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.
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Gene/Complex | Qualifier | Gene Ontology Term | Annotation Extension | Evidence | Source | Assigned On |
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RAD6-RAD18 ubiquitin ligase complex | involved in | error-free postreplication DNA repair | BSR | ComplexPortal | 2018-03-09 | |
RAD6-RAD18 ubiquitin ligase complex | enables | ATP binding | IPI | ComplexPortal | 2018-03-09 | |
RAD6-RAD18 ubiquitin ligase complex | enables | ubiquitin-protein transferase activity | IDA | ComplexPortal | 2018-03-09 | |
RAD6-RAD18 ubiquitin ligase complex | involved in | protein monoubiquitination | IDA | ComplexPortal | 2018-03-09 | |
RAD18 | contributes to | single-stranded DNA binding | has input single-stranded DNA | IDA | SGD | 2019-12-09 |
RAD18 | part of | Rad6-Rad18 complex | IDA | SGD | 2013-10-11 | |
RAD18 | contributes to | single-stranded DNA helicase activity | IDA | SGD | 2019-12-09 | |
RAD6 | enables | ubiquitin-protein transferase activity | IDA | SGD | 2013-08-07 | |
RAD6 | part of | Rad6-Rad18 complex | IDA | SGD | 2013-10-11 | |
RAD6 | contributes to | single-stranded DNA binding | has input single-stranded DNA | IDA | SGD | 2019-12-09 |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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Interactor | Interactor | Assay | Annotation | Action | Modification | |
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RAD18 | RAD6 | Affinity Capture-Western | manually curated | Hit-Bait | No Modification | |
bound in the absence or presence of single stranded DNA | RAD18 | RAD6 | Reconstituted Complex | manually curated | Hit-Bait | No Modification |
RAD18 | RAD6 | Co-purification | manually curated | Hit-Bait | No Modification | |
Rad6 formed a thioester adduct with GST-ubiquitin in an in vitro assay with purified Uba1, Rad6, ATP and GST-ub; source of ubiquitin is unknown | UBA1 | RAD6 | Biochemical Activity | manually curated | Bait-Hit | ubiquitinylated lysine |
likely ubiquitin conjugate | UBA4 | RAD6 | Reconstituted Complex | manually curated | Hit-Bait | No Modification |