Translesion synthesis (TLS) is the major pathway by which mammalian cells replicate across DNA lesions. Upon DNA damage, ubiquitination of proliferating cell nuclear antigen (PCNA) induces bypass of the lesion by directing the replication machinery into the TLS pathway. Yet, how this modification is recognized and interpreted in the cell remains unclear. Here we describe the identification of two ubiquitin (Ub)-binding domains (UBM and UBZ), which are evolutionarily conserved in all Y-family TLS polymerases (pols). These domains are required for binding of poleta and poliota to ubiquitin, their accumulation in replication factories, and their interaction with monoubiquitinated PCNA. Moreover, the UBZ domain of poleta is essential to efficiently restore a normal response to ultraviolet irradiation in xeroderma pigmentosum variant (XP-V) fibroblasts. Our results indicate that Ub-binding domains of Y-family polymerases play crucial regulatory roles in TLS.

Y-family DNA polymerases can replicate past a variety of damaged bases in vitro but, with the exception of DNA polymerase h (polh), which is defective in xeroderma pigmentosum variants, there is little information on the functions of these polymerases in vivo. Here, we show that DNA polymerase i (poli), like polh, associates with the replication machinery and accumulates at stalled replication forks following DNA-damaging treatment. We show that polh and poli foci form with identical kinetics and spatial distributions, suggesting that localization of these two polymerases is tightly co-ordinated within the nucleus. Furthermore, localization of poli in replication foci is largely dependent on the presence of polh. Using several different approaches, we demonstrate that polh and poli interact with each other physically and that the C-terminal 224 amino acids of poli are suf®cient for both the interaction with polh and accumulation in replication foci. Our results provide strong evidence that polh targets poli to the replication machinery, where it may play a general role in maintaining genome integrity as well as participating in translesion DNA synthesis. Keywords: DNA polymerase/replication foci/UV light/ xeroderma pigmentosum variants Introduction DNA damage occurs ubiquitously in all cells. In order to maintain the stability of the genome, cells have evolved mechanisms not only to repair all types of DNA damage, but also to replicate DNA from which the damage has not been removed (post-replication repair). In the case of human cells, a major mechanism for carrying out postreplication repair involves translesion synthesis (TLS) past damaged sites. TLS is de®cient in the variant form of the sun-sensitive cancer-prone disorder xeroderma pigmentosum (XP). The gene defective in these XP variants (XP-V) encodes a DNA polymerase, polh (Johnson et al., 1999;Masutani et al., 1999), which is able to replicate undamaged templates or those containing cyclobutane pyrimidine dimers (CPDs, the major UV photoproduct) with equal ef®ciencies (Masutani et al., 1999). TLS by polh is the principal mechanism for bypassing CPDs in human cells. Although the lack of polh in XP-V cells does not confer substantial hypersensitivity to killing by UV light, UV hypermutability is increased to levels approaching those in classical XP cells, which are de®cient in nucleotide excision repair (Maher et al., 1976).Polh is a member of the recently discovered Y-family of DNA polymerases (Ohmori et al., 2001), which have been best characterized for their lesion-bypassing properties (reviewed in Goodman, 2002). There are, however, few studies to date to indicate how these polymerases function inside cells. In previous work, we showed that in S-phase cells, polh localizes in replication foci. On exposure to DNA-damaging treatments, we observed an accumulation of polh-containing foci. These appear to represent replication factories in which replication forks are stalled at lesions (Kannouche et al., 2001). The C-terminal 70 amino acids of polh are requ...