Yeast DNA Repair Proteins Rad6 and Rad18 Form a Heterodimer That Has Ubiquitin Conjugating, DNA Binding, and ATP Hydrolytic Activities

• Published in: The Journal of biological chemistry 1997-09, Vol.272 (37), p.23360-23365
• Main Authors: Bailly, Veronique, Lauder, Scott, Prakash, Satya, Prakash, Louise
• Format: Article
• Language: English
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; Adenosine Triphosphatases - metabolism; ADENOSINETRIPHOSPHATASE; ADN; ATP; ATPASA; ATPASE; BINDING PROTEINS; Dimerization; DNA; DNA Repair; DNA, Single-Stranded - metabolism; DNA-Binding Proteins - genetics; DNA-Binding Proteins - isolation & purification; DNA-Binding Proteins - metabolism; ENZYMIC ACTIVITY; HIDROLISIS; HYDROLYSE; HYDROLYSIS; INTERACTIONS; Ligases - genetics; Ligases - isolation & purification; Ligases - metabolism; Protein Binding; PROTEINAS; PROTEINAS AGLUTINANTES; PROTEINE; PROTEINE DE LIAISON; PROTEINS; Recombinant Proteins - metabolism; SACCHAROMYCES CEREVISIAE; Saccharomyces cerevisiae Proteins; SINGLE STRANDED DNA; UBIQUITIN CONJUGATING ENZYME; Ubiquitin-Conjugating Enzymes; Ubiquitins - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.37.23360

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.