An onset model of mutually catalytic self-replicative systems formed by an assembly of polynucleotides

Self-replicability is the unique attribute observed in all the living organisms and the question how the life was physically initiated could be equivalent to the question how self-replicating informative polymers were formed in the abiotic material world. It has been suggested that the present DNA and proteins world was preceded by RNA world in which genetic information of RNA molecules was replicated by the mutual catalytic function of RNA molecules. However, the important question how the transition occurred from a material world to the very early pre-RNA world remains unsolved experimentally nor theoretically. We present an onset model of mutually catalytic self-replicative systems formed in an assembly of polynucleotides. A quantitative expression of the critical condition for the onset of growing fluctuation towards self-replication in this model is obtained by analytical and numerical calculations.Comment: 13 pages, 10 figures. Accepted for publication in Physical Review

A global profile of replicative polymerase usage

Three eukaryotic DNA polymerases are essential for genome replication. Polymerase (Pol) α–primase initiates each synthesis event and is rapidly replaced by processive DNA polymerases: Polɛ replicates the leading strand, whereas Polδ performs lagging-strand synthesis. However, it is not known whether this division of labor is maintained across the whole genome or how uniform it is within single replicons. Using Schizosaccharomyces pombe, we have developed a polymerase usage sequencing (Pu-seq) strategy to map polymerase usage genome wide. Pu-seq provides direct replication-origin location and efficiency data and indirect estimates of replication timing. We confirm that the division of labor is broadly maintained across an entire genome. However, our data suggest a subtle variability in the usage of the two polymerases within individual replicons. We propose that this results from occasional leading-strand initiation by Polδ followed by exchange for Polɛ

Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy

Development of single-molecule localization microscopy techniques has allowed nanometre scale localization accuracy inside cells, permitting the resolution of ultra-fine cell structure and the elucidation of crucial molecular mechanisms. Application of these methodologies to understanding processes underlying DNA replication and repair has been limited to defined in vitro biochemical analysis and prokaryotic cells. In order to expand these techniques to eukaryotic systems, we have further developed a photo-activated localization microscopy-based method to directly visualize DNA-associated proteins in unfixed eukaryotic cells. We demonstrate that motion blurring of fluorescence due to protein diffusivity can be used to selectively image the DNA-bound population of proteins. We designed and tested a simple methodology and show that it can be used to detect changes in DNA binding of a replicative helicase subunit, Mcm4, and the replication sliding clamp, PCNA, between different stages of the cell cycle and between distinct genetic backgrounds

Optimisation of the Schizosaccharomyces pombe urg1 expression system

The ability to study protein function in vivo often relies on systems that regulate the presence and absence of the protein of interest. Two limitations for previously described transcriptional control systems that are used to regulate protein expression in fission yeast are: the time taken for inducing conditions to initiate transcription and the ability to achieve very low basal transcription in the "OFF-state". In previous work, we described a Cre recombination-mediated system that allows the rapid and efficient regulation of any gene of interest by the urg1 promoter, which has a dynamic range of approximately 75-fold and which is induced within 30-60 minutes of uracil addition. In this report we describe easy-to-use and versatile modules that can be exploited to significantly tune down P urg1 "OFF-levels" while maintaining an equivalent dynamic range. We also provide plasmids and tools for combining P urg1 transcriptional control with the auxin degron tag to help maintain a null-like phenotype. We demonstrate the utility of this system by improved regulation of HO-dependent site-specific DSB formation, by the regulation Rtf1-dependent replication fork arrest and by controlling Rhp18(Rad18)-dependent post replication repair

PCNA ubiquitylation ensures timely completion of unperturbed DNA replication in fission yeast

PCNA ubiquitylation on lysine 164 is required for DNA damage tolerance. In many organisms PCNA is also ubiquitylated in unchallenged S phase but the significance of this has not been established. Using Schizosaccharomyces pombe, we demonstrate that lysine 164 ubiquitylation of PCNA contributes to efficient DNA replication in the absence of DNA damage. Loss of PCNA ubiquitylation manifests most strongly at late replicating regions and increases the frequency of replication gaps. We show that PCNA ubiquitylation increases the proportion of chromatin associated PCNA and the co-immunoprecipitation of Polymerase δ with PCNA during unperturbed replication and propose that ubiquitylation acts to prolong the chromatin association of these replication proteins to allow the efficient completion of Okazaki fragment synthesis by mediating gap filling

Polymerase δ replicates both strands after homologous recombination-dependent fork restart

To maintain genetic stability DNA must be replicated only once and replication completed even when individual replication forks are inactivated. Because fork inactivation is common, the passive convergence of an adjacent fork is insufficient to rescue all inactive forks. Thus, eukaryotic cells have evolved homologous recombination-dependent mechanisms to restart persistent inactive forks. Completing DNA synthesis via Homologous Recombination Restarted Replication (HoRReR) ensures cell survival, but at a cost. One such cost is increased mutagenesis caused by HoRReR being more error prone than canonical replication. This increased error rate implies that the HoRReR mechanism is distinct from that of a canonical fork. Here we exploit the fission yeast Schizosaccharomyces pombe to demonstrate that a DNA sequence duplicated by HoRReR during S phase is replicated semi-conservatively, but that both the leading and lagging strands are synthesised by DNA polymerase delta

Action Spectrum Analysis of UVR Genotoxicity for Skin: The Border Wavelengths between UVA and UVB Can Bring Serious Mutation Loads to Skin

UVR causes erythema, which has been used as a standardized index to evaluate the risk of UVR for human skin.However, the genotoxic significance of erythema has not been elucidated clearly. Here, we characterized thewavelength dependence of the genotoxic and erythematic effects of UVR for the skin by analyzing the inductionkinetics of mutation and inflammation in mouse skin using lacZ-transgenic mice and monochromatic UVRsources. We determined their action spectra and found a close correlation between erythema and an epidermisspecific antigenotoxic response, mutation induction suppression (MIS), which suppressed the mutant frequencies (MFs) to a constant plateau level only 2–3-fold higher than the background MF at the cost of apoptotic cell death, suggesting that erythema may represent the threshold beyond which the antigenotoxic but tissuedestructive MIS response commences. However, we unexpectedly found that MIS attenuates remarkably at the border wavelengths between UVA and UVB around 315 nm, elevating the MF plateaus up to levels B40-foldhigher than the background level. Thus, these border wavelengths can bring heavier mutation loads to the skinthan the otherwise more mutagenic and erythematic shorter wavelengths, suggesting that erythema-based UVRrisk evaluation should be reconsidered

Principals of RCME and plasmids created.

<p>(<b>A</b>). Schematic showing the process of RCME (Watson 2008): (i) starting with a base strain in which the <i>urg1</i> ORF is replaced by an antibiotic marker (each of <i>hphMX6</i>, <i>natMX6</i> and <i>kanMX6</i> are available) that is flanked by (incompatible) loxP and loxM3 sites, a plasmid (ii) is introduced. This plasmid contains the cloned gene of interest and any tagging sequences positioned between loxP and loxM3 sites. It also expresses Cre recombinase. Site-directed recombination next exchanges the sequences between the plasmid and the chromosome (iii). Successful exchange can easily be identified by loss of the antibiotic marker, typically seen in greater than 50% of cells. Plasmid loss in these colonies is then confirmed by replica plating to verify colonies are leu⁻. In our experience, all of these are successful integrants. (<b>B</b>). Plasmid for expression of untagged sequences (NO DSR) as previously published <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083800#pone.0083800-Watson1" target="_blank">[7]</a>. Shown is a schematic of the sequence between loxP (P) and loxM3 (M3) for pAW8E<i>Nde</i>I. A start codon is formed from an <i>Nde</i>I site. (<b>C</b>) Equivalent schematic of pAW8E<i>Nde</i>I containing various DSR sequences. (<b>D</b>) Schematic of plasmid used to express proteins with either a yEGFP tag, a 3xHA tag or an HA combined with an IAA17 degron tag (HAIAA17) (all with NO DSR). L  =  poly-tyrosine–glycine–serine (TGS) linker: TAG  =  yEGFP, 3xHA or HAIAA17 protein tag. (<b>E</b>) Equivalent schematic of pAW8E<i>Nde</i>I C-terminal tagging plasmids that also contain various DSR sequences. HA  =  human influenza hemagglutinin protein tag, yEGFP  =  yeast codon optimised green fluorescent protein, HAIAA17  =  Degron from Arabidopsis thaliana transcription repressor.</p