BrdU Incorporation and Labeling of Nascent DNA to Investigate Archaeal Replication Using Super-Resolution Imaging

Roxane Lestini (1) , Yoann Collien (1) , Debora Olivier (1) , Nicolas Olivier (1) , Hannu Myllykallio (1)

1 LOB - Laboratoire d'Optique et Biosciences

The labeling and specific detection of nascent DNA by the incorporation of thymidine analogs provide crucial information about DNA replication dynamics without requiring the intracellular expression of fluorescent proteins. After cell fixation and permeabilization, specific detection of thymidine analogs by antibodies can be performed using super-resolution imaging techniques. Here we describe a protocol to label nascent DNA using 5'-bromo-2'-deoxyuridine (BrdU) in Haloferax volcanii cells and generate super-resolved images of neo-synthesized DNA foci either by 3D Structured illumination microscopy (3D-SIM) or Stochastic Optical Reconstruction Microscopy (STORM).

Mots clés: 3D structured illuminated microscopy (3D-SIM); Archaea; BrdU; Haloferax volcanii; Nascent DNA labeling; Replication; Stochastic optical reconstruction microscopy (STORM); Super-resolution imaging

Domaines: Sciences du Vivant [q-bio]

Roxane Lestini, Yoann Collien, Debora Olivier, Nicolas Olivier, Hannu Myllykallio. BrdU Incorporation and Labeling of Nascent DNA to Investigate Archaeal Replication Using Super-Resolution Imaging. Archaea, 2522, Springer US, pp.419-434, 2022, Methods in Molecular Biology, ⟨10.1007/978-1-0716-2445-6_29⟩. ⟨hal-03833442⟩