Polκ κ κ κ is one of many DNA polymerases involved in translesion DNA synthesis (TLS). It belongs to the Y-family of polymerases along with Polη η η η, Polι ι ι ι and hREV1. Unlike Polη η η η encoded by the xeroderma pigmentosum variant (XPV ) gene, Polκ κ κ κ is unable to bypass UV-induced DNA damage in vitro, but it is able to bypass benzo[a]pyrene (B[a]P)-adducted guanines accurately and efficiently. In an attempt to identify factor(s) targeting Polκ κ κ κ to its cognate DNA lesion(s), we searched for Polκ κ κ κ-interacting proteins by using the yeast two-hybrid assay. We found that Polκ κ κ κ interacts with a C-terminal region of hREV1. Polη η η η and Polι ι ι ι were also found to interact with the same region of hREV1. The interaction between Polκ κ κ κ and hREV1 was confirmed by pull-down and coimmunoprecipitation assays. The C-terminal region of hREV1 is known to interact with hREV7, a non-catalytic subunit of Polζ ζ ζ ζ that is another structurally unrelated TLS enzyme, and we show that Polκ κ κ κ and hREV7 bind to the same C-terminal region of hREV1. Thus, our results suggest that hREV1 plays a pivotal role in the multi-enzyme, multi-step process of translesion DNA synthesis.

Translesion synthesis (TLS) is a DNA damage tolerance mechanism that allows continued DNA synthesis, even in the presence of damaged DNA templates. Mammals have multiple DNA polymerases specialized for TLS, including Pol, Pol, and Pol. These enzymes show preferential bypass for different lesions. Proliferating cell nuclear antigen (PCNA), which functions as a sliding clamp for the replicative polymerase Pol␦, also interacts with the three TLS polymerases. Although many PCNA-binding proteins have a highly conserved sequence termed the PCNA-interacting protein box (PIP-box), Pol, Pol, and Pol have a noncanonical PIP-box sequence. In response to DNA damage, Lys-164 of PCNA undergoes ubiquitination by the RAD6-RAD18 complex, and the ubiquitination is considered to facilitate TLS. Consistent with this, these three TLS polymerases have one or two ubiquitin binding domains and are recruited to replication forks via interactions with ubiquitinated PCNA involving the noncanonical PIP-box and ubiquitin binding domain. However, it is unclear how these TLS polymerases interact with PCNA. To address the structural basis for interactions between different TLS polymerases and PCNA, we determined crystal structures of PCNA bound to peptides containing the noncanonical PIP-box of these polymerases. We show that the three PIP-box peptides interact with PCNA in different ways, both from one another and from canonical PIP-box peptides. Especially, the PIP-box of Pol adopts a novel structure. Furthermore, these structures enable us to speculate how these TLS polymerases interact with Lys-164-monoubiquitinated PCNA. Our results will provide clues to understanding the mechanism of preferential recruitment of TLS polymerases to the stalled forks.Genomic DNA carrying genetic information is constantly damaged by various internal and external agents. Most types of DNA damage are removed by multiple DNA repair mechanisms, but some of them, especially those generating relatively small distortion of the DNA double helix structure, may escape DNA repair and persist in S-phase. When a replicative DNA polymerase encounters such a persisting lesion, it often stalls there. One way to continue replication past the lesion site is to replace the stalled replicative polymerase with a DNA polymerase specialized for translesion synthesis (TLS) 4 that is able to incorporate nucleotides opposite DNA lesions. Because TLS polymerases have low fidelity and processivity, they are subsequently replaced by replicative polymerases after the lesion bypass is completed. To date, several TLS polymerases have been found in mammals, including Pol, Pol, Pol, and REV1, which are all classified as Y-family DNA polymerases on the basis of similarity in their primary sequences (1).Human Pol, Pol, and Pol have been purified and extensively studied by in vitro experiments with DNA containing one of various types of lesion on the template strand (reviewed by Vaisman et al. (2) and Ohmori et al. (3)). Pol can efficiently incorporate two adenines opposite a thymine-thymine...

When a replicative DNA polymerase (Pol) is stalled by damaged DNA, a 'polymerase switch' recruits specialized translesion synthesis (TLS) DNA polymerase(s) to sites of damage. Mammalian cells have several TLS DNA polymerases, including the four Y-family enzymes (Polη, Polι, Polκ and REV1) that share multiple primary sequence motifs, but show preferential bypass of different DNA lesions. REV1 interacts with Polη, Polι, and Polκ and therefore appears to play a central role during TLS in vivo. Here we have investigated the molecular basis for interactions between REV1 and Polκ. We have identified novel REV1-interacting regions (RIRs) present in Polκ, Polι and Polη. Within the RIRs, the presence of two consecutive phenylalanines (FF) is essential for REV1-binding. PCNA-binding motifs found in many proteins also contain FF with some conserved residues N-terminal to FF, as frequently represented by Q-x-x-(I,L,M)-x-x-F-F (x, any residue). In contrast, our results show that the critical FF in RIR motifs are not flanked by specific conserved residues. Instead, the consensus sequence for REV1-binding is denoted by x-F-F-y-y-y-y (y, any residue but not proline). Our results identify structural requirements that are necessary for FF-flanking residues to confer interactions with REV1. A Polκ mutant lacking REV1-binding activity did not complement the genotoxin-sensitivity of Polk-null mouse embryonic fibroblast cells, thereby demonstrating that the REV1-interaction is essential for Polκ function in vivo.

Mammalian DNA polymerase (pol ), a member of the UmuC/DinB nucleotidyl transferase superfamily, has been implicated in spontaneous mutagenesis. Here we show that human pol copies undamaged DNA with average single-base substitution and deletion error rates of 7 ؋ 10 ؊3 and 2 ؋ 10 ؊3 , respectively. These error rates are high when compared to those of most other DNA polymerases. pol also has unusual error specificity, producing a high proportion of T⅐CMP mispairs and deleting and adding non-reiterated nucleotides at extraordinary rates. Unlike other members of the UmuC/ DinB family, pol can processively synthesize chains of 25 or more nucleotides. This moderate processivity may reflect a contribution of C-terminal residues, which include two zinc clusters. The very low fidelity and moderate processivity of pol is novel in comparison to any previously studied DNA polymerase, and is consistent with a role in spontaneous mutagenesis.The recently discovered UmuC/DinB nucleotidyl transferase superfamily of DNA polymerases (1-3) includes a subfamily whose members share extensive amino acid sequence homology with the Escherichia coli dinB gene product. The dinB gene is required for untargeted mutagenesis of phage (4), and overexpression of dinB in E. coli increases the spontaneous mutation rate in plasmids, especially for single-base deletions in a run of guanine residues (5). The dinB gene encodes DNA polymerase (pol) IV, a distributive enzyme that lacks detectable 3Ј35Ј exonuclease activity (6). pol IV has limited ability to bypass UV radiation-induced photoproducts, but misinserts nucleotides at undamaged template sites at rates that are higher than those observed for DNA pol III, the major replicative enzyme in E. coli (7). Moreover, when incubated in the presence of a template-primer with a terminal mismatch with a potential for misalignment, pol IV generates DNA products that are one nucleotide shorter than expected (6). This is consistent with the Ϫ1 frameshift mutations seen when dinB is overexpressed (5).Orthologs of E. coli dinB have been identified in eukaryotes (2, 8). The human DINB1 gene is localized at chromosome 5q13 and encodes an 870-amino acid DNA polymerase (9 -11), which we refer to here as DNA polymerase (pol ).1 The product of the hDINB1 gene has also been designated DNA polymerase (10), a designation used earlier (12) for the human homolog of the Drosophila melanogaster mus308 gene. pol has several properties in common with E. coli pol IV. When purified from insect cells expressing the full-length polymerase fused to glutathione S-transferase (GST) (11) or purified as a catalytically active fragment of amino acids 1-560 (9), pol lacks detectable 3Ј35Ј exonuclease activity. The purified full-length GST fusion protein has optimal activity at 37°C over the pH range 6.5-7.5, it is insensitive to inhibition by aphidicolin or dideoxynucleotides, and Mg 2ϩ is preferred over Mn 2ϩ as the essential divalent cation (11). Neither the full-length GST-enzyme purified from yeast cells (10) nor truncated pol (9)...

Several recently discovered human DNA polymerases are associated with translesion synthesis past DNA adducts. These include human DNA polymerase kappa (pol kappa), a homologue of Escherichia coli pol IV, which enhances the frequency of spontaneous mutation. Using a truncated form of pol kappa (pol kappa Delta C), translesion synthesis past dG-(+)- or dG-(-)-anti-N(2)-BPDE (7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene) adducts was explored. Site-specifically-modified oligodeoxynucleotides containing a single stereoisomeric dG-N(2)-BPDE lesion were used as DNA templates for primer extension reactions catalyzed by pol kappa Delta C. Primer extension was retarded one base prior to the dG-N(2)-BPDE lesion; when incubated for longer times or with higher concentration of enzyme, full primer extension was observed. Quantitative analysis of fully extended products showed preferential incorporation of dCMP, the correct base, opposite all four stereoisomeric dG-N(2)-BPDE lesions. (+)-trans-dG-N(2)-BPDE, a major BPDE-DNA adduct, promoted small amounts of dTMP, dAMP, and dGMP misincorporation opposite the lesion (total 2.7% of the starting primers) and deletions (1.1%). Although (+)-cis-dG-N(2)-BPDE was most effective in blocking translesion synthesis, its miscoding properties were similar to other dG-N(2)-BPDE isomers. Steady-state kinetic data indicate that dCMP is efficiently inserted opposite all dG-N(2)-BPDE adducts and extended past these lesions. The relative frequency of translesion synthesis (F(ins) x F(ext)) of dC.dG-N(2)-BPDE pairs was 2-6 orders of magnitude higher than that of other mismatched pairs. Pol kappa may play an important role in translesion synthesis by incorporating preferentially the correct base opposite dG-N(2)-BPDE. Its relatively low contribution to mutagenicity suggests that other newly discovered DNA polymerase(s) may be involved in mutagenic events attributed to dG-N(2)-BPDE adducts in human cells.