Yeast DNA repair proteins Rad6 and Rad18 form a heterodimer that has ubiquitin conjugating, DNA binding, and ATP hydrolytic activities.
The RAD6 and RAD18 genes of Saccharomyces cerevisiae are required for postreplicative bypass of ultraviolet (UV)-damaged DNA and for UV mutagenesis. The RAD6 encoded protein is a ubiquitin conjugating enzyme, and RAD18 encodes a protein containing a RING finger motif and a nucleotide binding motif. Rad18 can be co-immunoprecipitated with Rad6, indicating that the two proteins exist in a complex in vivo. Here, we co-overproduce the two proteins using a yeast multicopy plasmid, purify the Rad6-Rad18 complex to near homogeneity, and show that the complex is heterodimeric. The Rad6-Rad18 heterodimer has ubiquitin conjugating activity, binds single-stranded DNA, and possesses single-stranded DNA-dependent ATPase activity. The Rad6-Rad18 complex provides the first example wherein a ubiquitin conjugating activity is physically associated with DNA binding and ATPase activities provided by an associated protein factor. The co-existence of these activities should provide the complex with the ability to recognize single-stranded DNA resulting from stalling of the replication machinery at DNA damage sites and to recognize the components of the DNA replication machinery for ubiquitination by Rad6.
Bailly
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Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555-1061, USA.
Lauder
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Prakash
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S
Prakash
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L
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019281:Dimerization; D008025:Ligases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D044763:Ubiquitin-Conjugating Enzymes; D014452:Ubiquitins / Q000378:metabolism*
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C063607:RAD18 protein, S cerevisiae; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014452:Ubiquitins; C471606:RAD6 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; D000251:Adenosine Triphosphatases; D008025:Ligases