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...

We have discovered that 3,3',5-triiodothyronine (T3) inhibits binding of a PIP-box sequence peptide to proliferating cell nuclear antigen (PCNA) protein by competing for the same binding site, as evidenced by the co-crystal structure of the PCNA-T3 complex at 2.1 Å resolution. Based on this observation, we have designed a novel, non-peptide small molecule PCNA inhibitor, T2 amino alcohol (T2AA), a T3 derivative that lacks thyroid hormone activity. T2AA inhibited interaction of PCNA/PIP-box peptide with an IC(50) of ~1 μm and also PCNA and full-length p21 protein, the tightest PCNA ligand protein known to date. T2AA abolished interaction of PCNA and DNA polymerase δ in cellular chromatin. De novo DNA synthesis was inhibited by T2AA, and the cells were arrested in S-phase. T2AA inhibited growth of cancer cells with induction of early apoptosis. Concurrently, Chk1 and RPA32 in the chromatin are phosphorylated, suggesting that T2AA causes DNA replication stress by stalling DNA replication forks. T2AA significantly inhibited translesion DNA synthesis on a cisplatin-cross-linked template in cells. When cells were treated with a combination of cisplatin and T2AA, a significant increase in phospho(Ser(139))histone H2AX induction and cell growth inhibition was observed.

Background: HLTF is responsible for template-switching of DNA damage tolerance; HLTF has a novel DNA-binding HIRAN domain, but its function is unknown. Results: The structure of HIRAN domain bound to DNA reveals that the domain recognizes 3Ј-end of DNA. Conclusion: HLTF is recruited to a damaged site via interaction of the HIRAN domain with 3Ј-end. Significance: The structure provides a structural basis for the mechanism of template-switching.