In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 109, No. 37 ( 2012-09-11)
Kurzfassung:
The DprA family currently is defined on the basis of primary sequence identity by its C-terminal moiety (Pfam02481) ( 5 ), which turns to overlap the RF ( Fig. P1 ). Our comparisons of the structure presented here indicate that DprA now can be defined as comprising a SAM (as revealed by the atomic structure of DprA from S. pneumoniae ) and an extended RF ( Fig. P1 ). Furthermore, this analysis establishes that the potential for DprA to form dimers and interface residues is evolutionarily conserved. The conclusions drawn from the structural and functional analysis of DprA from S. pneumoniae can thus be extended to all members of the DprA family, highlighting the crucial role of the dimerization of DprA in the loading of RecA onto ssDNA during genetic transformation. The positioning of DprA residues involved in dimerization and in interaction with RecA on the solved structure highlighted a striking overlap between the corresponding DprA–DprA and DprA–RecA interaction surfaces ( Fig. P1 ). Site-directed mutagenesis of selected residues conserved in the DprA family demonstrated an even more extended overlap. This overlap strongly suggests that RecA interaction triggers a rearrangement or disruption of the DprA dimer to allow the nucleation of RecA and the subsequent formation of nucleofilaments ( Fig. P1 ). The structure of full-length DprA from S. pneumoniae reveals the association of two known structural folds, an N-terminal sterile alpha motif (SAM) domain and a C-terminal Rossman fold (RF) ( Fig. P1 ). The formation of DprA dimers involves interactions between the C-terminal regions of two monomers. We isolated DprA mutants that are unable to dimerize but retain full capacity to associate in a stable complex with RecA. Biochemical analysis of these monomeric DprA mutants revealed that they fail to form stable nucleocomplexes, and genetic studies showed that they display drastically reduced transformation efficiencies. DprA mutants specifically impaired in their interaction with RecA also were isolated and characterized. Despite normal formation of nucleocomplexes in vitro and homodimerization in vitro and in vivo, these mutants also were defective for transformation. Thus, these data establish that transformation requires both dimerization and nucleocomplex formation and the interaction of RecA and DprA. The RecA recombinase, as well as its eukaryotic Rad51 and archeal RadA counterparts, mediate DNA integration by spreading along ssDNA, catalyzing the search for homology and promoting exchange of homologous DNA strands. Nucleation of the recombinase [i.e., binding of its first subunit(s) to ssDNA] is the rate-limiting step in this process, hence the need for cofactors. We reported that transformation-dedicated DprA fulfills this role in vitro ( 5 ). Further, DprA binds ssDNA and interacts physically with RecA ( 5 ). However, these activities had not been shown to be required for genetic transformation in vivo. Thus, the mechanistic aspects of the functional interplay between DprA and RecA remained unknown. Therefore we combined structural, biochemical, and genetic studies to identify and characterize surfaces or amino acid residues, or both, that are crucial for the role of DprA in transformation, including residues important for interaction with RecA. Sexual reproduction, which is crucial for generating genetic diversity in eukaryotes, is lacking in bacteria. However, natural genetic transformation is regarded as a substitute ( 1 ). This widespread process, originally discovered in Streptococcus pneumoniae ( 2 ), promotes the uptake of exogenous DNA and its integration into the recipient genome, leading to the generation of new genotypes. Transformation contributes to genetic plasticity ( 3 ), potentially leading to the acquisition of antibiotic resistance and escape from vaccines ( 4 ). Exogenous DNA is taken up by the cell and integrates into the chromosome by homologous recombination. The search for homologous cellular DNA sequences is catalyzed by the universal bacterial recombinase RecA, assisted by the transformation-dedicated DNA- processing A (DprA) protein ( 5 ). Here we show that two properties of the DprA protein are crucial to transformation, allowing us to propose a mechanism of DprA-mediated delivery of RecA onto DNA.
Materialart:
Online-Ressource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.1205638109
Sprache:
Englisch
Verlag:
Proceedings of the National Academy of Sciences
Publikationsdatum:
2012
ZDB Id:
209104-5
ZDB Id:
1461794-8
SSG:
11
SSG:
12
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