The mechano-chemistry of a monomeric reverse transcriptase

Abstract

Retroviral reverse transcriptase catalyses the synthesis of an integration-competent dsDNA molecule, using as a substrate the viral RNA. Using optical tweezers, we follow the Murine Leukemia Virus reverse transcriptase as it performs strand-displacement polymerization on a template under mechanical force. Our results indicate that reverse transcriptase functions as a Brownian ratchet, with dNTP binding as the rectifying reaction of the ratchet. We also found that reverse transcriptase is a relatively passive enzyme, able to polymerize on structured templates by exploiting their thermal breathing. Finally, our results indicate that the enzyme enters the recently characterized backtracking state from the pre-translocation complex.

Synergy between RecBCD subunits is essential for efficient DNA unwinding

“…Thus, we used a different experimental geometry (Figure 3a), as was previously used for other helicases and polymerases (see for example Refs. (Dumont et al, 2006; Johnson et al, 2007; Lionnet et al, 2007; Malik et al, 2017b; Malik et al, 2017a; Manosas et al, 2010; Manosas et al, 2012; Manosas et al, 2013; Morin et al, 2012; Qi et al, 2013)). Here, a DNA hairpin is held under tension between two handles attached to beads trapped in optical tweezers.…”

“…In addition, for a processive helicase performing multiple enzymatic cycles without dissociation, the complete kinetic cycle must include also a translocation step, at which the helicase moves forward by a single step (Rn→Rn+1, where n denotes the number of steps carried out by the helicase on the DNA substrate). To characterize how this translocation step is incorporated into the enzyme’s ATPase cycle, two questions need to be addressed (Malik et al, 2017a): First, it is necessary to determine the location of the translocation step within the chemical steps comprising the ATPase cycle. For instance, the translocation step for the XPD helicase takes place after ATP binding (Qi et al, 2013).…”

“…However, the force on the tether affects the helicase velocity by modulating the stability of the unwinding fork: since translocation depends on the existence of an open fork, and DNA breathing fluctuations are very fast (Betterton and Jülicher, 2005), the open fork can be considered as a 'substrate' of the translocation reaction. Therefore, a step that involves forward translocation will be modulated by the factorPopen, equal to the probability of finding an open fork that is at least the size of the helicase step (Malik et al, 2017a). Popen is a function of the inherent stability of the DNA fork, a potential destabilization energy provided by the helicase, and the destabilization by the applied force.…”

Decoupling conservative forces and hydrodynamic interactions between optically trapped spheres

“…We follow the fluctuations of two identical 2-μm silica beads [ Fig. 1(a)], using a dual-trap optical tweezers setup as previously described [37,38], but using two separate lasers to minimize interference at short distances. Briefly, the collimated beams (w 0 = 4 mm) from two fiber-coupled lasers (852.2 and 855.2 nm; TA PRO, Toptica) were directed to two separate mirrors, one of which is mounted on a nanometer scale mirror mount (Nano-MTA, Mad City Labs), and combined with a polarizing beam splitter (PBS).…”

Displacement of Slow-Turnover DNA Glycosylases by Molecular Traffic on DNA

“…However, DNA polymerases varied in their ability to elongate the primer into the footprint of the cross-linked protein, some of them being able to proceed to the very point of the cross-link. This indicates that translocation of an elongating polymerase, which uses the energy of dNTP hydrolysis to move ahead [75,76], may generate enough force to distort the cross-linked protein globule. Would it be sufficient to displace a non-covalently bound protein?…”