5′CAG and 5′CTG Repeats Create Differential Impediment to the Progression of a Minimal Reconstituted T4 Replisome Depending on the Concentration of dNTPs

Instability of repetitive sequences originates from strand misalignment during repair or replicative DNA synthesis. To investigate the activity of reconstituted T4 replisomes across trinucleotide repeats (TNRs) during leading strand DNA synthesis, we developed a method to build replication miniforks containing a TNR unit of defined sequence and length. Each minifork consists of three strands, primer, leading strand template, and lagging strand template with a 5′ single-stranded (ss) tail. Each strand is prepared independently, and the minifork is assembled by hybridization of the three strands. Using these miniforks and a minimal reconstituted T4 replisome, we show that during leading strand DNA synthesis, the dNTP concentration dictates which strand of the structure-forming 5′CAG/5′CTG repeat creates the strongest impediment to the minimal replication complex. We discuss this result in the light of the known fluctuation of dNTP concentration during the cell cycle and cell growth and the known concentration balance among individual dNTPs

Polypurine reverse-Hoogsteen (PPRH) oligonucleotides can form triplexes with their target sequences even under conditions where they fold into G-quadruplexes

Polypurine reverse-Hoogsteen (PPRH) oligonucleotides are non-modified DNA molecules composed of two mirror-symmetrical polypurine stretches linked by a five-thymidine loop. They can fold into reverse-Hoogsteen hairpins and bind to their polypyrimidine target sequence by Watson-Crick bonds forming a three-stranded structure. They have been successfully used to knockdown gene expression and to repair single-point mutations in cells. In this work, we provide an in vitro characterization (UV and fluorescence spectroscopy, gel electrophoresis and nuclease assays) of the structure and stability of two repair-PPRH oligonucleotides and of the complexes they form with their single-stranded targets. We show that one PPRH oligonucleotide forms a hairpin, while the other folds, in potassium, into a guanine-quadruplex (G4). However, the hairpin-prone oligonucleotide does not form a triplex with its single-stranded target, while the G4-prone oligonucleotide converts from a G4 into a reverse-Hoogsteen hairpin forming a triplex with its target sequence. Our work proves, in particular, that folding of a PPRH oligonucleotide into a G4 does not necessarily impair sequence-specific DNA recognition by triplex formation. It also illustrates an original example of DNA structural conversion of a G4 into a reverse-Hoogsteen hairpin driven by triplex formation; this kind of conversion might occur at particular loci of genomic DNA

A Newly Identified Essential Complex, Dre2-Tah18, Controls Mitochondria Integrity and Cell Death after Oxidative Stress in Yeast

A mutated allele of the essential gene TAH18 was previously identified in our laboratory in a genetic screen for new proteins interacting with the DNA polymerase delta in yeast [1]. The present work shows that Tah18 plays a role in response to oxidative stress. After exposure to lethal doses of H2O2, GFP-Tah18 relocalizes to the mitochondria and controls mitochondria integrity and cell death. Dre2, an essential Fe/S cluster protein and homologue of human anti-apoptotic Ciapin1, was identified as a molecular partner of Tah18 in the absence of stress. Moreover, Ciapin1 is able to replace yeast Dre2 in vivo and physically interacts with Tah18. Our results are in favour of an oxidative stress-induced cell death in yeast that involves mitochondria and is controlled by the newly identified Dre2-Tah18 complex

Probing hyper-negatively supercoiled mini-circles with nucleases and DNA binding proteins.

It is well accepted that the introduction of negative supercoils locally unwinds the DNA double helix, influencing thus the activity of proteins. Despite the use of recent methods of molecular dynamics simulations to model the DNA supercoiling-induced DNA deformation, the precise extent and location of unpaired bases induced by the negative supercoiling have never been investigated at the nucleotide level. Our goals in this study were to use radiolabeled double-stranded DNA mini-circles (dsMCs) to locate the unpaired bases on dsMCs whose topology ranged from relaxed to hyper-negatively supercoiled states, and to characterize the binding of proteins involved in the DNA metabolism. Our results show that the Nuclease SI is nearly ten times more active on hyper-negatively supercoiled than relaxed DNA. The structural changes responsible for this stimulation of activity were mapped for the first time with a base pair resolution and shown to be subtle and distributed along the entire sequence. As divalent cations modify the DNA topology, our binding studies were conducted with or without magnesium. Without magnesium, the dsMCs topoisomers mostly differ by their twist. Under these conditions, the Escherichia coli topoisomerase I weakly binds relaxed dsMCs and exhibits a stronger binding on negatively and hyper-negatively supercoiled dsMCs than relaxed dsMCs, with no significant difference in the binding activity among the supercoiled topoisomers. For the human replication protein A (hRPA), the more negatively supercoiled is the DNA, the better the binding, illustrating the twist-dependent binding activity for this protein. The presence of magnesium permits the dsMCs to writhe upon introduction of negative supercoiling and greatly modifies the binding properties of the hRPA and Escherichia coli SSB on dsMCs, indicating a magnesium-dependent DNA binding behavior. Finally, our experiments that probe the topology of the DNA in the hRPA-dsMC complexes show that naked and hRPA-bound dsMCs have the same topology

Use of double-stranded DNA mini-circles to characterize the covalent topoisomerase-DNA complex

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Single-stranded DNA binding proteins unwind the newly synthesized double-stranded DNA of model miniforks.

International audienceSingle-stranded DNA binding (SSB) proteins are essential proteins of DNA metabolism. We characterized the binding of the bacteriophage T4 SSB, Escherichia coli SSB, human replication protein A (hRPA), and human hSSB1 proteins onto model miniforks and double-stranded-single-stranded (ds-ss) junctions exposing 3' or 5' ssDNA overhangs. T4 SSB proteins, E. coli SSB proteins, and hRPA have a different binding preference for the ss tail exposed on model miniforks and ds-ss junctions. The T4 SSB protein preferentially binds substrates with 5' ss tails, whereas the E. coli SSB protein and hRPA show a preference for substrates with 3' ss overhangs. When interacting with ds-ss junctions or miniforks, the T4 SSB protein, E. coli SSB protein, and hRPA can destabilize not only the ds part of a ds-ss junction but also the daughter ds arm of a minifork. The T4 SSB protein displays these unwinding activities in a polar manner. Taken together, our results position the SSB protein as a potential key player in the reversal of a stalled replication fork and in gap repair-mediated repetitive sequence expansion