L’initiation de la réplication dans les bactéries (Jean-Luc Ferat)

Dans les bactéries, la réplication est initiée à une origine unique par la protéine initiatrice, DnaA. La liaison ordonnée de DnaA à l’origine conduit à l’ouverture local de la double helice d’ADN et au recrutement des hélicases réplicatives directement sur l’ADN simple brin. Cette étape est fortement régulée et assistée par une co-hélicase, qui synchronise le chargement des hélicases réplicatives, assurant ainsi l’initiation bidirectionnelle de la réplication (BRI). Nous cherchons à comprendre 1) comment la co-hélicase DciA active de manière synchrone les hélicases réplicatives pendant l’initiation de la réplication et 2) comment la réplication initiée en absence de DciA conduit à la dégradation pathologique du chromosome entier. Nous avons développé un large éventail d’approches expérimentales (cytologiques, génétique, génomique, bioinformatique) pour répondre à ces deux questions. Nous utilisons  Escherichia coli et Vibrio cholerae comme organismes modèles.

Fig.1 Distribution of domesticated dnaC and dciA in the bacterial domain. Distribution of the bacterial phyla investigated on a 16S rRNA-based circular phylogenetic tree (Methods). When the presence of dnaC/I or dciA within a phylum (bold) was not homogeneous, classes (underlined) or orders (normal) are specified. Numbers boxed refer to the number of species’ genomes (and not strains) containing domesticated dnaC/I (fuchsia), dciA (turquoise) or neither (black). fuchsia / turquoise circles: orders in which dciA and dnaC/I genes were identified. The order Thiotrichales is identified with a green/black circle because most species lack dciA and dnaC/I. ① Gammaproteobacteria, ② Firmicutes, ③ Aquificae, ④ Chloroflexi and ⑤ Tenericutes.

In bacteria, replication is initiated at a single origin by the initiator protein, DnaA. The ordered binding of DnaA at the origin creates a local unwinding of the double helix that enables the recruitment of the replicative helicase, DnaB. Replisomes are then assembled on this nucleoprotein platform to trigger Bidirectional Replication Initiation. Contrary to eukaryotes, bacteria load directly their helicase on single strand DNA during replication initiation. This process is highly regulated and assisted by co-helicases in the three domains of life. Bacterial co-helicases are diverse. The first family that was studied, DnaC/I (DnaC in Escherichia coli and DnaI in Bacillus subtilis), is present in only a few orders of bacteria and results from the domestication of a phage gene (Brézellec et al., 2016). The main family of co-helicase is DciA. It was discovered only a few years ago although it is present in the vas majority of bacterial orders (Figure 1). We established that DciA, is the ancestral bacterial co-helicase (Brézellec et al., 2016, 2017).

We conducted a biochemical investigation of DciA together with its cognate replicative helicase to compare its activity to that of DnaC of E. coli. This study revealed surprisingly that contrary to DnaC/I the loading of the replicative helicase is not dependent on DciA. In other words, the replicative helicase of dciA-sepcies can self-load on DNA. At most, DciA improves the loading efficiency (Figure 2) (Marsin et al., 2021).

Fig.2 VcDciA stimulates the loading of VcDnaB on DNA. a, SPR analysis of the loading of VcDnaB on DNA in presence of VcDciA. Experiments were conducted as described in Figure 1B in presence of VcDnaB at 625nM. Different VcDnaB/VcDciA ratios (from 6/0 to 6/6 in subunits) were analyzed. ΔB represents the mass of VcDciA proteins that dissociated immediately after the loading of the VcDciA•VcDnaB complex on DNA. b, DNA unwinding kinetics in presence of VcDciA. The experiments were carried out as described in Figure 1D, except that VcDciA (variable concentrations) was added to VcDnaB (625 nM). The types of migration products are indicated on the right. c, DNA unwinding quantification in presence of VcDciA. The procedure is the same as that described in Figure 1E. The error bars correspond to the mean +/- SEM of three independent experiments. d, Helicase loading in absence of Mg²⁺. The experiment is carried out in absence of Mg to prevent the activation of the helicase and its translocation on DNA in the 5’ → 3’ direction until its release. ATP was added for it is required for the hexamerization of the helicase.

Flow cytometry analysis of [dciA^–] cells confirmed that the replicative helicase of V. cholerae can load itself in vivo without DciA. A genomic approach intended to assessing the replication status of [dciA^–] cells revealed that DciA is essential to ensure Bidirectional Replication Initiation (BRI), likely by synchronizing the loading of the replicative helicase during replication initiation. We showed that in absence of DciA, replication is initiated unidirectionally and that this Unidirectional Replication Initiation (URI) is infallibly followed by a catastrophic sequence of events ending in chromosome degradation (Besombes et al. submitted) (Figure 3).

Fig.3 Replication is initiated unidirectionally at ori1 on Chr1 in [dciA^–] cells. a) MF profiles of ori1⁺ dciA⁺ (top )and ori1⁺ [dciA^–] populations (bottom) maintained in continuous growth in minimal medium at 30°C for 12h before DNA extraction and sequence. The reads were aligned against V. cholerae reference genome (using Burrows-Wheeler Aligner) to assign a number of reads per genome position (marker frequency).  The value for each position of the genome corresponds to the marker frequency average of 1000 consecutive positions (1kb) surrounding it. log2 of these values are used to correct for the exponential growth of the cells (MF). b) same as in a), except that the origin of replication was inverted (ori^INV). The left and right replichore of Chr1 and Chr2 are colored in blue, purple, green and orange, respectively.

Our research program is aimed now at understanding how unprogrammed URI leads to chromosome degradation and how DciA loads and activates synchronously the replicative helicases during replication initiation. We address these questions 1) by inventorying the diverse routes followed by [dciA^–] cells and dissecting them frame by frame through a large diversity of approaches (fluorescent microscopy, digital PCR, PFGE, NGS, etc.), 2) by deciphering the functional network of DciA (two hybrid, Tn-seq) and 3) by creating a strain of V. cholerae in which dciA is replaced by dnaC to trace the modifications and their succession during adaptation to a new co-helicase and eventually to understand the mechanism underlying BRI.

We use two bacterial model organisms (Vibrio cholerae and Escherichia coli) to address these two questions, and developed a large spectrum of experimental approaches (cytological, genetic, genomic, bioinformatic).

References

Besombes A, Adam Y, Possoz C, Junier Y, Barre FX, Ferat JL. (submitted) Bidirectional replication initiation prevents chromosome degradation

Marsin S, Adam Y, Cargemel C, Andreani J, Baconnais S, Legrand P, Li de la Sierra-Gallay I, Humbert A, Aumont-Nicaise M, Velours C, Ochsenbein F, Durand D, Le Cam E, Walbott H, Possoz C, Quevillon-Cheruel S, Ferat JL. (2021) Study of the DnaB:DciA interplay reveals insights into the primary mode of loading of the bacterial replicative helicase. Nucleic Acids Res. 49:6569-6586

Brézellec P, Petit MA, Pasek S, Vallet-Gely I, Possoz C, Ferat JL. (2017) Domestication of Lambda Phage Genes into a Putative Third Type of Replicative Helicase Matchmaker. Genome Biol Evol. 9:1561-1566

Brézellec P, Vallet-Gely I, Possoz C, Quevillon-Cheruel S, Ferat JL. (2016) DciA is an ancestral replicative helicase operator essential for bacterial replication initiation. Nat Commun. 7:13271

Responsable :

Marie-Noëlle PRIOLEAU
Téléphone : +33 (0)157278102
Email : marie-noelle.prioleau(at)ijm.fr

Membres de l’équipe :

Caroline DONCARLI, Ingénieure en biologie

Jean-Luc FERAT, Professeur à l’université  Paris Cité

Kathrin MARHEINEKE, Directrice de recherche CNRS

Thibault CORNELOUP, Post-doctorant

Amélie BESOMBES, Doctorante

Aurélie MASSON, Doctorante

Juliette MANDELBROJT, Doctorante

Théo BARET, Ingénieur de recherche

Publications

Revues

– « Rôle de l’insulateur 5’HS4 du poulet dans la régulation de la réplication » soutenue en Juin 2009 par Vahideh Hassan-Zadeh.

– « Mécanismes moléculaires de l’initiation de la réplication » soutenue en Décembre 2010 par Françoise Meisch.

– « Identification de séquences cis-nucléiques nécessaires à l’initiation de la réplication chez les vertébrés » soutenue en Juin 2014 par Anne-Laure Valton.

– « Construction d’un domaine synthétique de réplication précoce et impact sur la structure chromatinienne et la permissivité transcriptionnelle » soutenue en Septembre 2015 par Caroline Tonnerre-Doncarli Brossas.

– « Mécanismes moléculaires impliqués dans la régulation du moment de déclenchement des origines de réplication » soutenue en Novembre 2016 par Antonin Counillon.

– « Rôle des G-quadruplexes dans la spécification des origines de réplication chez les vertébrés » soutenue en Septembre 2016 par Marc Laurent.

– « Etude moléculaire des éléments cis-régulateurs et de l’organisation de la chromatine des origines de réplication » soutenue en Septembre 2020 par Jérémy Poulet-Benedetti.

Nationales :

– Imagerie cellulaire et cytométrie en flux : Plateforme Imago-Seine, Institut Jacques Monod

– Organoïdes corticaux : Plateforme enSCORE, Labex Who am I ?

– Bio-informatique et bio-statistique : LBBE, UCB Lyon 1 (Laurent Duret) et ENS de Lyon (Franck Picard)

– G-quadruplex : Inserm U565, CNRS UMR 7196, MNHN, Paris, France (Patrizia Alberti, Carole Saintomé et Jean-François Riou)

– Sites Fragiles Communs : ^: Institut Gustave Roussy, Villejuif (Michelle Debatisse et Stéphane Koundrioukoff)

Internationales :

– Organisation tri-dimensionnelle du noyau : University of Massachusetts Medical School, Worcester, Etats-Unis (Anne-Laure Valton et Job Dekker)

– Rôle des G4s dans le positionnement des nucléosomes : Fox Chase Cancer center, Philadelphie, Etats-Unis et Moscow State University, Moscow, Russie (Vasily Studitsky).