG and 5'CTG Repeats Create Differential Impediment to the Progression of a Minimal Reconstituted T4 Replisome Depending on the Concentration of dNTPs.

Instability of repetitive sequences originates from strand misalignment during repair or replicative DNA synthesis. To investigate the activity of reconstituted T4 replisomes across trinucleotide repeats (TNRs) during leading strand DNA synthesis, we developed a method to build replication miniforks containing a TNR unit of defined sequence and length. Each minifork consists of three strands, primer, leading strand template, and lagging strand template with a 5′ single-stranded (ss) tail. Each strand is prepared independently, and the minifork is assembled by hybridization of the three strands. Using these miniforks and a minimal reconstituted T4 replisome, we show that during leading strand DNA synthesis, the dNTP concentration dictates which strand of the structure-forming 5′CAG/5′CTG repeat creates the strongest impediment to the minimal replication complex. We discuss this result in the light of the known fluctuation of dNTP concentration during the cell cycle and cell growth and the known concentration balance among individual dNTPs.