5-Methyl-cytosine stabilizes DNA but hinders DNA hybridization revealed by magnetic tweezers and simulations.

5-Methyl-cytosine (5mC) is one of the most important DNA modifications and plays versatile biological roles. It is well known that 5mC stabilizes DNA duplexes. However, it remains unclear how 5mC affects the kinetics of DNA melting and hybridization. Here, we studied the kinetics of unzipping and rezipping using a 502-bp DNA hairpin by single-molecule magnetic tweezers. Under constant loading rates, 5mC increases the unzipping force but counterintuitively decreases the rezipping force at various salt and temperature conditions. Under constant forces, the non-methylated DNA hops between metastable states during unzipping and rezipping, which implies low energy barriers. Surprisingly, the 5mC DNA can't rezip after fully unzipping unless much lower forces are applied, where it rezips stochastically in a one-step manner, which implies 5mC kinetically hinders DNA hybridization and high energy barriers in DNA hybridization. All-atom molecular dynamics simulations reveal that the 5mC kinetically hinders DNA hybridization due to steric effects rather than electrostatic effects caused by the additional methyl groups of cytosines. Considering the possible high speed of DNA unzipping and zipping during replication and transcription, our findings provide new insights into the biological roles of 5mC.

Single-molecule probing the duplex and G4 unwinding patterns of a RecD family helicase.

RecD family helicases play an important role in prokaryotic genome stability and serve as the structural models for studying superfamily 1B (SF1B) helicases. However, RecD-catalyzed duplex DNA unwinding behavior and the underlying mechanism are still elusive. RecD family helicases share a common proto-helicase with eukaryotic Pif1 family helicases, which are well known for their outstanding G-quadruplex (G4) unwinding ability. However, there are still controversial points as to whether and how RecD helicases unfold G4 structures. Here, single-molecule fluorescence resonance energy transfer (smFRET) and magnetic tweezers (MT) were used to study Deinococcus radiodurans RecD2 (DrRecD2)-mediated duplex DNA unwinding and resolution of G4 structures. A symmetric, repetitive unwinding phenomenon was observed on duplex DNA, revealed from the strand switch and translocation of one monomer. Furthermore, we found that DrRecD2 was able to unwind both parallel and antiparallel G4 structures without obvious topological preferences. Surprisingly, the unwinding properties of RecD on duplex and G4 DNA are different from those of Pif1. The findings provide an example, in which the patterns of two molecules derived from a common ancestor deviate during evolution, and they are of significance for understanding the unwinding mechanism and function of SF1B helicases.

A Universal Assay for Making DNA, RNA, and RNA-DNA Hybrid Configurations for Single-Molecule Manipulation in Two or Three Steps without Ligation.

Despite having a great variety of topologies, most DNA, RNA, and RNA-DNA hybrid (RDH) configurations for single-molecule manipulation are composed of several single-stranded (ss) DNA and ssRNA strands, with functional labels at the two ends for surface tethering. On this basis, we developed a simple, robust, and universal amplification-annealing (AA) assay for making all these configurations in two or three steps without inefficient digestion and ligation reactions. As examples, we made ssDNA, short ssDNA with double-stranded (ds) DNA handles, dsDNA with ssDNA handles, replication-fork shaped DNA/RDH/RNA, DNA holiday junction, three-site multiple-labeled and nicked DNA, torsion-constrained RNA/RDH, and short ssRNA with RDH handles. In addition to single-molecule manipulation techniques including optical tweezers, magnetic tweezers, and atomic force microscopy, these configurations can be applied in other surface-tethering techniques as well.

S-DNA and RecA/RAD51-Mediated Strand Exchange in Vitro.

S-DNA (stretched DNA) is an elongated base-paired DNA conformation under high tension. Because the RecA/Rad51 family DNA recombinases form helical filaments on DNA and mediate the formation of the DNA triplex (D-loop), in which the DNA is stretched, and because the extension of these nucleoprotein filaments is similar to the extension of S-DNA, S-DNA has long been hypothesized as a possible state of DNA that participants in RecA/Rad51-mediated DNA strand exchange in homologous recombination. Such a hypothesis, however, is still lacking direct experimental studies. In this work, we have studied the polymerization and strand exchange on S-DNA mediated by Escherichia coli RecA, human Rad51, and Saccharomyces cerevisiae Rad51 by single-molecule magnetic tweezers. We report that RecA/Rad51 polymerizes faster on S-DNA than on B-DNA with the same buffer conditions. Furthermore, the RecA/Rad51-mediated DNA triplex forms faster from S-DNA than from B-DNA together with the homologous single-stranded DNA. These results provide evidence that S-DNA can interact with RecA and Rad51 and shed light on the possible functions of S-DNA.