Duplex DNA Molecules Research Articles

Counting proteins bound to a single DNA molecule

Chromosomes contain DNA covered with proteins performing functions such as architectural organization and transcriptional regulation. The ability to count the number of proteins bound to various regions of the genome is essential for understanding both architectural and regulatory functions. We present a straightforward method of counting gfp-conjugated proteins bound to an individual duplex DNA molecule by calibrating to a commercially available fluorescence standard using wide-field fluorescence microscopy. We demonstrate our method using the E. coli nucleoid-associated protein Fis.

Investigating Deinococcus radiodurans RecA Protein Filament Formation on Double-Stranded DNA by a Real-Time Single-Molecule Approach

With the aid of an efficient, precise, and almost error-free DNA repair system, Deinococcus radiodurans can survive hundreds of double-strand breaks inflicted by high doses of irradiation or desiccation. RecA of D. radiodurans (DrRecA) plays a central role both in the early phase of repair by an extended synthesis-dependent strand annealing process and in the later more general homologous recombination phase. Both roles likely require DrRecA filament formation on duplex DNA. We have developed single-molecule tethered particle motion experiments to study the assembly dynamics of RecA proteins on individual duplex DNA molecules by observing changes in DNA tether length resulting from RecA binding. We demonstrate that DrRecA nucleation on double-stranded DNA is much faster than that of Escherichia coli RecA protein (EcRecA), but the extension is slower. This combination of attributes would tend to increase the number and decrease the length of DrRecA filaments relative to those of EcRecA, a feature that may reflect the requirement to repair hundreds of genomic double-strand breaks concurrently in irradiated Deinococcus cells.

Coupling dTTP Hydrolysis with DNA Unwinding by the DNA Helicase of Bacteriophage T7

The DNA helicase encoded by gene 4 of bacteriophage T7 assembles on single-stranded DNA as a hexamer of six identical subunits with the DNA passing through the center of the toroid. The helicase couples the hydrolysis of dTTP to unidirectional translocation on single-stranded DNA and the unwinding of duplex DNA. Phe(523), positioned in a β-hairpin loop at the subunit interface, plays a key role in coupling the hydrolysis of dTTP to DNA unwinding. Replacement of Phe(523) with alanine or valine abolishes the ability of the helicase to unwind DNA or allow T7 polymerase to mediate strand-displacement synthesis on duplex DNA. In vivo complementation studies reveal a requirement for a hydrophobic residue with long side chains at this position. In a crystal structure of T7 helicase, when a nucleotide is bound at a subunit interface, Phe(523) is buried within the interface. However, in the unbound state, it is more exposed on the outer surface of the helicase. This structural difference suggests that the β-hairpin bearing the Phe(523) may undergo a conformational change during nucleotide hydrolysis. We postulate that upon hydrolysis of dTTP, Phe(523) moves from within the subunit interface to a more exposed position where it contacts the displaced complementary strand and facilitates unwinding.

Orientation of Cyanine Fluorophores Terminally Attached to DNA via Long, Flexible Tethers

Cyanine fluorophores are commonly used in single-molecule FRET experiments with nucleic acids. We have previously shown that indocarbocyanine fluorophores attached to the 5′-termini of DNA and RNA via three-carbon atom linkers stack on the ends of the helix, orienting their transition moments. We now investigate the orientation of sulfoindocarbocyanine fluorophores tethered to the 5′-termini of DNA via 13-atom linkers. Fluorescence lifetime measurements of sulfoindocarbocyanine 3 attached to double-stranded DNA indicate that the fluorophore is extensively stacked onto the terminal basepair at 15°C, with properties that depend on the terminal sequence. In single molecules of duplex DNA, FRET efficiency between sulfoindocarbocyanine 3 and 5 attached in this manner is modulated with helix length, indicative of fluorophore orientation and consistent with stacked fluorophores that can undergo lateral motion. We conclude that terminal stacking is an intrinsic property of the cyanine fluorophores irrespective of the length of the tether and the presence or absence of sulfonyl groups. However, compared to short-tether indocarbocyanine, the mean rotational relationship between the two fluorophores is changed by ∼60° for the long-tether sulfoindocarbocyanine fluorophores. This is consistent with the transition moments becoming approximately aligned with the long axis of the terminal basepair for the long-linker species.

The Allure of Synthetic Biology

![Figure][1] CREDIT: ISTOCKPHOTO.COM Biologists have been manipulating genomes ever since Paul Berg first described a method to covalently join duplex DNA molecules in 1972. Despite key fundamental insights, a thriving biotechnology industry, and a growing number of medical applications, there have been limits to what has been possible. Now, synthetic biology goes beyond engineering individual genes to the construction of DNA-encoded circuits that can be programmed to control cell behavior. This emerging field brings together biologists, physicists, chemists, and engineers who seek both to understand life and to build new biological functions. For example, Harvard's George Church wants to redesign the genetic code (p. [1236][2]). The potential of synthetic biology has also attracted artists who want to critique it and make use of its techniques (p. [1242][3]), as well as do-it-yourself biologists, some of whom have set up community labs (p. [1240][4]). Nandagopal and Elowitz (p. [1244][5]) describe how building circuits and studying their behavior in cells can provide insight into biological design principles. Initially, the focus was on creating autonomous circuits, but recently there has been a move toward integration of endogenous and synthetic circuits. This can allow the “rewiring” of cellular circuitry to control biological processes—a goal not only of scientists who seek to understand these processes but also of biotechnicians and clinicians who would like to direct cell behavior to their advantage. Ruder, Lu, and Collins (p. [1248][6]) discuss specific constructs that highlight the potential for moving toward clinical applications. They envision synthetic circuits that detect unhealthy cellular phenotypes and take corrective action. Service (p. [1238][7]) covers progress in algal biofuels, for which synthetic biology has helped to make possible rapid improvements that may ultimately make algae a viable alterative fuel source. In these examples, the assembly of the synthetic systems is within an existing organism. Schwille (p. [1252][8]) paints a more radical view of synthetic biology, envisioning cells built entirely from synthetic modules. Here, the question is not how a biological system actually functions, but rather what minimal set of elements would support function. With the application of synthetic biology come ethical challenges. Erickson et al. (p. [1254][9]) present the industry point of view about the potential of synthetic biology and a possible regulatory framework. This special section, together with the three related research Reports and the profile in Science Careers, captures the range of expertise now entering this field and highlights how the construction of new biological systems might be harnessed to serve humanity. [1]: pending:yes [2]: http://www.sciencemag.org/content/333/6047/1236.full [3]: http://www.sciencemag.org/content/333/6047/1242.full [4]: http://www.sciencemag.org/content/333/6047/1240.full [5]: http://www.sciencemag.org/content/333/6047/1244.full [6]: http://www.sciencemag.org/content/333/6047/1248.full [7]: http://www.sciencemag.org/content/333/6047/1238.full [8]: http://www.sciencemag.org/content/333/6047/1252.full [9]: http://www.sciencemag.org/content/333/6047/1254.full

RecA K72R Filament Formation Defects Reveal an Oligomeric RecA Species Involved in Filament Extension

Using an ensemble approach, we demonstrate that an oligomeric RecA species is required for the extension phase of RecA filament formation. The RecA K72R mutant protein can bind but not hydrolyze ATP or dATP. When mixed with other RecA variants, RecA K72R causes a drop in the rate of ATP hydrolysis and has been used to study disassembly of hydrolysis-proficient RecA protein filaments. RecA K72R filaments do not form in the presence of ATP but do so when dATP is provided. We demonstrate that in the presence of ATP, RecA K72R is defective for extension of RecA filaments on DNA. This defect is partially rescued when the mutant protein is mixed with sufficient levels of wild type RecA protein. Functional extension complexes form most readily when wild type RecA is in excess of RecA K72R. Thus, RecA K72R inhibits hydrolysis-proficient RecA proteins by interacting with them in solution and preventing the extension phase of filament assembly.

Enhancement of DNA compaction by negatively charged nanoparticles. Application to reversible photocontrol of DNA higher-order structure

We report for the first time that negatively charged silica nanoparticles (NPs) enhance the ability of cationic surfactants to induce genomic DNA compaction. Single-chain compaction of duplex DNA molecules was studied by fluorescence microscopy in the presence of dodecyltrimethylammonium bromide (DTAB) and NPs. We found that very small amounts of NPs (∼10−4 to 10−2 wt%) significantly decreased the concentration of the surfactant at which DNA is compacted. This effect was maximal at intermediate NP concentration (here, 1.5 × 10−3 wt%) where the concentration of DTAB necessary for DNA compaction was 5-fold smaller than that in the absence of NPs. As a possible mechanism, we suggest that negatively charged NPs, by inducing the aggregation of DTAB molecules through electrostatic interactions, promote cooperative binding to DNA and thus enhance the ability of DTAB to compact DNA. By applying this phenomenon to a photosensitive cationic surfactant (AzoTAB), we could achieve reversible control of DNA higher-order structure using light at a much lower AzoTAB concentration than what has been reported up to now.

Hop2-Mnd1 Condenses DNA to Stimulate the Synapsis Phase of DNA Strand Exchange

Hop2-Mnd1 is a meiotic recombination mediator that stimulates DNA strand invasion by both Dmc1 and Rad51. To understand the biochemical mechanism of this stimulation, we directly visualized the heterodimer acting on single molecules of duplex DNA using optical tweezers and video fluorescence microscopy. The results show that the Hop2-Mnd1 heterodimer efficiently condenses double-stranded DNA via formation of a bright spot or DNA condensate. The condensation of DNA is Hop2-Mnd1 concentration-dependent, reversible, and specific to the heterodimer, as neither Hop2 nor Mnd1 acting alone can facilitate this reaction. The results also show that the rate-limiting nucleation step of DNA condensation is overcome in the presence of divalent metal ions, with the following order of preference: Mn2+>Mg2+>Ca2+. Hop2-Mnd1/Dmc1/single-stranded DNA nucleoprotein filaments also condense double-stranded DNA in a heterodimer concentration-dependent manner. Of importance, the concentration dependence parallels that seen in DNA strand exchange. We propose that rapid DNA condensation is a key factor in stimulating synapsis, whereas decondensation may facilitate the invasion step and/or the ensuing branch migration process.

The Full-length Saccharomyces cerevisiae Sgs1 Protein Is a Vigorous DNA Helicase That Preferentially Unwinds Holliday Junctions

The highly conserved RecQ family of DNA helicases has multiple roles in the maintenance of genome stability. Sgs1, the single RecQ homologue in Saccharomyces cerevisiae, acts both early and late during homologous recombination. Here we present the expression, purification, and biochemical analysis of full-length Sgs1. Unlike the truncated form of Sgs1 characterized previously, full-length Sgs1 binds diverse single-stranded and double-stranded DNA substrates, including DNA duplexes with 5'- and 3'-single-stranded DNA overhangs. Similarly, Sgs1 unwinds a variety of DNA substrates, including blunt-ended duplex DNA. Significantly, a substrate containing a Holliday junction is unwound most efficiently. DNA unwinding is catalytic, requires ATP, and is stimulated by replication protein A. Unlike RecQ homologues from multicellular organisms, Sgs1 is remarkably active at picomolar concentrations and can efficiently unwind duplex DNA molecules as long as 23,000 base pairs. Our analysis shows that Sgs1 resembles Escherichia coli RecQ protein more than any of the human RecQ homologues with regard to its helicase activity. The full-length recombinant protein will be invaluable for further investigation of Sgs1 biochemistry.

Investigating the Nucleation and Extension Rates of E.coli and Deinococcus RecA Along Duplex DNA

RecA is a protein which promotes the exchange between two homologous DNA molecules in homologous recombination process. When individual RecA molecules assemble on DNA, the DNA is stretched and underwound to form a nucleoprotein filament with its rigidity and end-to-end length increased. We have developed single-molecule tethered particle motion (TPM) experiments to study the assembly dynamics of RecA proteins on individual duplex DNA molecules. The TPM method is capable of measuring the changes in DNA length by observing the bead's Brownian motion, thus allowing us to monitor RecA nucleation and extension in real-time. Using much shorter DNA (a few hundreds basepairs), TPM experiments offer improved sensitivity, since the DNA length change can be readily detected as soon as a few RecA bounded to duplex DNA molecules. Our experiments indicated a faster nucleation rate compared to the previous reports (Galletto et al., 2006). Moreover, we have compared the nucleation and extension rates of E. coli RecA with the RecA from Deinococcus radiodurans, UV-resistant bacteria, under different nucleotide states, ATP and ATPγS. Deinococcus radiodurans RecA are found to nucleate faster (∼1.6×10-2 bp-1min-1) but extend slower (∼0.3-1.5 RecA/sec) under ATP.This difference reflects the physiological role of Dr. RecA when extensive UV-damaged DNA molecules are present.

Intersubunit Regulation Between Nuclease and Helicase Domains of Recbcd Enzyme

The Escherichia coliRecBCD helicase/nuclease initiates homologous recombinational repair of damaged blunt-end duplex DNA molecules. RecBCD, a multifunctional enzyme complex, contains two DNA motors as well as a nuclease domain to process duplex DNA and generate single-stranded DNA molecules. We used single-molecule tethered particle motion (TPM) experiments to investigate the regulation mechanism between the nuclease domain and two helicase domains of RecBCD enzyme using calcium ions, which specifically inhibit nuclease activity.

Novel Method to Detect DNA Methylation Using Gold Nanoparticles Coupled with Enzyme-Linkage Reactions

DNA methylation, catalyzed by methylases, plays a critical role in many biological processes, and methylases have been regarded as promising targets for antimicrobial drugs. In this paper, we propose a simple and sensitive colorimetric assay method to detect the activity of methylases so as to monitor DNA methylation using DNA-modified gold nanoparticles (AuNPs) coupled with enzyme-linkage reactions. The duplex DNA molecules modified on the surface of AuNPs are first methylated by DNA adenine methylation (Dam) methyltransferase (MTase) and then cut by methylation-sensitive restriction endonuclease Dpn I. Removal of duplex from the AuNP surfaces by the methylation/cleavage process will destabilize the nanoparticles, resulting in aggregation of AuNPs and a red-to-blue color change. Consequently, the enzyme activity of Dam MTase can be assayed and DNA methylation can be detected. Furthermore, this study may provide a sensitive platform to screen inhibitors for Dam MTase.

The first complete mitochondrial genome sequences of Amblypygi (Chelicerata: Arachnida) reveal conservation of the ancestral arthropod gene order

Amblypygi (whip spiders) are terrestrial chelicerates inhabiting the subtropics and tropics. In morphological and rRNA-based phylogenetic analyses, Amblypygi cluster with Uropygi (whip scorpions) and Araneae (spiders) to form the taxon Tetrapulmonata, but there is controversy regarding the interrelationship of these three taxa. Mitochondrial genomes provide an additional large data set of phylogenetic information (sequences, gene order, RNA secondary structure), but in arachnids, mitochondrial genome data are missing for some of the major orders. In the course of an ongoing project concerning arachnid mitochondrial genomics, we present the first two complete mitochondrial genomes from Amblypygi. Both genomes were found to be typical circular duplex DNA molecules with all 37 genes usually present in bilaterian mitochondrial genomes. In both species, gene order is identical to that of Limulus polyphemus (Xiphosura), which is assumed to reflect the putative arthropod ground pattern. All tRNA gene sequences have the potential to fold into structures that are typical of metazoan mitochondrial tRNAs, except for tRNA-Ala, which lacks the D arm in both amblypygids, suggesting the loss of this feature early in amblypygid evolution. Phylogenetic analysis resulted in weak support for Uropygi being the sister group of Amblypygi.

Studying RecBCD Helicase Translocation Along χ-DNA Using Tethered Particle Motion with a Stretching Force

Escherichia coli RecBCD helicase unwinds blunt-end duplex DNA to repair damaged DNA molecules in the homologous recombination pathway. Previous single-molecule experiments showed that RecBCD recognizes an 8 nt DNA sequence, χ, and lowers its unwinding rate afterward under saturating ATP condition. We have developed a single-molecule force-tethered particle motion (FTPM) method, which is modified from the conventional TPM method, and applied it to study RecBCD motion in detail. In the FTPM experiment, a stretching force is applied to the DNA-bead complex that suppresses the bead's Brownian motion, resulting in an improved spatial resolution at long DNA substrates. Based on the equipartition theorem, the mean-square displacement of the bead's Brownian motion measured by FTPM correlates linearly to DNA extension length with a predicted slope, circumventing the difficulties of conventional TPM experiments, such as nonlinearity and low resolution of long DNA substrates. The FTPM method offers the best resolution in the presence of only a small stretching force (0.20 pN). We used the FTPM method to investigate RecBCD helicase motion along 4.1 kb long χ-containing duplex DNA molecules, and observed that the translocation rate of RecBCD changes after the χ sequence under limited ATP concentrations. This suggests that χ recognition by RecBCD does not require saturating ATP conditions, contrary to what was previously reported.

Studying RecBCD Helicase Translocation along Chi-DNA Using Tethered Particle Motion with a Stretching Force

E. coli RecBCD helicase unwinds blunt-end duplex DNA to repair damaged DNA molecules in the homologous recombination pathway. Previous single-molecule experiments show that RecBCD recognizes an 8 nt DNA sequence, chi, and lowers its unwinding rate afterwards under saturating ATP condition. We have developed a single-molecule Force Tethered Particle Motion (FTPM) method, which is modified from the conventional TPM method, and applied it to study RecBCD motion in details. In the FTPM experiment, a stretching force is applied to the DNA-bead complex, and suppressed bead's Brownian motion, resulting in an improved spatial resolution at long DNA substrates. Based on the equipartition theorem, the mean square displacement (MSD) of the bead Brownian motion measured by FTPM correlates linearly to DNA extension length with a predicted slope, circumventing the difficulties, such as non-linearality and low resolution of long DNA substrates in conventional TPM experiments. The FTPM method offers the best resolution (56 bp at 433 bp long DNA) in the presence of only a small stretching force (0.20 pN). We have used the FTPM method to investigate the RecBCD helicase motion along 4.1 kb long chi-containing duplex DNA molecules, and observed that translocation rate of RecBCD changes after chi sequence under limited ATP concentrations. This suggests that chi recognition by RecBCD does not require saturating ATP conditions, contrary to what have been previously reported.

Observation of Oligonucleotide Dynamics by means of Fluorescent Nucleoside analog 6MI

To improve current understanding of the structural recognition mechanism of architectural DNA binding proteins such as HU and IHF, we are investigating the structure and dynamics of different DNA substrates. We are able to make these observations on both global and local levels by incorporating the fluorescent guanosine nucleoside analog 6-methylisoxanthopterin (6-MI), with H-bonds with cytosine similar to guanosine. We have previously shown this probe does not significantly perturb the global structures of duplex DNA molecules. 6-MI was systematically incorporated into a 34 base oligonucleotide. Initial characterization of local DNA environment included time resolved fluorescence and rotational correlation measurements of the duplex oligomers relative to 6-MI monomer and single stranded DNA. Analysis of time-resolved fluorescence decay yields 3 lifetime components of 0.4 ns, 4 ns and 6.5 ns. The largest lived component is similar to that of 6-MI monomer, 7 ns. The position of the probe shifts the fluorescent populations from 0.4 ns to 6.5ns upon formation of duplex, which implies that 6-MI local environment in these positions resembles that of the solvent exposed monomer. However, no direct correlation between adjacent base sequence and the fluorescent properties of 6MI was observed. To further investigate the increase in fluorescence upon duplex formation, we characterized the local and global structure of several oligonucleotides through temperature melts, quantum yield calculations, quenching assays, and Raman spectroscopy. The results suggest that, the position of 6-MI in the duplex sequence, helical turn, and surrounding base sequence determines the dynamics of 6-MI. This potentially leads to the formation of a fixed geometry of 6-MI which stacks poorly with adjacent bases. The lack of stacking interactions causes 6-MI to exhibit fluorescent properties of the monomer.

Assay for Human Rad51-Mediated DNA Displacement Loop Formation

Homologous recombination is an important mechanism for the repair of damaged chromosomes, for preventing the demise of damaged replication forks, and for several other aspects of chromosome metabolism and maintenance. The homologous recombination reaction is mediated by the Rad51 recombinase. In the presence of ATP, Rad51 polymerizes on single-stranded DNA (ssDNA) to form a nucleoprotein filament that is commonly referred to as the "presynaptic filament." The presynaptic filament is capable of locating a homologous duplex DNA molecule and catalyzing invasion of the duplex to form a DNA displacement loop called the "D-loop." This protocol describes an in vitro D-loop assay that uses a radiolabeled ssDNA oligonucleotide and a nonlabeled homologous supercoiled duplex DNA as substrates, and agarose gel electrophoresis together with PhosphorImaging for product analysis. To enhance the efficiency of the D-loop reaction, an ancillary factor (the Hop2-Mnd1 complex or Rad54) is included in the reaction. This reconstituted system provides researchers a biochemical means to dissect the mechanisms of the homologous recombination machinery.

Homologous recombination properties of OsRad51, a recombinase from rice

cDNA corresponding to OsRad51 protein was isolated from cDNA library of rice flowers (Oryza sativa, Indica cultivar group) and cloned in to pET28a expression vector. The protein was over expressed in E. coli BL21 (DE3) and purified. Purified OsRad51 could bind single and double stranded DNA, however it showed higher affinity for single stranded DNA. Transmission Electron Microscopy (TEM) studies of OsRad51-DNA complexes showed that this protein formed ring like structures and bound DNA forming filaments. OsRad51 protein promoted renaturation of complementary single strands in to duplex DNA molecules and also showed ATPase activity, which was stimulated by single strand DNA. Fluorescence resonance energy transfer (FRET) assays revealed that OsRad51 promoted homology dependent renaturation as well as strand exchange reactions. Renaturation activity was ATP dependent; however strand exchange activity was ATP independent. This is the first report on in vitro characterization of Rad51 protein from crop plants.

Characterization and Application of Controllable Local Chemical Changes Produced by Reagent Delivery from a Nanopipet

We introduce a versatile method that allows local and repeatable delivery (or depletion) of any water-soluble reagent from a nanopipet in ionic solution to make localized controlled changes in reagent concentration at a surface. In this work, Na(+) or OH(-) ions were dosed from the pipet using pulsed voltage-driven delivery. Total internal reflection fluorescence from CoroNa Green dye in the bath for Na(+) ions or fluorescein in the bath for pH quantified the resulting changes in local surface concentration. These changes had a time response as short as 10 ms and a radius of 1-30 microm and depended on the diameter of the pipet used, the applied voltage, and the pipet-surface separation. After the pipet dosing was characterized in detail, two proof-of-concept experiments on single cells and single molecules were then performed. We demonstrated local control of the sodium-sensitive flagellar motor in single Escherichia coli chimera on the time scale of 1 s by dosing sodium and monitoring the rotation of a 1 microm diameter bead fixed to the flagellum. We also demonstrated triggered single-molecule unfolding by dosing acid from the pipet to locally melt individual molecules of duplex DNA, as observed using fluorescent resonance energy transfer.

Mechanism of R‐Loop formation at Immunoglobulin Class Switch sequences

Received 12 July 2007/Returned for modification 21 August 2007/Accepted 9 October 2007 R-loops have been described in vivo at the immunoglobulin class switch sequences and at prokaryotic and mitochondrial origins of replication. However, the biochemical mechanism and determinants of R-loop formation are unclear. We find that R-loop formation is nearly eliminated when RNase T1 is added during transcription but not when it is added afterward. Hence, rather than forming simply as an extension of the RNA-DNA hybrid of normal transcription, the RNA must exit the RNA polymerase and compete with the nontemplate DNA strand for an R-loop to form. R-loops persist even when transcription is done in Li or Cs, which do not support G-quartet formation. Hence, R-loop formation does not rely on G-quartet formation. R-loop formation efficiency decreases as the number of switch repeats is decreased, although a very low level of R-loop formation occurs at even one 49-bp switch repeat. R-loop formation decreases sharply as G clustering is reduced, even when G density is kept constant. The critical level for R-loop formation is approximately the same point to which evolution drove the G clustering and G density on the nontemplate strand of mammalian switch regions. This provides an independent basis for concluding that the primary function of G clustering, in the context of high G density, is R-loop formation. R-loops are nucleic acid structures in which an RNA strand displaces one strand of DNA for a limited length in an otherwise duplex DNA molecule. R-loops were named by analogy to D-loops, which is where all three strands are DNA. R-loops form in vivo at sequences that generate a G-rich transcript at the prokaryotic origins of replication (20), mitochondrial origins of replication (18), and mammalian immunoglobulin (Ig) class switch sequences (reviewed in reference 45). In addition, R-loop formation occurs in vivo at some G-rich transcript locations that are distinctly high for mitotic recombination in Saccharomyces cerevisiae, and this high recombination rate is reduced upon overexpression of S. cerevisiae RNase H1 (14). When prokaryotes lack topoisomerase activity, R-loops can form at a wider variety of sequences, and the lethality associated with this can be remedied by overexpression of Escherichia coli RNase H1 (7). In an avian lymphoid cell line, lack of the ASF2/SF2 RNA-binding protein favors R-loop formation at G-rich transcript locations in the genome, and expression of human RNase H1 can abolish the R-loop (19).