Reconstitution of yeast nucleotide excision repair with purified Rad proteins, replication protein A, and transcription factor TFIIH.
Nucleotide excision repair (NER) functions to remove DNA damage caused by ultraviolet light and by other agents that distort the DNA helix. The NER machinery has been conserved in structure and function from yeast to humans, and in humans, defective NER is the underlying cause of the cancer-prone disease xeroderma pigmentosum. Here, we reconstitute the incision reaction of NER in Saccharomyces cerevisiae using purified protein factors. The Rad14 protein, the Rad4-Rad23 complex, the Rad2 nuclease, the Rad1-Rad10 nuclease, replication protein A, and the RNA polymerase II transcription factor TFIIH were purified to near homogeneity from yeast. We show that these protein factors are both necessary and sufficient for dual incision of DNA damaged by either ultraviolet light or N-acetoxy-2-aminoacetylfluorene. Incision in the reconstituted system requires ATP, which cannot be substituted by adenosine 5'-O-(3-thiotriphosphate), suggesting that the hydrolysis of ATP is indispensable for the incision reaction. The excision DNA fragments formed as a result of dual incision are in the 24-27-nucleotide range.
Guzder
S N
SN
Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston 77555-1061, USA.
Habraken
Y
Y
Sung
P
P
Prakash
L
L
Prakash
S
S
D015073:2-Acetylaminofluorene / Q000633:toxicity; D004249:DNA Damage; D004260:DNA Repair; D045643:DNA Repair Enzymes; D004261:DNA Replication; D004274:DNA, Recombinant / Q000187:drug effects / Q000302:isolation & purification / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism*; D051716:Replication Protein A; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D035381:TATA-Binding Protein Associated Factors; D035362:Transcription Factor TFIID; D051758:Transcription Factor TFIIH; D014157:Transcription Factors / Q000378:metabolism*; D019473:Transcription Factors, TFII; D014466:Ultraviolet Rays
D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C072975:RAD14 protein, S cerevisiae; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins; C490039:TAF6 protein, S cerevisiae; D035381:TATA-Binding Protein Associated Factors; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D019473:Transcription Factors, TFII; D051758:Transcription Factor TFIIH; D015073:2-Acetylaminofluorene; D045643:DNA Repair Enzymes