Differential RPA-1 and RAD-51 recruitment in vivo throughout the C. elegans germline, as revealed by laser microirradiation

Nucleic Acids Res. 2018 Jan 25;46(2):748-764. doi: 10.1093/nar/gkx1243.

Abstract

Studies of the repair pathways associated with DNA double strand breaks (DSBs) are numerous, and provide evidence for cell-cycle specific regulation of homologous recombination (HR) by the regulation of its associated proteins. Laser microirradiation is a well-established method to examine in vitro kinetics of repair and allows for live-imaging of DSB repair from the moment of induction. Here we apply this method to whole, live organisms, introducing an effective system to analyze exogenous, microirradiation-induced breaks in the Caenorhabditis elegans germline. Through this method we observed the sequential kinetics of the recruitment of ssDNA binding proteins RPA-1 and RAD-51 in vivo. We analyze these kinetics throughout different regions of the germline, and thus throughout a range of developmental stages of mitotic and meiotic nuclei. Our analysis demonstrates a largely conserved timing of recruitment of ssDNA binding proteins to DSBs throughout the germline, with a delay of RAD-51 recruitment at mid-pachytene nuclei. Microirradiated nuclei are viable and undergo a slow kinetics of resolution. We observe RPA-1 and RAD-51 colocalization for hours post-microirradiation throughout the germline, suggesting that there are mixed RPA-1/RAD-51 filaments. Finally, through live imaging analysis we observed RAD-51 foci movement with low frequency of coalescence.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Animals, Genetically Modified
  • Caenorhabditis elegans Proteins / genetics
  • Caenorhabditis elegans Proteins / metabolism*
  • Cell Nucleus / genetics
  • Cell Nucleus / metabolism
  • Cell Nucleus / radiation effects
  • DNA Breaks, Double-Stranded
  • Germ Cells / metabolism*
  • Germ Cells / radiation effects
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Kinetics
  • Lasers
  • Luminescent Measurements / methods
  • Rad51 Recombinase / genetics
  • Rad51 Recombinase / metabolism
  • Recombinational DNA Repair
  • Replication Protein A / genetics
  • Replication Protein A / metabolism*
  • Time-Lapse Imaging / methods

Substances

  • Caenorhabditis elegans Proteins
  • Replication Protein A
  • Green Fluorescent Proteins
  • Rad51 Recombinase
  • rad-51 protein, C elegans