Asami Hishiki

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 Polη, Polι, and Polκ. These enzymes show preferential bypass for different lesions. Proliferating cell nuclear antigen (PCNA), which functions as a sliding clamp for the replicative polymerase Polδ, 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), Polη, Polι, and Polκ 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 Polι 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. 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 Polη, Polι, and Polκ. These enzymes show preferential bypass for different lesions. Proliferating cell nuclear antigen (PCNA), which functions as a sliding clamp for the replicative polymerase Polδ, 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), Polη, Polι, and Polκ 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 Polι 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) 4The abbreviations used are: TLS, translesion synthesis; PCNA, proliferating cell nuclear antigen; IDCL, interdomain connecting loop; PIP-box, PCNA-interacting protein box; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; UBD, ubiquitin-binding domain; SPR, surface plasmon resonance; WT, wild type; GST, glutathione S-transferase. 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 Polη, Polι, Polκ, and REV1, which are all classified as Y-family DNA polymerases on the basis of similarity in their primary sequences (1Ohmori H. Friedberg E.C. Fuchs R.P. Goodman M.F. Hanaoka F. Hinkle D. Kunkel T.A. Lawrence C.W. Livneh Z. Nohmi T. Prakash L. Prakash S. Todo T. Walker G.C. Wang Z. Woodgate R. Mol. Cell.. 2001; 8: 7-8Google Scholar). Human Polη, Polι, and Polκ 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. (2Vaisman A. Lehmann A.R. Woodgate R. Adv. Protein Chem... 2004; 69: 205-228Google Scholar) and Ohmori et al. (3Ohmori H. Ohashi E. Ogi T. Adv. Protein Chem... 2004; 69: 265-278Google Scholar)). Polη can efficiently incorporate two adenines opposite a thymine-thymine (T-T) cyclobutane pyrimidine dimer (4Masutani C. Kusumoto R. Iwai S. Hanaoka F. EMBO J... 2000; 19: 3100-3109Google Scholar), one of the major photoproducts induced by UV irradiation. This function is consistent with the fact that defects in Polη result in a sunlight-sensitive and cancer-prone syndrome, xeroderma pigmentosum variant (5Masutani C. Kusumoto R. Yamada A. Dohmae N. Yokoi M. Yuasa M. Araki M. Iwai S. Takio K. Hanaoka F. Nature.. 1999; 399: 700-704Google Scholar, 6Johnson R.E. Kondratick C.M. Prakash S. Prakash L. Science.. 1999; 285: 263-265Google Scholar). Polι efficiently incorporates an adenine opposite the 3′-T of the T-T (6-4)-photoproduct (7Tissier A. Frank E.G. McDonald J.P. Iwai S. Hanaoka F. Woodgate R. EMBO J... 2000; 19: 5259-5266Google Scholar, 8Vaisman A. Frank E.G. Iwai S. Ohashi E. Ohmori H. Hanaoka F. Woodgate R. DNA Repair.. 2003; 2: 991-1006Google Scholar). Although Polκ is unable to bypass a T-T cyclobutane pyrimidine dimer or (6-4)-photoproduct, it bypasses benzo[a]pyrene diol epoxide-adducted guanines by inserting the correct cytosine opposite the bulky lesion (9Suzuki N. Ohashi E. Kolbanovskiy A. Geacintov N.E. Grollman A.P. Ohmori H. Shibutani S. Biochemistry.. 2002; 41: 6100-6106Google Scholar, 10Zhang Y. Yuan F. Wu X. Wang M. Rechkoblit O. Taylor J.S. Geacintov N.E. Wang Z. Nucleic Acids Res... 2000; 28: 4138-4146Google Scholar-11Zhang Y. Wu X. Guo D. Rechkoblit O. Wang Z. DNA Repair.. 2002; 1: 559-569Google Scholar). Mouse cells lacking Polκ show high sensitivity to treatment with benzo[a]pyrene (12Ogi T. Shinkai Y. Tanaka K. Ohmori H. Proc. Natl. Acad. Sci. U. S. A... 2002; 99: 15548-15553Google Scholar, 13Bi X. Slater D.M. Ohmori H. Vaziri C. J. Biol. Chem... 2005; 280: 22343-22355Google Scholar). Thus, TLS polymerases have different lesion specificity. Proliferating cell nuclear antigen (PCNA) is a ring-shaped homotrimeric protein that encircles DNA and tethers DNA polymerases to the primer terminus, thereby functioning as the sliding processivity factor of DNA replication. Each PCNA monomer is composed of two similar domains (N- and C-terminal domains) linked by a long loop termed the interdomain connecting loop (IDCL). PCNA interacts with a large number of proteins involved in replication, repair, cell cycle, chromatin assembly, and sister-chromatid cohesion (14Moldovan G.L. Pfander B. Jentsch S. Cell.. 2007; 129: 665-679Google Scholar). Most of these proteins have a conserved sequence, the so-called "PCNA-interacting protein box" (PIP-box) (15Warbrick E. BioEssays.. 1998; 20: 195-199Google Scholar). The canonical PIP-box is composed of eight amino acid residues, QXXhXXaa, where the residue at position 1 (p1) is a conserved Gln, "h" at p4 is a such as or at and is an such as or and is the structures of PCNA bound to a from some such as the protein Z. J. M. J. Cell.. Scholar), or bound to the protein of S. K. H. K. K. K. M. E. H. T. EMBO J... 2005; Scholar) have been both of which a canonical structures that the interactions of canonical with PCNA are In the of the conserved at in the canonical PIP-box a in PCNA the in from p4 to in the canonical PIP-box a in which the residue at p4 and the two at and a and a in PCNA the in In to these interactions of the PIP-box with PCNA, the C-terminal the PIP-box in Z. J. M. J. Cell.. Scholar) and S. K. H. K. K. K. M. E. H. T. EMBO J... 2005; Scholar) an with the IDCL, those of the of Y. 2004; Scholar) and Wu Taylor C. Proc. Natl. Acad. Sci. U. S. A... 2005; Scholar), which also a canonical PIP-box, are long to interact extensively with the have a PCNA-binding sequence in Polη L. R.E. B. J. Prakash L. Prakash S. Mol. 2001; Scholar), Polι L. Kondratick C.M. Prakash S. Prakash L. Mol. Cell.. 2001; 8: Scholar, Lehmann A.R. Woodgate R. J. Biol. Chem... 2004; Scholar), and Polκ L. R.E. J. Prakash L. Prakash S. Proc. Natl. Acad. Sci. U. S. A... 2001; Scholar, T. Lehmann A.R. J. 2005; Scholar), of which a canonical PIP-box for the conserved at is replaced with in Polη and in Polι and Polκ. these three TLS to have sequence similar to the the the to interact with PCNA C. E. S. Friedberg E.C. Mol. Cell.. Scholar). The fact that Y-family DNA polymerases have a canonical PIP-box may consistent with the that TLS which are have for PCNA replicative and TLS polymerases are recruited to the replication Polη and interacts with PCNA, and it a sequence similar to the PIP-box at terminus, is the residue in the protein in which been to the with PCNA in vitro L. R.E. B. J. Prakash L. Prakash S. Mol. 2001; Scholar). In vitro the DNA of Polκ is also by the of PCNA, factor and protein from similarity to the Polη C-terminal sequence, the Polκ sequence is the residue in the at the been to a PCNA-binding site L. R.E. J. Prakash L. Prakash S. Mol. 2002; Scholar). Although Polκ found to nuclear that with PCNA in cells after UV or the with an in the PIP-box sequence of the amino acid or the such T. Lehmann A.R. J. 2005; Scholar). Thus, PCNA binding is considered to one of the for the of nuclear of Polκ and Polη in cells T. Lehmann A.R. J. 2005; Scholar, A.R. B. C. D. Woodgate R. Lehmann A.R. EMBO J... 2002; Scholar). Polη and Polκ, which a PIP-box at the terminus, the PCNA-binding site of Polι is amino is in a of the protein; two the PIP-box to by that the Polι with PCNA Lehmann A.R. Woodgate R. J. Biol. Chem... 2004; Scholar, L. N. R.E. J. Prakash L. Prakash S. Mol. 2005; Scholar). Furthermore, Polι nuclear in cells after UV but a with Lehmann A.R. Woodgate R. J. Biol. Chem... 2004; Scholar). PCNA to ubiquitination at Lys-164 by the RAD6-RAD18 complex, which is ubiquitin in cells C. Pfander B. G.L. Jentsch S. Nature.. 2002; ubiquitination is considered to an for DNA synthesis by TLS polymerase at a site of DNA damage in both and cells Nature.. 2003; Scholar, J. Lehmann A.R. Mol. Cell.. 2004; K. S. M. T. H. M. EMBO J... 2004; Scholar). Consistent with this, all Y-family TLS polymerases one or two ubiquitin-binding domains M. C.M. N. F. B. M. Lehmann A.R. K. Science.. 2005; Scholar, A.R. McDonald J.P. S. Woodgate R. EMBO J... Scholar). we the crystal structures of PCNA bound to the noncanonical PIP-box peptides of Polη, Polι, and Polκ and that those noncanonical interact with PCNA in different ways, both from one another and from canonical PIP-box peptides. Our results that the PIP-box of Polκ for PCNA those of Polη and Furthermore, and structural that the PIP-box sequence of Polι from that by one residue and that it a novel with multiple We how the different interactions between PCNA and the noncanonical of the three TLS polymerases with interactions between PCNA and the of these polymerases. PCNA in a K. H. S. S. E. T. J. Biol. Chem... Scholar). The protein purified by and as for the PCNA A. T. A. Ohmori H. M. H. F. Biol. Scholar). peptides used in The with peptides on the of the of the by to the of PCNA protein in the surface at a of at The and and determined from the The the cell The to by of at used in the experiments are in of Polη, Polκ, and Polι peptides with PCNA and PIP-box and the of the in a PCNA to Polη of the with an used for the with PCNA of PCNA bound to the by in at by of the of of and of a containing and to a and for PCNA to Polκ of the with to the termed with PCNA of PCNA bound to the by in at by of the of and of a containing and to a and for PCNA to Polι bound to the and the as A. T. T. Ohmori H. M. H. Biol. Scholar). The used for an at K. for PCNA bound to the by an on at and those for PCNA bound to the and peptides by an on at the by the Z. Scholar). are in The structures by with the A. A. J. Scholar) PCNA Wu Taylor C. Proc. Natl. Acad. Sci. U. S. A... 2005; as a The structures with the K. Biol. 2004; Scholar) and with the J.S. J. M. T. G.L. Biol. 1998; Scholar) and Biol. Scholar). of the structures with the J. Scholar). are in and have been in the Protein and by the on the and all by the and and in are those for the or for PCNA bound to the Polη, Polκ, or Polι a and where is by the of the which and where is by the of the which Most Protein and where is by the of the which in a of the Polη or Polι with PCNA and of PIP-box structures bound to PCNA and PCNA is by a surface and of PCNA that interact with the are PIP-box are by of PCNA that interact with the (p1) residue of Polη in the are structures of Polη, Polκ, and Polι bound to PCNA are in and PIP-box are by and some PIP-box of Polι are DNA Polη and Polι and K. C. Protein Scholar), in and E. a to and by and and and purified by the at protein induced by at and the cells to for at at cells by for at and the in and 1 containing by and cell by at for The with containing The with containing and in containing Polη and Polι with containing and The with and by containing To of or with of wild PCNA or the PCNA in of containing The for at and on of the three with 1 of containing and the bound proteins with of containing and The proteins by and to with or of of Human Polη, Polκ, and interactions of a carrying the PIP-box of Polη, Polκ, or Polι with PCNA in we an We a which the of Polη an for to the for binding to PCNA. a we used a carrying the PIP-box of the protein that is to have a for PCNA. The for the us to the as which is in with the of by E. Biochemistry.. 2000; Scholar). The a for PCNA with an of Polη with for PCNA that the between PCNA and the In to the Polη we for PCNA binding with a or a carrying the C-terminal PIP-box of Polκ. a in which to Gln, the residue conserved at in PIP-box show an for PCNA However, we found that the of the to the C-terminal of Polη, to the of the for PCNA, to the of of the C-terminal from the binding to PCNA. These results that the presence of several is for the noncanonical to interact with PCNA and that the PIP-box sequence of Polκ a for PCNA as with that of In a containing the PCNA-binding sequence of Polι for PCNA with an of which similar to that of the but also in the in binding to PCNA. Furthermore, also binding to PCNA, a in PCNA These results that the two in the noncanonical PIP-box of Polι are but that a In the sequence is replaced with or in the sequence of the or of Polι When we a carrying the PIP-box sequence of or Polι or with for binding to PCNA by SPR, we a PCNA-binding with the Polι similar to that of the Polι but with the Polι results from structural that the noncanonical PIP-box of Polι is and as in that an residue is in at in all of the three TLS polymerases. of PCNA in with TLS PCNA bound to three peptides is in the One of the three Polη peptides and is bound to of the PCNA and of the are in the for the C-terminal residue The in the which for is linked to of the PCNA but the structures of the of the and are the to PCNA binding to the one PCNA is bound to three peptides in the and one of the three Polι peptides and is bound to of the PCNA and of and of Polι peptides are also the the Polκ peptides binding to PCNA, but the of to the of the Polκ in a in the for PCNA. Thus, we PCNA bound to a with to the terminus, The two PCNA one and the and and both are bound to three peptides. The peptides binding to two of and a from the in a in the of and of the we the structures of the of the and peptides as structures to the with PCNA. These PIP-box peptides in the are as the Polη, Polι, and Polκ of the Polη and Polκ with of the noncanonical PIP-box of the Polη bound to PCNA is similar to that of the canonical PIP-box and but the PCNA with the residue at between the noncanonical and canonical In the of (p1) in the noncanonical PIP-box of Polη the and interacts with the and of PCNA in a and the conserved at in the canonical PIP-box interacts with the in a Z. J. M. J. Cell.. Scholar, S. K. H. K. K. K. M. E. H. T. EMBO J... 2005; Scholar, Y. 2004; Wu Taylor C. Proc. Natl. Acad. Sci. U. S. A... 2005; Scholar). a Polη with an for PCNA the wild Polη 1 and and the the of (p1) in the noncanonical PIP-box of the Polκ is highly thereby that (p1) is to interact with the This is consistent with the that the of is large to the In the Polκ of and residue is replaced by which a bulky and is also unable to the as interactions by the in the of Polη and Polκ are similar to and to those of the in the canonical PIP-box These and a by three forks to the of and in Polη or and in Polκ a the and of PCNA and and and in the Polη binding to PCNA the of the interactions between the and PCNA. The structures of the the of Polη and Polκ peptides are from those of the of and The of and an with the of PCNA. However, the of the Polη or Polκ is those of and and one or a with the IDCL, as in the of the and peptides that have the and of Polη in the via with and in the IDCL, In of Polη is in both the and of in the Polη results in a with PCNA that these C-terminal to the between of Polη and of PCNA. This is consistent with the of with PCNA. the C-terminal residue in the Polκ sequence, interacts with and the interact with PCNA that the function to the between of Polκ and of PCNA by of the of as in of the Polι with the noncanonical PIP-box of Polι to Lehmann A.R. Woodgate R. J. Biol. Chem... 2004; Scholar, L. N. R.E. J. Prakash L. Prakash S. Mol. 2005; Scholar). Our that and of Polι a of and as from the PIP-box and that the noncanonical PIP-box of Polι In results show that the of the noncanonical PIP-box of Polι from those of the of Polη and Polκ in the (p1) of Polι interact with the as by with (p1) of Polκ. the of the (p1) residue an with of and the of the of (p1) is in with the of Furthermore, the of the residue also an with the of thereby a structure. that for PCNA that the between the of (p1) and of is for the with PCNA. the of Polι with PCNA but from the interactions of Polη, Polκ, and the canonical The of in the of Polι the of the the of is that of or Thus, the of of the and to with in the of a Polι with a for PCNA as with the This structural of Polι the of the the PIP-box, as The of the Polι also in the the PIP-box from those of the Polη and Polκ as as the and the C-terminal of the Polη or Polκ one or a with the and similar to the and peptides. the the PIP-box of Polι in an internal as to the terminus, as in Polη and Polκ, we that the the PIP-box of Polι an with IDCL, as in and the an with the and are thereby to and In Polη, Polκ, and canonical the of the residue interacts with the of in the and in and However, the structural of the of the residue of Polι from a with the of in the an is between the of the residue and the of in the a the the PIP-box of the Polι an with the IDCL, in to interactions of Polι with PCNA to as with Polη and the Polι an for PCNA with that of the Polη which is in The of the noncanonical of Polη, Polκ, and Polι that they an residue at of these residues, of Polη, of Polκ, and of Polι, an with of PCNA an is also in the complex, where the residue interacts with S. K. H. K. K. K. M. E. H. T. EMBO J... 2005; Scholar, Y. 2004; Scholar). Our that a PCNA protein with an binding to the Polη as with the PCNA protein a Polη with an binding to or the PCNA protein Furthermore, to the at to the between PCNA and Polη or Polι, we a and PCNA in with WT, the of binding of PCNA with Polη or Polι to These results that at to between PCNA and these TLS polymerases. in the residue is highly that such an is a in the noncanonical PIP-box of these TLS polymerases in We have determined the crystal structures of PCNA bound to three containing the noncanonical PIP-box of Polη, Polι, or Polκ. The structures that the interactions of these with PCNA are from one another and also from those of peptides containing a canonical PIP-box and To structural are the to the of a noncanonical PIP-box with PCNA, and they many novel of PCNA interactions with The structural of TLS polymerase conserved in the noncanonical PIP-box of TLS polymerase (p1) of Polη interacts with the of PCNA in a which is in We found that results in a in binding to PCNA that (p1) in the binding of Polη for PCNA such that the DNA polymerase with replicative polymerases in the replication This also to the for the Polη, which at Because is to the the of Polη for PCNA is to that of which is the PCNA-interacting of and the canonical Thus, of for PCNA by the residue at in the of Polη Furthermore, we have that the PIP-box sequence of Polι is The of (p1) of Polι and (p1) of Polκ interact with the that of that residue p4 in the Polι and Polκ peptides a with PCNA and that the number of between the Polι and PCNA is that between the Polη and PCNA, how the Polι interact with PCNA with an with that of the Polη Our that Polι multiple and which is in with PCNA. In the high of Polι also by of the which an of the of between two protein J. Mol. Scholar). and have of and a of these are consistent with for PCNA. Although the number of interactions of the Polι with PCNA is that of the Polη the of a Polι with PCNA is by multiple interactions such that the Polι a high for PCNA with that of the Polη The results in also that the PIP-box of Polκ for PCNA as with Polη or PCNA is at Lys-164 by the RAD6-RAD18 DNA damage in and cells C. Pfander B. G.L. Jentsch S. Nature.. 2002; Scholar, Nature.. 2003; Scholar, J. Lehmann A.R. Mol. Cell.. 2004; K. S. M. T. H. M. EMBO J... 2004; Scholar). This is to for recruitment of TLS polymerase at a site of DNA how ubiquitination at Lys-164 of PCNA with a TLS polymerase containing a The clues to the of the of the three TLS polymerases with Lys-164-monoubiquitinated PCNA. Polη and Polκ have one and two the in both Polη and at the of their PIP-box in the in Polη and one of the two domains in Polκ to the are to interact with the ubiquitin linked to Lys-164 with the of PCNA bound to the PIP-box to interact with the ubiquitin linked to Lys-164 with one of the the between the and PIP-box are long in Polη and in Polι two domains in Polι and at the C-terminal of the PIP-box the the PIP-box of Polι are in an with the of PCNA. This enable the to to interact with the ubiquitin linked to Lys-164 with the bound to the PIP-box, as in Although the between the of the and the PIP-box between these TLS the PIP-box and of TLS polymerase interact the of the PCNA. the of of Polη determined by EMBO 2007; 8: Scholar). The in the between the and ubiquitin to to which is similar to the of many L. 2005; Scholar) but that of the Polη PIP-box to PCNA This that the PIP-box a to the between Polη and ubiquitinated PCNA the thereby the binding of Polη to ubiquitinated PCNA ubiquitinated proteins that are in In that the noncanonical of Polη, Polι, and Polκ interact with PCNA from one Polη, Polι, and Polκ a for PCNA replicative polymerase with a canonical Our results also a structural basis to Polη is recruited to of DNA damage, in to Polκ. This a novel of the Polι PIP-box, with position to the that binding to ubiquitinated PCNA for TLS. We N. K. H. M. and M. for at and Y. N. N. and S. for at We also T. and C. Vaziri for to the with

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.

Small molecule inhibitors of proliferating cell nuclear antigen (PCNA)/PCNA interacting protein box (PIP-Box) interactions, including T2 amino alcohol (T2AA), inhibit translesion DNA synthesis. The crystal structure of PCNA in complex with T2AA revealed that T2AA bound to the surface adjacent to the subunit interface of the homotrimer of PCNA in addition to the PIP-box binding cavity. Because this site is close to Lys-164, which is monoubiquitinated by RAD18, we postulated that T2AA would affect monoubiquitinated PCNA interactions. Binding of monoubiquitinated PCNA and a purified pol η fragment containing the UBZ and PIP-box was inhibited by T2AA in vitro. T2AA decreased PCNA/pol η and PCNA/REV1 chromatin colocalization but did not inhibit PCNA monoubiquitination, suggesting that T2AA hinders interactions of pol η and REV1 with monoubiquitinated PCNA. Interstrand DNA cross-links (ICLs) are repaired by mechanisms using translesion DNA synthesis that is regulated by monoubiquitinated PCNA. T2AA significantly delayed reactivation of a reporter plasmid containing an ICL. Neutral comet analysis of cells receiving T2AA in addition to cisplatin revealed that T2AA significantly enhanced formation of DNA double strand breaks (DSBs) by cisplatin. T2AA promoted colocalized foci formation of phospho-ATM and 53BP1 and up-regulated phospho-BRCA1 in cisplatin-treated cells, suggesting that T2AA increases DSBs. When cells were treated by cisplatin and T2AA, their clonogenic survival was significantly less than that of those treated by cisplatin only. These findings show that the inhibitors of monoubiquitinated PCNA chemosensitize cells by inhibiting repair of ICLs and DSBs.

HLTF (helicase-like transcription factor) is a yeast RAD5 homolog found in mammals. HLTF has E3 ubiquitin ligase and DNA helicase activities, and plays a pivotal role in the template-switching pathway of DNA damage tolerance. HLTF has an N-terminal domain that has been designated the HIRAN (HIP116 and RAD5 N-terminal) domain. The HIRAN domain has been hypothesized to play a role in DNA binding; however, the structural basis of, and functional evidence for, the HIRAN domain in DNA binding has remained unclear. Here we show for the first time the crystal structure of the HIRAN domain of human HLTF in complex with DNA. The HIRAN domain is composed of six β-strands and two α-helices, forming an OB-fold structure frequently found in ssDNA-binding proteins, including in replication factor A (RPA). Interestingly, this study reveals that the HIRAN domain interacts with not only with a single-stranded DNA but also with a duplex DNA. Furthermore, the structure unexpectedly clarifies that the HIRAN domain specifically recognizes the 3′-end of DNA. These results suggest that the HIRAN domain functions as a sensor to the 3′-end of the primer strand at the stalled replication fork and that the domain facilitates fork regression. HLTF is recruited to a damaged site through the HIRAN domain at the stalled replication fork. Furthermore, our results have implications for the mechanism of template switching.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 3′-end of DNA.Conclusion: HLTF is recruited to a damaged site via interaction of the HIRAN domain with 3′-end.Significance: The structure provides a structural basis for the mechanism of template-switching. HLTF (helicase-like transcription factor) is a yeast RAD5 homolog found in mammals. HLTF has E3 ubiquitin ligase and DNA helicase activities, and plays a pivotal role in the template-switching pathway of DNA damage tolerance. HLTF has an N-terminal domain that has been designated the HIRAN (HIP116 and RAD5 N-terminal) domain. The HIRAN domain has been hypothesized to play a role in DNA binding; however, the structural basis of, and functional evidence for, the HIRAN domain in DNA binding has remained unclear. Here we show for the first time the crystal structure of the HIRAN domain of human HLTF in complex with DNA. The HIRAN domain is composed of six β-strands and two α-helices, forming an OB-fold structure frequently found in ssDNA-binding proteins, including in replication factor A (RPA). Interestingly, this study reveals that the HIRAN domain interacts with not only with a single-stranded DNA but also with a duplex DNA. Furthermore, the structure unexpectedly clarifies that the HIRAN domain specifically recognizes the 3′-end of DNA. These results suggest that the HIRAN domain functions as a sensor to the 3′-end of the primer strand at the stalled replication fork and that the domain facilitates fork regression. HLTF is recruited to a damaged site through the HIRAN domain at the stalled replication fork. Furthermore, our results have implications for the mechanism of template switching. 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 3′-end of DNA. Conclusion: HLTF is recruited to a damaged site via interaction of the HIRAN domain with 3′-end. Significance: The structure provides a structural basis for the mechanism of template-switching.

Post-replication DNA repair in eukaryotes is regulated by ubiquitination of proliferating cell nuclear antigen (PCNA). Monoubiquitination catalyzed by RAD6-RAD18 (an E2-E3 complex) stimulates translesion DNA synthesis, whereas polyubiquitination, promoted by additional factors such as MMS2-UBC13 (a UEV-E2 complex) and HLTF (an E3 ligase), leads to template switching in humans. Here, using an in vitro ubiquitination reaction system reconstituted with purified human proteins, we demonstrated that PCNA is polyubiquitinated predominantly via en bloc transfer of a pre-formed ubiquitin (Ub) chain rather than by extension of the Ub chain on monoubiquitinated PCNA. Our results support a model in which HLTF forms a thiol-linked Ub chain on UBC13 (UBC13∼Ubn) and then transfers the chain to RAD6∼Ub, forming RAD6∼Ubn+1. The resultant Ub chain is subsequently transferred to PCNA by RAD18. Thus, template switching may be promoted under certain circumstances in which both RAD18 and HLTF are coordinately recruited to sites of stalled replication.