Single-Stranded DNA Curtains for Studying Homologous Recombination.

Homologous recombination is an important pathway involved in the repair of double-stranded DNA breaks. Genetic studies form the foundation of our knowledge on homologous recombination. Significant progress has also been made toward understanding the biochemical and biophysical properties of the proteins, complexes, and reaction intermediates involved in this essential DNA repair pathway. However, heterogeneous or transient recombination intermediates remain extremely difficult to assess through traditional ensemble methods, leaving an incomplete mechanistic picture of many steps that take place during homologous recombination. To help overcome some of these limitations, we have established DNA curtain methodologies as an experimental platform for studying homologous DNA recombination in real-time at the single-molecule level. Here, we present a detailed overview describing the preparation and use of single-stranded DNA curtains in applications related to the study of homologous DNA recombination with emphasis on recent work related to the study of the eukaryotic recombinase Rad51.

Assembly of DNA curtains using hydrogen silsesquioxane as a barrier to lipid diffusion.

We have established a single-molecule imaging experimental platform called "DNA curtains" in which DNA molecules tethered to a lipid bilayer are organized into patterns at nanofabricated metallic barriers on the surface of a microfluidic sample chamber. This technology has wide applications for real-time single-molecule imaging of protein-nucleic acid interactions. Here, we demonstrate that DNA curtains can also be made from hydrogen silsesquioxane (HSQ). HSQ offers important advantages over metallic barriers because it can be lithographically patterned directly onto fused silica slides without any requirement for further processing steps, thereby offering the potential for rapid prototype development and/or scale up for manufacturing.

Fabrication of Nanoscale "Curtain Rods" for DNA Curtains Using Nanoimprint Lithography.

We have developed a new lithographically-based patterning process which significantly increases the throughput of experiments which probe how repair proteins scan DNA molecules for errors. In this process, nanoscale barriers are formed to interrupt the flow of a lipid bilayer in which DNA is tethered to proteins in the bilayer. The barriers trap the DNA, which is then stretched out by hydrodynamic flow, resulting in the formation of "DNA curtains." Nanoimprint lithography is used to facilitate massively parallel data collection for protein diffusion experiments on DNA.

Developmentally programmed assembly of higher order telomerase complexes with distinct biochemical and structural properties.

In Euplotes crassus, telomerase is responsible for telomere maintenance during vegetative growth and de novo telomere synthesis during macronuclear development. Here we show that telomerase in the vegetative stage of the life cycle exists as a 280-kD complex that can add telomeric repeats only onto telomeric DNA primers. Following the initiation of macronuclear development, telomerase assembles into larger complexes of 550 kD, 1600 kD, and 5 MD. In the 1600-kDa and 5-MDa complexes, telomerase is more processive than in the two smaller complexes and can add telomeres de novo onto nontelomeric 3' ends. Assembly of higher order telomerase complexes is accompanied by an extended region of RNase V1 and RNase T1 protection in the telomerase RNA subunit that is not observed with telomerase from vegetatively growing cells. The protected residues encompass a highly conserved region previously proposed to serve as a platform for formation of higher order structures. These findings provide the first direct demonstration of developmentally regulated higher order telomerase complexes with unique biochemical and structural properties.

Flexible positioning of the telomerase-associated nuclease leads to preferential elimination of nontelomeric DNA.

In addition to a reverse transcriptase activity, telomerase is associated with a DNA endonuclease that removes nucleotides from a primer 3' terminus prior to telomere repeat addition. Here we examine the DNA specificity of the primer cleavage-elongation reaction carried out by the Euplotes crassus telomerase. We show that the primer cleavage activity copurified with the E. crassus telomerase polymerase, indicating that it either is an intrinsic property of telomerase or is catalyzed by a tightly associated factor. Using chimeric primers containing stretches of telomeric DNA that could be precisely positioned on the RNA template, we found that the cleavage site is more flexible than originally proposed. Primers harboring mismatches in dT tracts that aligned opposite nucleotides 37 to 40 in the RNA template were cleaved to eliminate the mismatched residues along with the adjacent 3' sequence. The cleaved product was then elongated to generate perfect telomeric repeats. Mismatches in dG tracts were not removed, implying that the nuclease does not track coordinately with the polymerase active site. Our data indicate that the telomerase-associated nuclease could provide a rudimentary proofreading function in telomere synthesis by eliminating mismatches between the DNA primer and the 5' region of the telomerase RNA template.

Developmentally regulated initiation of DNA synthesis by telomerase: evidence for factor-assisted de novo telomere formation.

Telomerase serves a dual role at telomeres, maintaining tracts of telomere repeats and forming telomeres de novo on broken chromosomes in a process called chromosome healing. In ciliates, both mechanisms are readily observed. Vegetatively growing cells maintain pre-existing telomeres, while cells undergoing macronuclear development fragment their chromosomes and form telomeres de novo. Here we provide the first evidence for developmentally regulated initiation of DNA synthesis by telomerase. In vitro assays were conducted with telomerase from vegetative and developing Euplotes macronuclei using chimeric primers that contained non-telomeric 3' ends and an upstream stretch of telomeric DNA. In developing macronuclei, chimeric primers had two fates: nucleotides were either polymerized directly onto the 3' terminus or residues were removed from the 3' end by endonucleolytic cleavage before polymerization began. In contrast, telomerase from vegetative macronuclei used only the cleavage pathway. Telomere repeat addition onto non-telomeric 3' ends was lost when developing macronuclei were lysed and the contents purified on glycerol gradients. However, when fractions from the glycerol gradient were added back to partially purified telomerase, telomere synthesis was restored. The data indicate that a dissociable chromosome healing factor (CHF) collaborates with telomerase to initiate developmentally programmed de novo telomere formation.

Processing of nontelomeric 3' ends by telomerase: default template alignment and endonucleolytic cleavage.

Telomerase is a specialized reverse transcriptase that maintains telomeres at chromosome ends by extending preexisting tracts of telomeric DNA and forming telomeres de novo on broken chromosomes. Whereas the interaction of telomerase with telomeric DNA has been studied in some detail, relatively little is known about how this enzyme processes nontelomeric DNA. In this study we recruited the Euplotes telomerase to nontelomeric 3' termini in vitro using chimeric DNA primers that carried one repeat of a telomeric sequence at various positions upstream of a nontelomeric 3' end. Such primers were processed in two distinct pathways. First, nontelomeric 3' ends could be elongated directly by positioning a primer terminus at a specific site on the RNA template. Delivery to this default site was precise, always resulting in the addition of 4 dG residues to the non-telomeric 3' ends. These same residues initiate new telomeres formed in vivo. Alternatively, 3' nontelomeric nucleotides were removed from primers prior to initiating the first elongation cycle. As with default positioning of nontelomeric 3' ends, the cleavage event was extremely precise and was followed by the addition of dG residues to the primer 3' ends. The specificity of the cleavage reaction was mediated by primer interaction with the RNA template and, remarkably, proceeded by an endonucleolytic mechanism. These observations suggest a mechanism for the precision of developmentally regulated de novo telomere formation and expand our understanding of the enzymatic properties of telomerase.