Vertebrate DNA damage tolerance requires the C-terminus but not BRCT or transferase domains of REV1

Abstract

REV1 is central to the DNA damage response of eukaryotes through an as yet poorly understood role in translesion synthesis. REV1 is a member of the Y-type DNA polymerase family and is capable of in vitro deoxycytidyl transferase activity opposite a range of damaged bases. However, non-catalytic roles for REV1 have been suggested by the Saccharomyces cerevisiae rev1-1 mutant, which carries a point mutation in the N-terminal BRCT domain, and the recently demonstrated ability of the mammalian protein to interact with each of the other translesion polymerases via its extreme C-terminus. Here, we show that a region adjacent to this polymerase interacting domain mediates an interaction with PCNA. These C-terminal domains of REV1 are necessary, although not sufficient, for effective tolerance of DNA damage in the avian cell line DT40, while the BRCT domain and transferase activity are not directly required. Together these data provide strong support for REV1 playing an important non-catalytic role in coordinating translesion synthesis. Further, unlike in budding yeast, rad18 is not epistatic to rev1 for DNA damage tolerance suggesting that REV1 and RAD18 play largely independent roles in the control of vertebrate translesion synthesis.

DNA polymerase zeta (pol ζ) in higher eukaryotes

“…This Cterminal region of mammalian REV1 is important for inter- BRCT UBM npg acting with many other translesion synthesis polymerases. By yeast two-hybrid and transient co-transfection, mouse and human pols i, h and k interacted with the C-terminus of REV1 [40,44,47]. This supports the theory that REV1 may act as a scaffolding protein, facilitating the interchange between different DNA translesion polymerases at a site of damage.…”

“…Site-directed mutagenesis of D569 and E570 to alanine within the catalytic domain of this form of human REV1 inactivates the dCMP transferase activity [42]. When examined in yeast and chicken REV1, the dCMP transferase activity is not critical for damage tolerance activity [43,44]. Other structural domains are known to be important.…”

The Fanconi Anemia/BRCA pathway: new faces in the crowd

“…Loss of the FA pathway results in a hypomutable phenotype following crosslinker treatment, further suggesting a relationship between the FA and TLS pathway (Papadopoulo et al 1990a,b). Furthermore, Rev1 deficient DT40 cells, in common with FA cells, demonstrate hypersensitivity to cisplatin (Ross et al 2005). Finally, as will be discussed later, recent evidence suggests that the FA pathway and TLS are terminated by the same deubiquitinating enzyme, USP1, further indicating their coordinate regulation (T. Huang and A.D. D'Andrea, unpubl.…”

The Vital Role of Polymerase ζ and REV1 in Mutagenic, but Not Correct, DNA Synthesis across Benzo[a]pyrene-dG and Recruitment of Polymerase ζ by REV1 to Replication-stalled Site

“…However, de Groote et al (44) recently argued against this assertion by showing the BRCT domain, together with a further N-terminal region, to bind to recessed, phosphorylated 5Ј ends of double-stranded DNA. REV1 probably has a PCNA-binding site(s) in its C-terminal region (12,46,47). Furthermore, although Akagi et al (48) have shown that the accumulation of human endogenous REV1 into locally UV-irradiated regions of nucleus depends on the interaction with pol , Andersen et al (45) have reported that the recruitment of the human endogenous REV1 is independent of the interaction with pol .…”

“…This enzyme activity plays a role in TLS across certain classes of lesions (10,11). REV1 also plays a noncatalytic role in TLS (12) and physically interacts with the other Y family polymerases (13,14) and the REV7 subunit of pol (15,16). The biological significance of these interactions remains to be determined.…”