Exposure of Escherichia coli to a variety of DNA‐damaging agents results in the induction of the global ‘SOS response’. Expression of many of the genes in the SOS regulon are controlled by the LexA protein. LexA acts as a transcriptional repressor of these unlinked genes by binding to specific sequences (LexA boxes) located within the promoter region of each LexA‐regulated gene. Alignment of 20 LexA binding sites found in the E. coli chromosome reveals a consensus of 5′‐TACTG(TA)5CAGTA‐3′. DNA sequences that exhibit a close match to the consensus are said to have a low heterology index and bind LexA tightly, whereas those that are more diverged have a high heterology index and are not expected to bind LexA. By using this heterology index, together with other search criteria, such as the location of the putative LexA box relative to a gene or to promoter elements, we have performed computational searches of the entire E. coli genome to identify novel LexA‐regulated genes. These searches identified a total of 69 potential LexA‐regulated genes/operons with a heterology index of < 15 and included all previously characterized LexA‐regulated genes. Probes were made to the remaining genes, and these were screened by Northern analysis for damage‐inducible gene expression in a wild‐type lexA+ cell, constitutive expression in a lexA(Def) cell and basal expression in a non‐inducible lexA(Ind−) cell. These experiments have allowed us to identify seven new LexA‐regulated genes, thus bringing the present number of genes in the E. coli LexA regulon to 31. The potential function of each newly identified LexA‐regulated gene is discussed.

Y‐family DNA polymerases have spacious active sites that can accommodate a wide variety of geometric distortions. As a consequence, they are considerably more error‐prone than high‐fidelity replicases. It is hardly surprising, therefore, that the in vivo activity of these polymerases is tightly regulated, so as to minimize their inadvertent access to primer‐termini. We report here that one such mechanism employed by human cells relies on a specific and direct interaction between DNA polymerases ι and η with ubiquitin (Ub). Indeed, we show that both polymerases interact noncovalently with free polyUb chains, as well as mono‐ubiquitinated proliferating cell nuclear antigen (Ub‐PCNA). Mutants of polι (P692R) and polη (H654A) were isolated that are defective in their interactions with polyUb and Ub‐PCNA, whilst retaining their ability to interact with unmodified PCNA. Interestingly, the polymerase mutants exhibit significantly lower levels of replication foci in response to DNA damage, thereby highlighting the biological importance of the polymerase–Ub interaction in regulating the access of the TLS polymerases to stalled replication forks in vivo.

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...