Rad10-YFP focus induction in response to UV depends on RAD14 in yeast.
Rad14 is a DNA damage recognition protein in yeast Nucleotide Excision Repair (NER) and believed to function early in the cascade of events. The function of Rad14 presumably precedes that of the Rad1-Rad10 endonuclease complex, which functions in a downstream step incising DNA 5' to the site of DNA damage. We investigated whether recruitment of Rad10 to UV-induced DNA damage sites in live cells is dependent on Rad14 using fluorescence microscopy. Experiments were carried out using Saccharomyces cerevisiae strains in which the gene for Rad14 was fused to Cyan Fluorescent Protein (Rad14-CFP) and that of Rad10 was fused to Yellow Fluorescent Protein (Rad10-YFP). Rad14-CFP forms nuclear localized CFP fluorescent foci in response to UV irradiation with the peak induction occurring 15min post-irradiation. In contrast, Rad10-YFP foci form in response to UV with the peak induction occurring 2h post-irradiation. Recruitment of Rad14-CFP is not dependent on the RAD10 gene but Rad10-YFP is recruited to UV-induced YFP foci in a RAD14-dependent fashion. Time-lapse experiments indicate that Rad14-CFP foci are transient, typically persisting less than 6min. Together these data support the model that yeast NER protein assembly is step-wise whereas Rad14 required to recruit Rad10 and Rad14 involvement is transient.
Mardiros
Armen
A
Department of Chemistry and Biochemistry, California State University Northridge, Northridge, CA 91330-8262, USA.
Benoun
Joseph M
JM
Haughton
Robert
R
Baxter
Kelly
K
Kelson
Eric P
EP
Fischhaber
Paula L
PL
D001426:Bacterial Proteins; D004249:DNA Damage / Q000528:radiation effects; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000235:genetics* / Q000378:metabolism; D004271:DNA, Fungal / Q000235:genetics* / Q000528:radiation effects; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000528:radiation effects; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000235:genetics* / Q000378:metabolism; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D004271:DNA, Fungal; D008164:Luminescent Proteins; C072975:RAD14 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; C064162:RAD10 protein, S cerevisiae; D015719:Single-Strand Specific DNA and RNA Endonucleases; D045643:DNA Repair Enzymes