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

Emily Koury; Kailey Harrell; Sarit Smolikove

doi:10.1093/nar/gkx1243

Back

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

Journal article

Open access

Peer reviewed

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

Emily Koury, Kailey Harrell and Sarit Smolikove

Nucleic acids research, Vol.46(2), pp.748-764

01/25/2018

DOI: 10.1093/nar/gkx1243

PMCID: PMC5778493

PMID: 29244155

Files and links (1)

url

https://doi.org/10.1093/nar/gkx1243View

Published (Version of record) Open Access

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.

Rad51 Recombinase - metabolism

Green Fluorescent Proteins - metabolism

Recombinational DNA Repair

Rad51 Recombinase - genetics

Animals, Genetically Modified

Caenorhabditis elegans Proteins - metabolism

Green Fluorescent Proteins - genetics

Replication Protein A - metabolism

DNA Breaks, Double-Stranded

Luminescent Measurements - methods

Animals

Cell Nucleus - metabolism

Cell Nucleus - genetics

Cell Nucleus - radiation effects

Lasers

Replication Protein A - genetics

Time-Lapse Imaging - methods

Germ Cells - metabolism

Kinetics

Caenorhabditis elegans Proteins - genetics

Germ Cells - radiation effects

Details

Title: Subtitle: Differential RPA-1 and RAD-51 recruitment in vivo throughout the C. elegans germline, as revealed by laser microirradiation
Creators: Emily Koury - Department of Biology, The University of Iowa, Iowa City, IA 52242, USA
Kailey Harrell - Department of Biology, The University of Iowa, Iowa City, IA 52242, USA
Sarit Smolikove - Department of Biology, The University of Iowa, Iowa City, IA 52242, USA
Resource Type: Journal article
Publication Details: Nucleic acids research, Vol.46(2), pp.748-764
DOI: 10.1093/nar/gkx1243
PMID: 29244155
PMCID: PMC5778493
NLM abbreviation: Nucleic Acids Res
ISSN: 0305-1048
eISSN: 1362-4962
Publisher: England
Grant note: R01 GM112657 / NIGMS NIH HHS P40 OD010440 / NIH HHS
Language: English
Date published: 01/25/2018
Academic Unit: Biology
Record Identifier: 9983991958202771

Metrics

36 Record Views

25 Times Cited - Web of Science