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

Abstract

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.Exposure of cells to ultraviolet (UV) light and to many other agents causes the formation of lesions in the DNA. During DNA replication, such lesions located in the template strand block the DNA replication machinery, resulting in a gap in the newly synthesized strand across from the damage site. A variety of postreplicational repair mechanisms have evolved to restore the continuity of the newly synthesized DNA strand (reviewed in Ref. 1).Genetic studies in the yeast Saccharomyces cerevisiae have been instrumental in identifying the genes involved in postreplicational repair. RAD6 and RAD18, members of the RAD6 epistasis group, play a prominent role in this repair process. Mutations in RAD6 cause extreme sensitivity to UV light and to other DNA damaging agents; rad6 mutants are highly deficient in postreplicational repair of UV-damaged DNA (2) and they exhibit no mutation induction in response to UV (3). RAD6encodes an ubiquitin conjugating enzyme of 172 residues (4, 5). The first 149 amino acids of Rad6 form a globular domain, while the distal 23 residues, which are predominantly acidic, constitute a freely extending tail domain (6). Mutational inactivation of the active site cysteine 88 residue in Rad6 has indicated that the ubiquitin conjugating activity is essential for all the biological functions of Rad6 (7). Mutants of RAD18 resemble those of RAD6 in their high degree of sensitivity to UV, defects in postreplicational repair of UV-damaged DNA (2), and defects in UV mutagenesis (8, 9). However, unlike RAD6, which is indispensable for sporulation, mutations in RAD18 do not affect sporulation (10).Other genes that belong to the RAD6 epistasis group include REV1, REV3, REV7, and RAD5. Although mutants of the REV genes show only a marginal increase in UV sensitivity, like rad6 and rad18 mutants, they are defective in UV mutagenesi...

RAD6B overexpression confers chemoresistance: RAD6 expression during cell cycle and its redistribution to chromatin during DNA damage-induced response

“…All these proteins have been shown to interact at least transiently (Broomfield et al, 1998;Ulrich and Jentsch, 2000;Torres-Ramos et al, 2002). RAD6-RAD18 dimers (Bailly et al, 1997;Xin et al, 2000) in the presence of REV3 or RAD30 have been implicated in error-prone lesion bypass (Roche et al, 1995;McDonald et al, 1997;Cejka et al, 2001). Similarly, RAD6 has been found to be a common component in complexes with PCNA (Hoege et al, 2002), Bre1 (Wood et al, 2002 and p53 (Lyakhovich and Shekhar, 2003); these data suggest that mechanisms regulating expression and activity of RAD6 may be crucial in determining the levels and type of complexes formed by RAD6 with key proteins that are critical for regulation and fidelity of cell cycle checkpoints and preservation of genomic integrity.…”

DNA Repair Defects Channel Interstrand DNA Cross-links into Alternate Recombinational and Error-prone Repair Pathways

“…RAD5 is a member of the error-free branch of the RAD6 PRR epistasis group (43,44). Rad6 is a ubiquitin-conjugating enzyme required for all forms of PRR; it forms a complex with the RING finger protein Rad18, which binds single-stranded DNA (45). The Rad6-Rad18 heterodimer monoubiquitinates the DNA replication and repair factor PCNA (46).…”

Eukaryotic DNA damage tolerance and translesion synthesis through covalent modifications of PCNA

“…Furthermore, the N-terminus of Rad6 is also required for N-end rule protein degradation [29, 37,38]: while the full-length Rad6 interacts with the E3 protein Ubr1, the Rad6 ∆1-9 protein is unable to form a complex with Ubr1 [37]. Rad6 is known to form a stable complex with Rad18 [39], and this complex displays Ub conjugation (from Rad6), ssDNA-binding and ATPase (from Rad18) activities [40]. However, Rad18 had not been defined as an E3 until the RING finger motif was discovered [41,42] and found in Rad18, and the physical interaction of Rad18 with the substrate Pol30 (PCNA) was demonstrated [43].…”