HMCES Maintains Genome Integrity by Shielding Abasic Sites in Single-Strand DNA.

Abasic sites are one of the most common DNA lesions. All known abasic site repair mechanisms operate only when the damage is in double-stranded DNA. Here, we report the discovery of 5-hydroxymethylcytosine (5hmC) binding, ESC-specific (HMCES) as a sensor of abasic sites in single-stranded DNA. HMCES acts at replication forks, binds PCNA and single-stranded DNA, and generates a DNA-protein crosslink to shield abasic sites from error-prone processing. This unusual HMCES DNA-protein crosslink intermediate is resolved by proteasome-mediated degradation. Acting as a suicide enzyme, HMCES prevents translesion DNA synthesis and the action of endonucleases that would otherwise generate mutations and double-strand breaks. HMCES is evolutionarily conserved in all domains of life, and its biochemical properties are shared with its E. coli ortholog. Thus, HMCES is an ancient DNA lesion recognition protein that preserves genome integrity by promoting error-free repair of abasic sites in single-stranded DNA.

Multi-copy single-stranded DNA in Escherichia coli.

Multi-copy single-stranded DNA (msDNA) is composed of covalently bound single-stranded DNA and RNA, and synthesized by retron-encoded reverse transcriptase. msDNA-synthesizing systems are thought to be a recent acquisition by Escherichia coli because, to date, only seven types of msDNA, which differ markedly in their primary nucleotide sequences, have been found in a small subset of E. coli strains. The wide use of E. coli in molecular research means that it is important to understand more about these stable, covalently bound, single-stranded DNA or RNA compounds. The present review provides insights into the molecular biosynthesis, distribution and function of E. coli msDNA to raise awareness about these special molecules.

Tripartite DNA Lesion Recognition and Verification by XPC, TFIIH, and XPA in Nucleotide Excision Repair.

Transcription factor IIH (TFIIH) is essential for both transcription and nucleotide excision repair (NER). DNA lesions are initially detected by NER factors XPC and XPE or stalled RNA polymerases, but only bulky lesions are preferentially repaired by NER. To elucidate substrate specificity in NER, we have prepared homogeneous human ten-subunit TFIIH and its seven-subunit core (Core7) without the CAK module and show that bulky lesions in DNA inhibit the ATPase and helicase activities of both XPB and XPD in Core7 to promote NER, whereas non-genuine NER substrates have no such effect. Moreover, the NER factor XPA activates unwinding of normal DNA by Core7, but inhibits the Core7 helicase activity in the presence of bulky lesions. Finally, the CAK module inhibits DNA binding by TFIIH and thereby enhances XPC-dependent specific recruitment of TFIIH. Our results support a tripartite lesion verification mechanism involving XPC, TFIIH, and XPA for efficient NER.

Breaking bad: R-loops and genome integrity.

R-loops, nucleic acid structures consisting of an RNA-DNA hybrid and displaced single-stranded (ss) DNA, are ubiquitous in organisms from bacteria to mammals. First described in bacteria where they initiate DNA replication, it now appears that R-loops regulate diverse cellular processes such as gene expression, immunoglobulin (Ig) class switching, and DNA repair. Changes in R-loop regulation induce DNA damage and genome instability, and recently it was shown that R-loops are associated with neurodegenerative disorders. We discuss recent developments in the field; in particular, the regulation and effects of R-loops in cells, their effect on genomic and epigenomic stability, and their potential contribution to the origin of diseases including cancer and neurodegenerative disorders.

Potent Intracellular Knock-Down of Influenza A Virus M2 Gene Transcript by DNAzymes Considerably Reduces Viral Replication in Host Cells.

Influenza A virus has been known to be an important respiratory pathogen and cause of several epidemics and devastating pandemics leading to loss of life and resources across the globe. The M2 ion channel protein is highly conserved and essentially required during the trafficking, assembly, and budding processes of virus, thus an attractive target for designing antiviral drugs. We designed several 10-23 DNAzymes (Dz) targeting different regions of the M2 gene and analyzed their ability to specifically cleave the target RNA in both cell-free system as well as in cell culture using transient transfections. Dz114, among several others, directed against the predicted single-stranded bulge regions showed 70% inhibition of M2 gene expression validated by PCR and Western blot analysis. The activity was dependent on Mg(2+) (10-50 mM) in a dose-dependent manner. The mutant-Dz against M2 gene showed no down-regulation thereby illustrating high level of specificity of designed Dz114 towards the target RNA. Our findings suggest that Dz may be used as potential inhibitor of viral RNA replication and can be explored further for development of an effective therapeutic agent against influenza infection. These catalytic nucleic acid molecules may further be investigated as an alternative to the traditional influenza vaccines that require annual formulation.

Diverse small circular single-stranded DNA viruses identified in a freshwater pond on the McMurdo Ice Shelf (Antarctica).

Antarctica has some of the harshest environmental conditions for existence of life on Earth. In this pilot study we recovered eight diverse circular single-stranded DNA (ssDNA) viral genome sequences (1904-3120 nts) from benthic mats dominated by filamentous cyanobacteria in a freshwater pond on the McMurdo Ice Shelf sampled in 1988. All genomes contain two to three major open reading frames (ORFs) that are uni- or bi-directionally transcribed and all have an ORF encoding a replication-associated protein (Rep). In one genome, the second ORF has similarity to a capsid protein (CP) of Nepavirus which is most closely related to geminiviruses. Additionally, all genomes have two intergenic regions that contain putative stem loop structures, six genomes have NANTATTAC as the nonanucleotide motif, while one has CCTTATTAC, and another has a non-canonical stem loop. In the large intergenic region, we identified iterative sequences flanking the putative stem-loop elements which are a hallmark of most circular ssDNA viruses encoding rolling circle replication (RCR) initiators of the HUH endonuclease superfamily. The Reps encoded by ssDNA viral genomes recovered in this study shared <38% pairwise identity to all other Reps of known ssDNA viruses. A previous study on Lake Limnopolar (Livingston Island, South Shetland Islands), using next-generation sequencing identified circular ssDNA viruses and their putative Reps share <35% pairwise identity to those from the viral genomes removed in this study. It is evident from our pilot study that the global diversity of ssDNA viruses is grossly underestimated and there is limited knowledge on ssDNA viruses in Antarctica.

Statistical mechanics of DNA unzipping under periodic force: scaling behavior of hysteresis loops.

A simple model of DNA based on two interacting polymers has been used to study the unzipping of a double stranded DNA subjected to a periodic force. We propose a dynamical transition where, without changing the physiological condition, it is possible to bring DNA from the zipped or unzipped state to a new dynamic (hysteretic) state by varying the frequency of the applied force. Our studies reveal that the area of the hysteresis loop grows with the same exponents as of the isotropic spin systems. These exponents are amenable to verification in the force spectroscopic experiments.

Efficient transcription initiation in bacteria: an interplay of protein-DNA interaction parameters.

As the first, and usually rate-limiting, step of transcription initiation, bacterial RNA polymerase (RNAP) binds to double stranded DNA (dsDNA) and subsequently opens the two strands of DNA (the open complex formation). The rate determining step in the open complex formation is opening of a short (6 bp) DNA called the -10 region, which interacts with RNAP in both dsDNA and single stranded (ssDNA) forms. Accordingly, formation of the open complex depends on (physically independent) domains of RNAP that interact with ssDNA and dsDNA, as well as on parameters of DNA melting and sequences of -10 regions. We here aim to understand how these different interactions are mutually related to ensure efficient open complex formation. To achieve this, we use a recently developed biophysical model of transcription initiation, which allows the calculation of the kinetic parameters of transcription initiation on the scale of whole genome. We consequently investigate kinetic properties of sequences derived from all E. coli intergenic regions, and from more than 300 experimentally confirmed E. coli σ(70) promoters. We find that interaction specificities of σ(70) DNA binding domains reduce the number of sequences where RNAP binds strongly, but forms the open complex too slowly to achieve functional transcription (so-called poised promoters). However, we find that, despite this reduction, there is still a significant number of such poised promoters in the intergenic regions, which may provide a major source of false positives in genome-wide searches of transcription start sites. Furthermore, we surprisingly find that sequences of -10 regions of the functional promoters increase the extent of RNAP poising, which we interpret in terms of an extension of a recently proposed model of promoter recognition ('mix-and-match model') to kinetic parameters. Overall, our results allow better understanding of the design of σ(70) DNA binding domains and promoter sequences, and place a fundamental limit on accuracy of methods for promoter detection that are based on strong RNAP binding (e.g. ChIP-chip).

Overcoming the chromatin barrier to end resection.

Repair of double-strand breaks by homologous recombination requires Repair of double-strand breaks by homologous recombination requires 5'-3' resection of the DNA ends to create 3' single-stranded DNA tails. While much progress has been made in identifying the proteins that directly participate in end resection, how this process occurs in the context of chromatin is not well understood. Two papers in Nature report that Fun30, a poorly characterized member of the Swi2/Snf2 family of chromatin remodelers, plays a role in end processing by facilitating the Exo1 and Sgs1-Dna2 resection pathways.

[Double strand break repair, one mechanism can hide another: alternative non-homologous end joining]. / Réparation des cassures double-brin de l'ADN, un mécanisme peut en cacher un autre : la ligature d'extrémités non homologues alternative.

DNA double strand breaks are major cytotoxic lesions encountered by the cells. They can be induced by ionizing radiation or endogenous stress and can lead to genetic instability. Two mechanisms compete for the repair of DNA double strand breaks: homologous recombination and non-homologous end joining (NHEJ). Homologous recombination requires DNA sequences homology and is initiated by single strand resection. Recently, advances have been made concerning the major steps and proteins involved in resection. NHEJ, in contrast, does not require sequence homology. The existence of a DNA double strand break repair mechanism, independent of KU and ligase IV, the key proteins of the canonical non homologous end joining pathway, has been revealed lately and named alternative non homologous end joining. The hallmarks of this highly mutagenic pathway are deletions at repair junctions and frequent use of distal microhomologies. This mechanism is also initiated by a single strand resection of the break. The aim of this review is firstly to present recent data on single strand resection, and secondly the alternative NHEJ pathway, including a discussion on the fidelity of NHEJ. Based on current knowledge, canonical NHEJ does not appear as an intrinsically mutagenic mechanism, but in contrast, as a conservative one. The structure of broken DNA ends actually dictates the quality repair of the alternative NHEJ and seems the actual responsible for the mutagenesis attributed beforehand to the canonical NHEJ. The existence of this novel DNA double strand breaks repair mechanism needs to be taken into account in the development of radiosensitizing strategies in order to optimise the efficiency of radiotherapy.

Study of gene-specific DNA repair in the comet assay with padlock probes and rolling circle amplification.

We used padlock probes to study the rate of gene specific repair of three genes, OGG1 (8-oxoguanine-DNA glycosylase-1), XPD (xeroderma pigmentosum group D), and HPRT (hypoxanthine-guanine phosphoribosyltransferase) in human lymphocytes, in relation to the repair rate of Alu repeats and total genomic DNA. Padlock probes offer highly specific detection of short target sequences by combining detection by ligation and signal amplification. In this approach only genes in sequences containing strand breaks, which become single-stranded in the tail, are available for hybridisation. Thus the total number of signals from the padlock probes per comet gives a direct measure of the amount of damage (strand-breaks) present and allows the repair process to be monitored. This method could provide insights on the organisation of genomic DNA in the comet tail. Alu repeat containing DNA was repaired rapidly in comparison with total genomic DNA, and the studied genes were generally repaired more rapidly than the Alu repeats.

Structure-function analyses point to a polynucleotide-accommodating groove essential for APOBEC3A restriction activities.

Members of the human APOBEC3 family of editing enzymes can inhibit various mobile genetic elements. APOBEC3A (A3A) can block the retrotransposon LINE-1 and the parvovirus adeno-associated virus type 2 (AAV-2) but does not inhibit retroviruses. In contrast, APOBEC3G (A3G) can block retroviruses but has only limited effects on AAV-2 or LINE-1. What dictates this differential target specificity remains largely undefined. Here, we modeled the structure of A3A based on its homology with the C-terminal domain of A3G and further compared the sequence of human A3A to those of 11 nonhuman primate orthologues. We then used these data to perform a mutational analysis of A3A, examining its ability to restrict LINE-1, AAV-2, and foreign plasmid DNA and to edit a single-stranded DNA substrate. The results revealed an essential functional role for the predicted single-stranded DNA-docking groove located around the A3A catalytic site. Within this region, amino acid differences between A3A and A3G are predicted to affect the shape of the polynucleotide-binding groove. Correspondingly, transferring some of these A3A residues to A3G endows the latter protein with the ability to block LINE-1 and AAV-2. These results suggest that the target specificity of APOBEC3 family members is partly defined by structural features influencing their interaction with polynucleotide substrates.

Functional roles of the N- and C-terminal regions of the human mitochondrial single-stranded DNA-binding protein.

Biochemical studies of the mitochondrial DNA (mtDNA) replisome demonstrate that the mtDNA polymerase and the mtDNA helicase are stimulated by the mitochondrial single-stranded DNA-binding protein (mtSSB). Unlike Escherichia coli SSB, bacteriophage T7 gp2.5 and bacteriophage T4 gp32, mtSSBs lack a long, negatively charged C-terminal tail. Furthermore, additional residues at the N-terminus (notwithstanding the mitochondrial presequence) are present in the sequence of species across the animal kingdom. We sought to analyze the functional importance of the N- and C-terminal regions of the human mtSSB in the context of mtDNA replication. We produced the mature wild-type human mtSSB and three terminal deletion variants, and examined their physical and biochemical properties. We demonstrate that the recombinant proteins adopt a tetrameric form, and bind single-stranded DNA with similar affinities. They also stimulate similarly the DNA unwinding activity of the human mtDNA helicase (up to 8-fold). Notably, we find that unlike the high level of stimulation that we observed previously in the Drosophila system, stimulation of DNA synthesis catalyzed by human mtDNA polymerase is only moderate, and occurs over a narrow range of salt concentrations. Interestingly, each of the deletion variants of human mtSSB stimulates DNA synthesis at a higher level than the wild-type protein, indicating that the termini modulate negatively functional interactions with the mitochondrial replicase. We discuss our findings in the context of species-specific components of the mtDNA replisome, and in comparison with various prokaryotic DNA replication machineries.

Analysis of replication profiles reveals key role of RFC-Ctf18 in yeast replication stress response.

Maintenance of genome integrity relies on surveillance mechanisms that detect and signal arrested replication forks. Although evidence from budding yeast indicates that the DNA replication checkpoint (DRC) is primarily activated by single-stranded DNA (ssDNA), studies in higher eukaryotes have implicated primer ends in this process. To identify factors that signal primed ssDNA in Saccharomyces cerevisiae, we have screened a collection of checkpoint mutants for their ability to activate the DRC, using the repression of late origins as readout for checkpoint activity. This quantitative analysis reveals that neither RFC(Rad24) and the 9-1-1 clamp nor the alternative clamp loader RFC(Elg1) is required to signal paused forks. In contrast, we found that RFC(Ctf18) is essential for the Mrc1-dependent activation of Rad53 and for the maintenance of paused forks. These data identify RFC(Ctf18) as a key DRC mediator, potentially bridging Mrc1 and primed ssDNA to signal paused forks.

DNA translocation and unzipping through a nanopore: some geometrical effects.

This article explores the role of some geometrical factors on the electrophoretically driven translocations of macromolecules through nanopores. In the case of asymmetric pores, we show how the entry requirements and the direction of translocation can modify the information content of the blocked ionic current as well as the transduction of the electrophoretic drive into a mechanical force. To address these effects we studied the translocation of single-stranded DNA through an asymmetric alpha-hemolysin pore. Depending on the direction of the translocation, we measure the capacity of the pore to discriminate between both DNA orientations. By unzipping DNA hairpins from both sides of the pores we show that the presence of single strand or double strand in the pore can be discriminated based on ionic current levels. We also show that the transduction of the electrophoretic drive into a denaturing mechanical force depends on the local geometry of the pore entrance. Eventually we discuss the application of this work to the measurement of energy barriers for DNA unzipping as well as for protein binding and unfolding.

Single-stranded DNA binding by F TraI relaxase and helicase domains is coordinately regulated.

Transfer of conjugative plasmids requires relaxases, proteins that cleave one plasmid strand sequence specifically. The F plasmid relaxase TraI (1,756 amino acids) is also a highly processive DNA helicase. The TraI relaxase activity is located within the N-terminal approximately 300 amino acids, while helicase motifs are located in the region comprising positions 990 to 1450. For efficient F transfer, the two activities must be physically linked. The two TraI activities are likely used in different stages of transfer; how the protein regulates the transition between activities is unknown. We examined TraI helicase single-stranded DNA (ssDNA) recognition to complement previous explorations of relaxase ssDNA binding. Here, we show that TraI helicase-associated ssDNA binding is independent of and located N-terminal to all helicase motifs. The helicase-associated site binds ssDNA oligonucleotides with nM-range equilibrium dissociation constants and some sequence specificity. Significantly, we observe an apparent strong negative cooperativity in ssDNA binding between relaxase and helicase-associated sites. We examined three TraI variants having 31-amino-acid insertions in or near the helicase-associated ssDNA binding site. B. A. Traxler and colleagues (J. Bacteriol. 188:6346-6353) showed that under certain conditions, these variants are released from a form of negative regulation, allowing them to facilitate transfer more efficiently than wild-type TraI. We find that these variants display both moderately reduced affinity for ssDNA by their helicase-associated binding sites and a significant reduction in the apparent negative cooperativity of binding, relative to wild-type TraI. These results suggest that the apparent negative cooperativity of binding to the two ssDNA binding sites of TraI serves a major regulatory function in F transfer.

The 10-23 DNA enzyme generated by a novel expression vector mediate inhibition of taco expression in macrophage.

The 10-23 DNA enzyme (10-23 DNAzyme), a single-stranded DNA (ssDNA) molecule, can efficiently and specifically cleave almost any target RNA molecules. Therefore, it is regarded as one of the promising tools in gene therapy. However, there are still some obstacles, such as low efficiency of cellular uptake and instability in vivo, in its application. Taking advantage of the mechanism of Moloney mouse leukemia virus (MMLV) reverse transcriptase (RT), we investigate the construction of a novel ssDNA expression vector in this study. In order to improve the expression efficiency, the mmlv-rt gene and ODN-PMT (an oligodeoxynucleotide including other essential sequences for generating ssDNA) were cloned into a single plasmid under the control of 2 separated promoters. The ability of the vector to generate specific 10-23 DNAzyme in mammalian cell was tested by constructing a tryptophan-aspartate-containing coat protein (taco) gene-specific 10-23 DNAzyme expression plasmid. The potential of the expressed 10-23 DNAzyme to suppress TACO expression was also investigated. Our results indicated that this vector generates desired 10-23 DNAzyme in mammalian cells. The expressed 10-23 DNAzyme targeting taco gene can reduce TACO expression both at mRNA level (by 78.26%) and at protein level (by 75.30%).

Characterization of the mycobacterial AdnAB DNA motor provides insights into the evolution of bacterial motor-nuclease machines.

Mycobacterial AdnAB exemplifies a family of heterodimeric motor-nucleases involved in processing DNA double strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal UvrD-like motor domain and a C-terminal RecB-like nuclease module. Here we conducted a biochemical characterization of the AdnAB motor, using a nuclease-inactivated heterodimer. AdnAB is a vigorous single strand DNA (ssDNA)-dependent ATPase (k(cat) 415 s(-1)), and the affinity of the motor for the ssDNA cofactor increases 140-fold as DNA length is extended from 12 to 44 nucleotides. Using a streptavidin displacement assay, we demonstrate that AdnAB is a 3' --> 5' translocase on ssDNA. AdnAB binds stably to DSB ends. In the presence of ATP, the motor unwinds the DNA duplex without requiring an ssDNA loading strand. We integrate these findings into a model of DSB unwinding in which the "leading" AdnB and "lagging" AdnA motor domains track in tandem, 3' to 5', along the same DNA single strand. This contrasts with RecBCD, in which the RecB and RecD motors track in parallel along the two separated DNA single strands. The effects of 5' and 3' terminal obstacles on ssDNA cleavage by wild-type AdnAB suggest that the AdnA nuclease receives and processes the displaced 5' strand, while the AdnB nuclease cleaves the displaced 3' strand. We present evidence that the distinctive "molecular ruler" function of the ATP-dependent single strand DNase, whereby AdnAB measures the distance from the 5'-end to the sites of incision, reflects directional pumping of the ssDNA through the AdnAB motor into the AdnB nuclease. These and other findings suggest a scenario for the descent of the RecBCD- and AddAB-type DSB-processing machines from an ancestral AdnAB-like enzyme.

Downstream of FGF during mesoderm formation in Xenopus: the roles of Elk-1 and Egr-1.

Signalling by members of the FGF family is required for induction and maintenance of the mesoderm during amphibian development. One of the downstream effectors of FGF is the SRF-interacting Ets family member Elk-1, which, after phosphorylation by MAP kinase, activates the expression of immediate-early genes. Here, we show that Xenopus Elk-1 is phosphorylated in response to FGF signalling in a dynamic pattern throughout the embryo. Loss of XElk-1 function causes reduced expression of Xbra at neurula stages, followed by a failure to form notochord and muscle and then the partial loss of trunk structures. One of the genes regulated by XElk-1 is XEgr-1, which encodes a zinc finger transcription factor: we show that phosphorylated XElk-1 forms a complex with XSRF that binds to the XEgr-1 promoter. Superficially, Xenopus tropicalis embryos with reduced levels of XEgr-1 resemble those lacking XElk-1, but to our surprise, levels of Xbra are elevated at late gastrula stages in such embryos, and over-expression of XEgr-1 causes the down-regulation of Xbra both in whole embryos and in animal pole regions treated with activin or FGF. In contrast, the myogenic regulatory factor XMyoD is activated by XEgr-1 in a direct manner. We discuss these counterintuitive results in terms of the genetic regulatory network to which XEgr-1 contributes.

SSB as an organizer/mobilizer of genome maintenance complexes.

When duplex DNA is altered in almost any way (replicated, recombined, or repaired), single strands of DNA are usually intermediates, and single-stranded DNA binding (SSB) proteins are present. These proteins have often been described as inert, protective DNA coatings. Continuing research is demonstrating a far more complex role of SSB that includes the organization and/or mobilization of all aspects of DNA metabolism. Escherichia coli SSB is now known to interact with at least 14 other proteins that include key components of the elaborate systems involved in every aspect of DNA metabolism. Most, if not all, of these interactions are mediated by the amphipathic C-terminus of SSB. In this review, we summarize the extent of the eubacterial SSB interaction network, describe the energetics of interactions with SSB, and highlight the roles of SSB in the process of recombination. Similar themes to those highlighted in this review are evident in all biological systems.