Specific complex formation between yeast RAD6 and RAD18 proteins: a potential mechanism for targeting RAD6 ubiquitin-conjugating activity to DNA damage sites.

Abstract

The RAD6 gene of Saccharomyces cerevisiae encodes a ubiquitin-conjugating enzyme that is required for postreplication repair of UV-damaged DNA, DNA damage induced mutagenesis, sporulation, and amino-end rule protein degradation. RAD6 interacts physically with the UBR1 gene product in carrying out the multiubiquitination of amino-end rule proteolytic substrates. In mediating postreplication repair, it has remained unclear whether RAD6 acts in a pleiotropic manner distal from the site of DNA damage or is targeted to the damage site via interaction with another repair component. Here, we show that RAD6 forms a specific complex with the product of the DNA repair gene RAD18. The biological significance of this interaction is attested by the observation that overproduction of the rad6 Ala-88 mutant protein, which lacks ubiquitin-conjugating activity but retains the ability to interact with RAD18 protein, confers a high level of UV sensitivity on wild-type PAD + cells that can be corrected by the concomitant overexpression of RAD18. We demonstrate that whereas RAD6 has no affinity for DNA, RAD18 binds single-stranded DNA. Thus, association of RAD6 with RAD18 could provide a means for targeting RAD6 to damage-containing DNA regions, where the RAD6 ubiquitin-conjugating function could modulate the activity of a stalled DNA replication machinery. We also show that RAD6 forms separate complexes with RAD18 and with UBR1, and the extremely conserved amino terminus of RAD6 that is required for complex formation with UBR1 is dispensable for complex formation with RAD18.

Ubiquitinated proliferating cell nuclear antigen activates translesion DNA polymerases η and REV1

“…The Rad18 subunit of Rad6͞Rad18 is a DNA-binding protein with a binding preference for ssDNA (34). Rad18 also interacts with PCNA (1).…”

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].…”

The error-free component of the RAD6/RAD18 DNA damage tolerance pathway of budding yeast employs sister-strand recombination

“…Rad6 is an E2 ubiquitin conjugase (12) which, as a heterodimer with Rad18 (13), monoubiquitinates lysine 164 of proliferating cell nuclear antigen (14), a modification that promotes translesion replication (15). The latter process employs DNA polymerase , whose two subunits are encoded by REV3 and REV7 (16,17), DNA polymerase (18), encoded by RAD30 (19), and Rev1, a deoxycytidyl transferase (20) that also has another function in translesion replication (21).…”