Replication is required for the RecA localization response to DNA damage in Bacillus subtilis.
In both prokaryotes and eukaryotes, proteins involved in DNA repair often organize into multicomponent complexes that can be visualized as foci in living cells. We used a RecA-GFP fusion to examine the subcellular cues that direct RecA-GFP to assemble as foci in response to DNA damage. We used two different methods to inhibit initiation of DNA replication and determined that DNA replication is required for the cell to establish RecA-GFP foci after exposure to DNA-damaging agents. Furthermore, use of endonuclease cleavage to generate a site-specific double-strand break demonstrated that the replication machinery (replisome) and DNA synthesis are required for assembly of RecA-GFP foci during repair of a double-strand break. We monitored the cellular levels of RecA and found that focus formation does not require further induction of protein levels, suggesting that foci result from a redistribution of existing protein to sites of damage encountered by the replisome. Taken together, our results support the model that existing RecA protein is recruited to ssDNA generated by the replisome at sites of DNA damage. These results provide insight into the mechanisms that the cell uses to recruit repair proteins to damaged DNA in living cells.
Simmons
Lyle A
LA
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Grossman
Alan D
AD
Walker
Graham C
GC
D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism; D015139:Blotting, Southern; D004249:DNA Damage; D004261:DNA Replication; D004269:DNA, Bacterial / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D011938:Rec A Recombinases / Q000378:metabolism*
D004269:DNA, Bacterial; D049452:Green Fluorescent Proteins; D011938:Rec A Recombinases