Tight Interplay between Replication Stress and Competence Induction in Streptococcus pneumoniae

Cells respond to genome damage by inducing restorative programs, typified by the SOS response of Escherichia coli. Streptococcus pneumoniae (the pneumococcus), with no equivalent to the SOS system, induces the genetic program of competence in response to many types of stress, including genotoxic drugs. The pneumococcal competence regulon is controlled by the origin-proximal, auto-inducible comCDE operon. It was previously proposed that replication stress induces competence through continued initiation of replication in cells with arrested forks, thereby increasing the relative comCDE gene dosage and expression and accelerating the onset of competence. We have further investigated competence induction by genome stress. We find that absence of RecA recombinase stimulates competence induction, in contrast to SOS response, and that double-strand break repair (RexB) and gap repair (RecO, RecR) initiation effectors confer a similar effect, implying that recombinational repair removes competence induction signals. Failure of replication forks provoked by titrating PolC polymerase with the base analogue HPUra, over-supplying DnaA initiator, or under-supplying DnaE polymerase or DnaC helicase stimulated competence induction. This induction was not correlated with concurrent changes in origin-proximal gene dosage. Our results point to arrested and unrepaired replication forks, rather than increased comCDE dosage, as a basic trigger of pneumococcal competence.

Tetrameric UvrD helicase is located at the E. coli replisome due to frequent replication blocks

ABSTRACTDNA replication in all organisms must overcome nucleoprotein blocks to complete genome duplication. Accessory replicative helicases in Escherichia coli, Rep and UvrD, help replication machinery overcome blocks by removing incoming nucleoprotein complexes or aiding the re-initiation of replication. Mechanistic details of Rep function have emerged from recent live cell studies, however, the activities of UvrD in vivo remain unclear. Here, by integrating biochemical analysis and super-resolved single-molecule fluorescence microscopy, we discovered that UvrD self-associates into a tetramer and, unlike Rep, is not recruited to a specific replisome protein despite being found at approximately 80% of replication forks. By deleting rep and DNA repair factors mutS and uvrA, perturbing transcription by mutating RNA polymerase, and antibiotic inhibition; we show that the presence of UvrD at the fork is dependent on its activity. This is likely mediated by the very high frequency of replication blocks due to DNA bound proteins, including RNA polymerase, and DNA damage. UvrD is recruited to sites of nucleoprotein blocks via distinctly different mechanisms to Rep and therefore plays a more important and complementary role than previously realised in ensuring successful DNA replication.

Why Do Centromeres Evolve So Fast: BIR Replication, Hypermutation, Transposition, and Molecular-Drive

Centromeres are among the fastest evolving genomic regions in a diverse array of organisms. The evolutionary process driving this rapid evolution has not been unambiguously established. Here I integrate diverse information to motivate a model in which centromeres evolve rapidly because of their intrinsic molecular phenotype: they tightly bind centromeric proteins throughout the cell cycle. DNA-bound proteins have been shown to cause stalling and collapse of DNA replication forks in many genomic regions, including centromeres. Collapsed replication forks generate one-sided double strand breaks (DSBs) that are repaired by the Break-Induced Repair (BIR) pathway. Here I show why this repair is expected to generate tandem repeat structure and three key features at centromeres: i) increased nucleotide substitution mutation rates, ii) out-of- register re-initiation of replication that leads to indels spanning one or more repeat units, and iii) elevated rates of large and small transpositions within centromeres and between genomic regions. These phenotypes lead to: i) a rapid rate of nucleotide substitutions within a clade of centromeric sequences, ii) continual turnover of monomers within centromeres that fosters molecular-drift and molecular-drive, and iii) recurrent quantum leaps in centromere sequence due to the formation of mosaic monomers and new sequences transposed into non-homologous centromeres. These features are plausibly the major reason centromeres evolve so rapidly. I also speculate on how the DNA sequence of centromeres might perpetually coevolve with the protein sequence of histone CENH3 –the major epigenetic mark of centromeres.

Chromosome segregation in B. subtilis is highly heterogeneous

Objective The bacterial cell cycle comprises initiation of replication and ensuing elongation, concomitant chromosome segregation (in some organisms with a delay termed cohesion), and finally cell division. By quantifying the number of origin and terminus regions in exponentially growing Bacillus subtilis cells, and after induction of DNA damage, we aimed at determining cell cycle parameters at different growth rates at a single cell level. Results B. subtilis cells are mostly mero-oligoploid during fast growth and diploid during slow growth. However, we found that the number of replication origins and of termini is highly heterogeneous within the cell population at two different growth rates, and that even at slow growth, a majority of cells attempts to maintain more than a single chromosome at all times of the cell cycle. Heterogeneity of chromosome copy numbers may reflect different subpopulations having diverging growth rates even during exponential growth conditions. Cells continued to initiate replication and segregate chromosomes after induction of DNA damage, as judged by an increase in origin numbers per cell, showing that replication and segregation are relatively robust against cell cycle perturbation.

Chromosome segregation in B. subtilis is highly heterogeneous

Objective: The bacterial cell cycle comprises initiation of replication and ensuing elongation, concomitant chromosome segregation (in some organisms with a delay termed cohesion), and finally cell division. By quantifying the number of origin and terminus regions in exponentially growing Bacillus subtilis cells, and after induction of DNA damage, we aimed at determining cell cycle parameters at different growth rates at a single cell level.Results: B. subtilis cells are mostly mero-oligoploid during fast growth and diploid during slow growth. However, we found that the number of replication origins and of termini is highly heterogeneous within the cell population at two different growth rates, and that even at slow growth, a majority of cells attempts to maintain more than a single chromosome at all times of the cell cycle. Heterogeneity of chromosome copy numbers may reflect different subpopulations having diverging growth rates even during exponential growth conditions. Cells continued to initiate replication and segregate chromosomes after induction of DNA damage, as judged by an increase in origin numbers per cell, showing that replication and segregation are relatively robust against cell cycle perturbation.

Chromosome segregation in B. subtilis is highly heterogeneous

Objective The bacterial cell cycle comprises initiation of replication and ensuing elongation, concomitant chromosome segregation (in some organisms with a delay termed cohesion), and finally cell division. By quantifying the number of origin and terminus regions in exponentially growing Bacillus subtilis cells, and after induction of DNA damage, we aimed at determining cell cycle parameters at different growth rates at a single cell level. Results B. subtilis cells are mostly mero-oligoploid during fast growth and diploid during slow growth. However, we found that the number of replication origins and of termini is highly heterogeneous within the cell population at two different growth rates, and that even at slow growth, a majority of cells attempts to maintain more than a single chromosome at all times of the cell cycle. Heterogeneity of chromosome copy numbers may reflect different subpopulations having diverging growth rates even during exponential growth conditions. Cells continued to initiate replication and segregate chromosomes after induction of DNA damage, as judged by an increase in origin numbers per cell, showing that replication and segregation are relatively robust against cell cycle perturbation.

CrispR method for efficient tagging in S2 cells

Here we present a modified technique for improved efficient tagging in Drosophila S2 cells. it is a combination of a technique developed by Forstemann lab (Mainly homologous recombination design using pMH3, pMH4 and modified pMH to include UAS sequence and Myc tag) and Bassett lab for the sgRNA maufacture using pAc-sgRNA-Cas9 vector and following their protocol. Design of the homologous recombination templatenew vectors for Homologous recombination have also been engineered in order to get new tags: Replacement of GFP by excision of GFP from pMH3 using and replacement by UAS sequence or myc tag. UAS tags have been eficiently inserted at a specific location in the genome in order to analyse the binding of replication proteins and their effect on initiation of replication activity.

Ανάπτυξη επισωματικών φορέων γονιδιακής μεταφοράς που βασίζονται σε χρωμοσωματικά στοιχεία του ανθρώπου

Η γονιδιακή θεραπεία αποτελεί μια θεραπευτική παρέμβαση στην οποία το φάρμακο είναι DNA ή RNA (διαγονίδιο) και υπόσχεται ίαση σε μια σειρά μονογονιδιακών ασθενειών και κακοηθειών. Η γονιδιακή μεταφορά του επιθυμητού διαγονιδίου επιτυγχάνεται με την χρήση φορέων κυρίως ιϊκών που καλούνται να υπερκεράσουν τους φυσικούς φραγμούς και να καθοδηγήσουν το γονίδιο στο πυρήνα του κυττάρου. Οι επισωματικοί φορείς αποτελούν μια εναλλακτική προσέγγιση στους ήδη καθιερωμένους ιϊκούς φορείς κυρίως στο ότι δεν ενσωματώνονται στο DNA των κυττάρων. Οι φορείς αυτοί τελευταία στηρίζονται στην ύπαρξη μιας χρωμοσωμικής αλληλουχίας που ονομάζεται S/MAR (scaffold/ matrix attachment region) για την συγκράτηση τους στον πυρήνα του κυττάρου. Περαιτέρω, ο συνδυασμός της αλληλουχίας S/MAR με ένα ακόμα χρωμοσωμικό στοιχείο το IR (Initiation of replication) φαίνεται να βελτιώνει τις ιδιότητες του φορέα σε κυτταρικές σειρές και σε αρχέγονα αιμοποιητικά κύτταρα. Στην παρούσα διατριβή δημιουργήσαμε ένα νέο φορέα (pEPI-IR/β-globin) ο οποίος φέρει ως διαγονίδιο το γονίδιο της β-σφαιρίνης της αιμοσφαιρίνης του ανθρώπου, τις ρυθμιστικές αλληλουχίες αυτού αλλά και τις δύο χρωμοσωμικές αλληλουχίες S/MAR και IR. Mελετήσαμε την λειτουργικότητα του φορέα αυτού και προσδιορίστηκε ότι ο φορέας pEPI-IR/β-globin μπορεί να διαμολύνει επιτυχώς κύτταρα της αιματολογικής κυτταρικής σειράς Κ562 με την διαδικασία της nucleofection. Είναι σε θέση να εγκαταστήσει σταθερή διαμόλυνση στα κύτταρα αυτά και να ανιχνεύεται μετά από τουλάχιστον 100 γενεές (σε 1,3 αντίγραφα ανά κύτταρο), ενώ κατά το διάστημα αυτό διατηρεί την επισωματική του κατάσταση. Διατηρείται η λειτουργικότητα της κασέτας του γονιδίου της β-σφαιρίνης και η έκφραση του ανιχνεύεται τόσο σε επίπεδο mRNA με real-time PCR όσο και σε επίπεδο πεπτιδίου με ανοσοφθορισμό in situ. Συμπερασματικά ο pEPI-IR/β-globin αποτελεί έναν νέο επισωματικό φορέα ο οποίος είναι λειτουργικός σε κύτταρα Κ562 και μπορεί να αποτελέσει την βάση ανάπτυξης επισωματικών φορέων της β-σφαιρίνης για περαιτέρω πειράματα για τους σκοπούς της γονιδιακής θεραπείας θαλασσαιμικών συνδρόμων.

Modeling of DNA replication in rapidly growing bacteria with one and two replication origins

In rapidly growing bacteria initiation of DNA replication occurs at intervals shorter than the time required for completing genome duplication, leading to overlapping rounds of replication. We propose a mathematical model of DNA replication defined by the periodicity of replication initiation. Our model predicts that a steeper gradient of the replication profile is to be expected in origin proximal regions due to the overlapping rounds of synthesis. By comparing our model with experimental data from a strain with an additional replication origin, we predict defined alterations to replication parameters: (i) a reduced fork velocity when there were twice as many forks as normal; (ii) a slower fork speed if forks move in a direction opposite to normal, in line with head-on replication-transcription collisions being a major obstacle for fork progression; (iii) slower cell doubling for a double origin strain compared to wild-type cells; and (iv) potentially an earlier initiation of replication at the ectopic origin than at the natural origin, which, however, does not a˙ect the overall time required to complete synthesis.