Structure–function analysis of pneumococcal DprA protein reveals that dimerization is crucial for loading RecA recombinase onto DNA during transformation

Abstract

Transformation promotes genome plasticity in bacteria via RecAdriven 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-totail 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 DprARecA 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.bacterial transformation | genetic exchange | recombinase loader | recombination mediator protein | horizontal gene transfer

DprA is required for natural transformation and affects pilin variation in Neisseria gonorrhoeae

“…2007; Quevillon- Cheruel et al, 2012;Yadav et al, 2013Yadav et al, , 2014; collectively, these investigations demonstrate that DprA localizes to DNA uptake machinery and binds to incoming ssDNA, presumably protecting the DNA from degradation by cytoplasmic nucleases. The ssDNA-DprA complex has been shown to interact with RecA to form filaments, which promote recombination by RecA.…”

“…DprA has an established role during transformation of many bacteria species, as mutants deficient in DprA are completely or partially deficient in transformation (Berge et al, 2003;Dwivedi et al, 2013;Karudapuram & Barcak, 1997;Mirouze et al, 2013;Mortier-Barriere et al, 2007;Quevillon-Cheruel et al, 2012;Smeets et al, 2000Smeets et al, , 2006Takata et al, 2005). B. subtilis and S. pneumoniae DprA have been extensively studied (Mortier-Barriere et al,…”

“…The role of DprA in transformation [reviewed in Johnston et al (2014) and Rotman & Seifert (2014)] has been studied in several species of naturally transforming bacteria including B. subtilis (Tadesse & Graumann, 2007;Yadav et al, 2014), S. pneumoniae (Mirouze et al, 2013;MortierBarriere et al, 2007;Quevillon-Cheruel et al, 2012), Heli. pylori (Smeets et al, 2000;Zhang & Blaser, 2012) and C. jejuni (Takata et al, 2005).…”

“…pylori, Bacillus subtilis and Campylobacter jejuni (Ando et al, 1999;Berge et al, 2003;Dwivedi et al, 2013;Quevillon-Cheruel et al, 2012;Smeets et al, 2000;Takata et al, 2005;Yadav et al, 2013Yadav et al, , 2014. Additionally, DprA was found to be required for transformation in N. meningitidis (Sun et al, 2005) which serves as a model for Neisseria transformation.…”

“…The mechanism of DprA binding to ssDNA and dsDNA has been investigated in Heli. pylori (Dwivedi et al, 2015) and S. pneumoniae (Quevillon-Cheruel et al, 2012). Interestingly, DprA localizes to the DNA uptake machinery during transformation in B. subtilis (Tadesse & Graumann, 2007) presumably to receive incoming DNA to shuttle to RecA for recombination.…”