The Human Specialized DNA Polymerases and Non-B DNA: Vital Relationships to Preserve Genome Integrity

In addition to the canonical right-handed double helix, DNA molecule can adopt several other non-B DNA structures. Readily formed in the genome at specific DNA repetitive sequences, these secondary conformations present a distinctive challenge for progression of DNA replication forks. Impeding normal DNA synthesis, cruciforms, hairpins, H DNA, Z DNA and G4 DNA considerably impact the genome stability and in some instances play a causal role in disease development. Along with previously discovered dedicated DNA helicases, the specialized DNA polymerases emerge as major actors performing DNA synthesis through these distorted impediments. In their new role, they are facilitating DNA synthesis on replication stalling sites formed by non-B DNA structures and thereby helping the completion of DNA replication, a process otherwise crucial for preserving genome integrity and concluding normal cell division. This review summarizes the evidence gathered describing the function of specialized DNA polymerases in replicating DNA through non-B DNA structures.     

Inverted repeats,stem-loops,and cruciforms: Significance for initiation of DNA replication

Inverted repeats occur nonrandomly in the DNA of most organisms. Stem-loops and cruciforms can form from inverted repeats. Such structures have been detected in pro- and eukaryotes. They may affect the supercoiling degree of the DNA, the positioning of nucleosomes, the formation of other secondary structures of DNA, or directly interact with proteins. Inverted repeats, stem-loops, and cruciforms are present at the replication origins of phage, plasmids, mitochondria, eukaryotic viruses, and mammalian cells. Experiments with anti-cruciform antibodies suggest that formation and stabilization of cruciforms at particular mammalian origins may be associated with initiation of DNA replication. Many proteins have been shown to interact with cruciforms, recognizing features like DNA crossovers, four-way junctions, and curved/bent DNA of specific angles. A human cruciform binding protein (CBP) displays a novel type of interaction with cruciforms and may be linked to initiation of DNA replication. © 1996 Wiley-Liss, Inc.     

Determination of the elastic properties of short ssDNA molecules by mechanically folding and unfolding DNA hairpins

The characterization of elastic properties of biopolymers is crucial to understand many molecular reactions determined by conformational bending fluctuations of the polymer. Direct measurement of such elastic properties using single‐molecule methods is usually hindered by the intrinsic tendency of such biopolymers to form high‐order molecular structures. For example, single‐stranded deoxyribonucleic acids (ssDNA) tend to form secondary structures such as local double helices that prevent the direct measurement of the ideal elastic response of the ssDNA. In this work, we show how to extract the ideal elastic response in the entropic regime of short ssDNA molecules by mechanically pulling two‐state DNA hairpins of different contour lengths. This is achieved by measuring the force dependence of the molecular extension and stiffness on mechanically folding and unfolding the DNA hairpin. Both quantities are fit to the worm‐like chain elastic model giving values for the persistence length and the interphosphate distance. This method can be used to unravel the elastic properties of short ssDNA and RNA sequences and, more generally, any biopolymer that can exhibit a cooperative two‐state transition between mechanically folded and unfolded states (such as proteins). © 2014 Wiley Periodicals, Inc. Biopolymers 101: 1193–1199, 2014.     

Diffusion of Human Replication Protein A along Single-Stranded DNA

Replication protein A (RPA) is a eukaryotic single-stranded DNA (ssDNA) binding protein that plays critical roles in most aspects of genome maintenance, including replication, recombination and repair. RPA binds ssDNA with high affinity, destabilizes DNA secondary structure and facilitates binding of other proteins to ssDNA. However, RPA must be removed from or redistributed along ssDNA during these processes. To probe the dynamics of RPA–DNA interactions, we combined ensemble and single-molecule fluorescence approaches to examine human RPA (hRPA) diffusion along ssDNA and find that an hRPA heterotrimer can diffuse rapidly along ssDNA. Diffusion of hRPA is functional in that it provides the mechanism by which hRPA can transiently disrupt DNA hairpins by diffusing in from ssDNA regions adjacent to the DNA hairpin. hRPA diffusion was also monitored by the fluctuations in fluorescence intensity of a Cy3 fluorophore attached to the end of ssDNA. Using a novel method to calibrate the Cy3 fluorescence intensity as a function of hRPA position on the ssDNA, we estimate a one-dimensional diffusion coefficient of hRPA on ssDNA of D₁ ~ 5000 nt² s^− 1 at 37 °C. Diffusion of hRPA while bound to ssDNA enables it to be readily repositioned to allow other proteins access to ssDNA.     

Presynaptic filament dynamics in homologous recombination and DNA repair

Homologous recombination (HR) is an essential genome stability mechanism used for high-fidelity repair of DNA double-strand breaks and for the recovery of stalled or collapsed DNA replication forks. The crucial homology search and DNA strand exchange steps of HR are catalyzed by presynaptic filaments-helical filaments of a recombinase enzyme bound to single-stranded DNA (ssDNA). Presynaptic filaments are fundamentally dynamic structures, the assembly, catalytic turnover, and disassembly of which must be closely coordinated with other elements of the DNA recombination, repair, and replication machinery in order for genome maintenance functions to be effective. Here, we reviewed the major dynamic elements controlling the assembly, activity, and disassembly of presynaptic filaments; some intrinsic such as recombinase ATP-binding and hydrolytic activities, others extrinsic such as ssDNA-binding proteins, mediator proteins, and DNA motor proteins. We examined dynamic behavior on multiple levels, including atomic- and filament-level structural changes associated with ATP binding and hydrolysis as evidenced in crystal structures, as well as subunit binding and dissociation events driven by intrinsic and extrinsic factors. We examined the biochemical properties of recombination proteins from four model systems (T4 phage, Escherichia coli, Saccharomyces cerevisiae, and Homo sapiens), demonstrating how their properties are tailored for the context-specific requirements in these diverse species. We proposed that the presynaptic filament has evolved to rely on multiple external factors for increased multilevel regulation of HR processes in genomes with greater structural and sequence complexity.     

Cellular functions of human RPA1. Multiple roles of domains in replication, repair, and checkpoints

In eukaryotes, the single strand DNA (ssDNA)-binding protein, replication protein A (RPA), is essential for DNA replication, repair, and recombination. RPA is composed of the following three subunits: RPA1, RPA2, and RPA3. The RPA1 subunit contains four structurally related domains and is responsible for high affinity ssDNA binding. This study uses a depletion/replacement strategy in human cells to reveal the contributions of each domain to RPA cellular functions. Mutations that substantially decrease ssDNA binding activity do not necessarily disrupt cellular RPA function. Conversely, mutations that only slightly affect ssDNA binding can dramatically affect cellular function. The N terminus of RPA1 is not necessary for DNA replication in the cell; however, this region is important for the cellular response to DNA damage. Highly conserved aromatic residues in the high affinity ssDNA-binding domains are essential for DNA repair and cell cycle progression. Our findings suggest that as long as a threshold of RPA-ssDNA binding activity is met, DNA replication can occur and that an RPA activity separate from ssDNA binding is essential for function in DNA repair.     

Characterization of DNA-binding and strand-exchange stimulation properties of y-RPA, a yeast single-strand-DNA-binding protein.

Single-stranded DNA binding proteins (SSBs) have been isolated from many organisms, including Escherichia coli, Saccharomyces cerevisiae and humans. Characterization of these proteins suggests they are required for DNA replication and are active in homologous recombination. As an initial step towards understanding the role of the eukaryotic SSBs in DNA replication and recombination, we examined the DNA binding and strand exchange stimulation properties of the S. cerevisiae single-strand binding protein y-RPA (yeast replication protein A). y-RPA was found to bind to single-stranded DNA (ssDNA) as a 115,000 M(r) heterotrimer containing 70,000, 36,000 and 14,000 M(r) subunits. It saturated ssDNA at a stoichiometry of one heterotrimer per 90 to 100 nucleotides and binding occurred with high affinity (K omega greater than 10(9) M-1) and co-operativity (omega = 10,000 to 100,000). Electron microscopic analysis revealed that y-RPA binding was highly co-operative and that the ssDNA present in y-RPA-ssDNA complexes was compacted fourfold, arranged into nucleosome-like structures, and was free of secondary structure. y-RPA was also tested for its ability to stimulate the yeast Sepl and E. coli RecA strand-exchange proteins. In an assay that measures the pairing of circular ssDNA with homologous linear duplex DNA, y-RPA stimulated the strand-exchange activity of Sepl approximately threefold and the activity of RecA protein to the same extent as did E. coli SSB. Maximal stimulation of Sepl occurred at a stoichiometry of one y-RPA heterotrimer per 95 nucleotides of ssDNA. y-RPA stimulated RecA and Sepl mediated strand exchange reactions in a manner similar to that observed for the stimulation of RecA by E. coli SSB; in both of these reactions, y-RPA inhibited the aggregation of ssDNA and promoted the co-aggregation of single-stranded and double-stranded linear DNA. These results demonstrate that the E. coli and yeast SSBs display similar DNA-binding properties and support a model in which y-RPA functions as an E. coli SSB-like protein in yeast.     

Evidence of Pervasive Biologically Functional Secondary Structures within the Genomes of Eukaryotic Single-Stranded DNA Viruses

Single-stranded DNA (ssDNA) viruses have genomes that are potentially capable of forming complex secondary structures through Watson-Crick base pairing between their constituent nucleotides. A few of the structural elements formed by such base pairings are, in fact, known to have important functions during the replication of many ssDNA viruses. Unknown, however, are (i) whether numerous additional ssDNA virus genomic structural elements predicted to exist by computational DNA folding methods actually exist and (ii) whether those structures that do exist have any biological relevance. We therefore computationally inferred lists of the most evolutionarily conserved structures within a diverse selection of animal- and plant-infecting ssDNA viruses drawn from the families Circoviridae, Anelloviridae, Parvoviridae, Nanoviridae, and Geminiviridae and analyzed these for evidence of natural selection favoring the maintenance of these structures. While we find evidence that is consistent with purifying selection being stronger at nucleotide sites that are predicted to be base paired than at sites predicted to be unpaired, we also find strong associations between sites that are predicted to pair with one another and site pairs that are apparently coevolving in a complementary fashion. Collectively, these results indicate that natural selection actively preserves much of the pervasive secondary structure that is evident within eukaryote-infecting ssDNA virus genomes and, therefore, that much of this structure is biologically functional. Lastly, we provide examples of various highly conserved but completely uncharacterized structural elements that likely have important functions within some of the ssDNA virus genomes analyzed here.     

Presynaptic filament dynamics in homologous recombination and DNA repair

Homologous recombination (HR) is an essential genome stability mechanism used for high-fidelity repair of DNA double-strand breaks and for the recovery of stalled or collapsed DNA replication forks. The crucial homology search and DNA strand exchange steps of HR are catalyzed by presynaptic filaments—helical filaments of a recombinase enzyme bound to single-stranded DNA (ssDNA). Presynaptic filaments are fundamentally dynamic structures, the assembly, catalytic turnover, and disassembly of which must be closely coordinated with other elements of the DNA recombination, repair, and replication machinery in order for genome maintenance functions to be effective. Here, we reviewed the major dynamic elements controlling the assembly, activity, and disassembly of presynaptic filaments; some intrinsic such as recombinase ATP-binding and hydrolytic activities, others extrinsic such as ssDNA-binding proteins, mediator proteins, and DNA motor proteins. We examined dynamic behavior on multiple levels, including atomic- and filament-level structural changes associated with ATP binding and hydrolysis as evidenced in crystal structures, as well as subunit binding and dissociation events driven by intrinsic and extrinsic factors. We examined the biochemical properties of recombination proteins from four model systems (T4 phage, Escherichia coli, Saccharomyces cerevisiae, and Homo sapiens), demonstrating how their properties are tailored for the context-specific requirements in these diverse species. We proposed that the presynaptic filament has evolved to rely on multiple external factors for increased multilevel regulation of HR processes in genomes with greater structural and sequence complexity.     

DnaA and ORC: more than DNA replication initiators

Mutations in DNA replication initiator genes in both prokaryotes and eukaryotes lead to a pleiotropic array of phenotypes, including defects in chromosome segregation, cytokinesis, cell cycle regulation and gene expression. For years, it was not clear whether these diverse effects were indirect consequences of perturbed DNA replication, or whether they indicated that DNA replication initiator proteins had roles beyond their activity in initiating DNA synthesis. Recent work from a range of organisms has demonstrated that DNA replication initiator proteins play direct roles in many cellular processes, often functioning to coordinate the initiation of DNA replication with essential cell-cycle activities. The aim of this review is to highlight these new findings, focusing on the pathways and mechanisms utilized by DNA replication initiator proteins to carry out a diverse array of cellular functions.     

A perfect palindrome in the Escherichia coli chromosome forms DNA hairpins on both leading- and lagging-strands

DNA palindromes are hotspots for DNA double strand breaks, inverted duplications and intra-chromosomal translocations in a wide spectrum of organisms from bacteria to humans. These reactions are mediated by DNA secondary structures such as hairpins and cruciforms. In order to further investigate the pathways of formation and cleavage of these structures, we have compared the processing of a 460 base pair (bp) perfect palindrome in the Escherichia coli chromosome with the same construct interrupted by a 20 bp spacer to form a 480 bp interrupted palindrome. We show here that the perfect palindrome can form hairpin DNA structures on the templates of the leading- and lagging-strands in a replication-dependent reaction. In the presence of the hairpin endonuclease SbcCD, both copies of the replicated chromosome containing the perfect palindrome are cleaved, resulting in the formation of an unrepairable DNA double-strand break and cell death. This contrasts with the interrupted palindrome, which forms a hairpin on the lagging-strand template that is processed to form breaks, which can be repaired by homologous recombination.     

The Archaeal Topoisomerase Reverse Gyrase Is a Helix-destabilizing Protein That Unwinds Four-way DNA Junctions

Four-way junctions are non-B DNA structures that originate as intermediates of recombination and repair (Holliday junctions) or from the intrastrand annealing of palindromic sequences (cruciforms). These structures have important functional roles but may also severely interfere with DNA replication and other genetic processes; therefore, they are targeted by regulatory and architectural proteins, and dedicated pathways exist for their removal. Although it is well known that resolution of Holliday junctions occurs either by recombinases or by specialized helicases, less is known on the mechanisms dealing with secondary structures in nucleic acids. Reverse gyrase is a DNA topoisomerase, specific to microorganisms living at high temperatures, which comprises a type IA topoisomerase fused to an SF2 helicase-like module and catalyzes ATP hydrolysis-dependent DNA positive supercoiling. Reverse gyrase is likely involved in regulation of DNA structure and stability and might also participate in the cell response to DNA damage. By applying FRET technology to multiplex fluorophore gel imaging, we show here that reverse gyrase induces unwinding of synthetic four-way junctions as well as forked DNA substrates, following a mechanism independent of both the ATPase and the strand-cutting activity of the enzyme. The reaction requires high temperature and saturating protein concentrations. Our results suggest that reverse gyrase works like an ATP-independent helix-destabilizing protein specific for branched DNA structures. The results are discussed in light of reverse gyrase function and their general relevance for protein-mediated unwinding of complex DNA structures.     

Conserved sequence preference in DNA binding among recombination proteins: an effect of ssDNA secondary structure.

Repetitive sequences have been proposed to be recombinogenic elements in eukaryotic chromosomes. We tested whether dinucleotide repeats sequences are preferential sites for recombination because of their high affinity for recombination enzymes. We compared the kinetics of the binding of the scRad51, hsRad51 and ecRecA proteins to oligonucleotides with repeats of dinucleotides GT, CA, CT, GA, GC or AT. Since secondary structures in single-stranded DNA (ssDNA) act as a barrier to complete binding we measured whether these oligonucleotides are able to form stable secondary structures. We show that the preferential binding of recombination proteins is conserved among the three proteins and is influenced mainly by secondary structures in ssDNA.     

RADX interacts with single‐stranded DNA to promote replication fork stability

Single‐stranded DNA (ssDNA) regions form as an intermediate in many DNA‐associated transactions. Multiple cellular proteins interact with ssDNA via the oligonucleotide/oligosaccharide‐binding (OB) fold domain. The heterotrimeric, multi‐OB fold domain‐containing Replication Protein A (RPA) complex has an essential genome maintenance role, protecting ssDNA regions from nucleolytic degradation and providing a recruitment platform for proteins involved in responses to replication stress and DNA damage. Here, we identify the uncharacterized protein RADX (CXorf57) as an ssDNA‐binding factor in human cells. RADX binds ssDNA via an N‐terminal OB fold cluster, which mediates its recruitment to sites of replication stress. Deregulation of RADX expression and ssDNA binding leads to enhanced replication fork stalling and degradation, and we provide evidence that a balanced interplay between RADX and RPA ssDNA‐binding activities is critical for avoiding these defects. Our findings establish RADX as an important component of cellular pathways that promote DNA replication integrity under basal and stressful conditions by means of multiple ssDNA‐binding proteins.