Natural transformation is a widespread mechanism driving genetic exchanges in bacteria. It proceeds by the capture and internalization of exogenous DNA in linear single strands, ultimately integrated in the genome by homologous recombination. It is unknown how the RecA-directed D-loop intermediate of this dedicated recombination pathway is processed. We report that resolution of the transformation D-loop depends on two endonucleases of opposing phylogenetic distribution in bacteria. One is YraN, which has co-evolved and interacts with the ComM helicase, known to extend DNA recombination at the transformation D-loop. The other is CoiA, which is restricted to the Bacillota. CoiA is shown to be a resolvase of the transformation D-loop, extended by the RadA helicase in these species. We demonstrate that both YraN and CoiA act synergistically with their cognate helicases. These findings reveal that bacteria have evolved two helicase/nuclease pairs for the maturation and recombination extension of the transformation D-loop. ### Competing Interest Statement The authors have declared no competing interest. Agence Nationale de la Recherche, https://ror.org/00rbzpz17, ANR-20-CE12-0004, ANR-10-BLAN-1331, ANR-17-CE13-0031, ANR-22-CE44-0044, ANR-10-LABX-62-IBEID, PIA/ANR-16-CONV-0005 Fondation pour la Recherche Médicale, https://ror.org/04w6kn183, FDT202001010890 European Union's Horizon research and innovation programme. Marie Skłodowska-Curie Postdoctoral Fellowships, 101208987 National Institute of Health, USA, R35GM128674

Author summary The environment in which bacteria live puts them under a great deal of stress, forcing them to adapt constantly, either temporarily or permanently. Streptococcus pneumoniae, a pathogeni...