Structure-function analysis of pneumococcal DprA protein reveals that dimerization is crucial for loading RecA recombinase onto DNA during transformation.
Transformation promotes genome plasticity in bacteria via RecA-driven homologous recombination. In the gram-positive human pathogen Streptococcus pneumoniae, the transformasome a multiprotein complex, internalizes, protects, and processes transforming DNA to generate chromosomal recombinants. Double-stranded DNA is internalized as single strands, onto which the transformation-dedicated DNA processing protein A (DprA) ensures the loading of RecA to form presynaptic filaments. We report that the structure of DprA consists of the association of a sterile alpha motif domain and a Rossmann fold and that DprA forms tail-to-tail dimers. The isolation of DprA self-interaction mutants revealed that dimerization is crucial for the formation of nucleocomplexes in vitro and for genetic transformation. Residues important for DprA-RecA interaction also were identified and mutated, establishing this interaction as equally important for transformation. Positioning of key interaction residues on the DprA structure revealed an overlap of DprA-DprA and DprA-RecA interaction surfaces. We propose a model in which RecA interaction promotes rearrangement or disruption of the DprA dimer, enabling the subsequent nucleation of RecA and its polymerization onto ssDNA.
Institut de Biochimie et de Biophysique Moléculaire et Cellulaire, Université de Paris-Sud, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8619, Bât 430, 91405 Orsay Cedex, France. sophie.quevillon-cheruel@u-psud.fr
Sophie
S
Quevillon-Cheruel
Nathalie
N
Campo
Nicolas
N
Mirouze
Isabelle
I
Mortier-Barrière
Mark A
MA
Brooks
Marion
M
Boudes
Dominique
D
Durand
Anne-Lise
AL
Soulet
Johnny
J
Lisboa
Philippe
P
Noirot
Bernard
B
Martin
Herman
H
van Tilbeurgh
Marie-Françoise
MF
Noirot-Gros
Jean-Pierre
JP
Claverys
Patrice
P
Polard
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D015153:Blotting, Western; D003460:Crystallization; D004247:DNA / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D019281:Dimerization; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011938:Rec A Recombinases / Q000378:metabolism*; D013296:Streptococcus pneumoniae / Q000378:metabolism*; D014169:Transformation, Bacterial / Q000235:genetics / Q000502:physiology*; D020798:Two-Hybrid System Techniques
D001426:Bacterial Proteins; D017931:DNA Primers; C093815:DprA protein, bacteria; D008565:Membrane Proteins; D004247:DNA; D011938:Rec A Recombinases