Structural basis for intra- and intermolecular interactions on RAD9 subunit of 9-1-1 checkpoint clamp implies functional 9-1-1 regulation by RHINO.

Eukaryotic DNA clamp is a trimeric protein featuring a toroidal ring structure that binds DNA on the inside of the ring and multiple proteins involved in DNA transactions on the outside. Eukaryotes have two types of DNA clamps: the replication clamp PCNA and the checkpoint clamp RAD9-RAD1-HUS1 (9-1-1). 9-1-1 activates the ATR-CHK1 pathway in DNA damage checkpoint, regulating cell cycle progression. Structure of 9-1-1 consists of two moieties: a hetero-trimeric ring formed by PCNA-like domains of three subunits and an intrinsically disordered C-terminal region of the RAD9 subunit, called RAD9 C-tail. The RAD9 C-tail interacts with the 9-1-1 ring and disrupts the interaction between 9-1-1 and DNA, suggesting a negative regulatory role for this intramolecular interaction. In contrast, RHINO, a 9-1-1 binding protein, interacts with both RAD1 and RAD9 subunits, positively regulating checkpoint activation by 9-1-1. This study presents a biochemical and structural analysis of intra- and inter-molecular interactions on the 9-1-1 ring. Biochemical analysis indicates that RAD9 C-tail binds to the hydrophobic pocket on the PCNA-like domain of RAD9, implying that the pocket is involved in multiple protein-protein interactions. The crystal structure of the 9-1-1 ring in complex with a RHINO peptide reveals that RHINO binds to the hydrophobic pocket of RAD9, shedding light on the RAD9-binding motif. Additionally, the study proposes a structural model of the 9-1-1-RHINO quaternary complex. Together, these findings provide functional insights into the intra- and inter-molecular interactions on the front side of RAD9, elucidating the roles of RAD9 C-tail and RHINO in checkpoint activation.

Uncommon Arrangement of Self-resistance Allows Biosynthesis of de novo Purine Biosynthesis Inhibitor that Acts as an Immunosuppressor.

(-)-FR901483 (1) isolated from the fungus Cladobotryum sp. No.11231 achieves immunosuppression via nucleic acid biosynthesis inhibition rather than IL-2 production inhibition as accomplished by FK506 and cyclosporin A. Recently, we identified the frz gene cluster for the biosynthesis of 1. It contains frzK, a gene homologous to phosphoribosyl pyrophosphate amidotransferase (PPAT)that catalyzes the initial step of de novo purine biosynthesis. We speculated that frzK encodes a PPAT that escapes inhibition by 1 and functions as a self-resistance enzyme (SRE) for the producing host. Nevertheless, details remained elusive. Here, we report the biochemical and structural analyses of FrzK and its Escherichia coli counterpart, PurF. Recombinantly produced FrzK exhibited PPAT activity, albeit weaker than PurF, but evaded strong inhibition by 1. These results confirmed that the target of 1 is PPAT, and FrzK acts as an SRE by maintaining the de novo purine biosynthetic capability in the presence of 1. To understand how FrzK evades inhibition by 1, we determined the crystal structure of PurF in the complex with 1 and constructed a homology model of FrzK. Sequence and structural analyses of various PPATs identified that many residues unique to FrzK occur near the Flexible Loop that remains disordered when inactive but becomes ordered and covers up the active site upon activation by substrate binding. Kinetic characterizations of mutants of the unique residues revealed that the resistance of FrzK against 1 may be conferred by structurally predisposing the Flexible Loop to the active, closed conformation even in the presence of 1.

The 9-1-1 DNA clamp subunit RAD1 forms specific interactions with clamp loader RAD17, revealing functional implications for binding-protein RHINO.

The RAD9-RAD1-HUS1 complex (9-1-1) is a eukaryotic DNA clamp with a crucial role at checkpoints for DNA damage. The ring-like structure of 9-1-1 is opened for loading onto 5' recessed DNA by the clamp loader RAD17 RFC-like complex (RAD17-RLC), in which the RAD17 subunit is responsible for specificity to 9-1-1. Loading of 9-1-1 is required for activation of the ATR-CHK1 checkpoint pathway and the activation is stimulated by a 9-1-1 interacting protein, RHINO, which interacts with 9-1-1 via a recently identified RAD1-binding motif. This discovery led to the hypothesis that other interacting proteins may contain a RAD1-binding motif as well. Here, we show that vertebrate RAD17 proteins also have a putative RAD1-binding motif in their N-terminal regions, and we report the crystal structure of human 9-1-1 bound to a human RAD17 peptide incorporating the motif at 2.1 Å resolution. Our structure confirms that the N-terminal region of RAD17 binds to the RAD1 subunit of 9-1-1 via specific interactions. Furthermore, we show that the RAD1-binding motif of RHINO disturbs the interaction of the N-terminal region of RAD17 with 9-1-1. Our results provide deeper understanding of how RAD17-RLC specifically recognizes 9-1-1 and imply that RHINO has a functional role in 9-1-1 loading/unloading and checkpoint activation.

Crystal structure of the sliding DNA clamp from the Gram-positive anaerobic bacterium Clostridioides difficile.

The sliding DNA clamp is a ring-shaped protein that encircles DNA within its central channel. It binds to multiple proteins, such as DNA polymerases and DNA repair enzymes, and stimulates their enzymatic activities, thereby playing a crucial role in cell survival and proliferation. Accordingly, the bacterial clamp DnaN is considered to be a promising target for bacterial infection therapy. In this regard, 3D structures of DnaN from pathogenic bacteria are essential for the development of chemical compounds with antimicrobial activity. Here, the crystal structure of DnaN from a Gram-positive bacterium Clostridioides difficile, a human pathogen causing infectious diarrhoea, has been determined at 2.13 Å resolution. A comparison of the structures of DnaN from other bacteria indicates that the structural features of DnaN in terms of overall organization are essentially conserved within Gram-positive and Gram-negative bacteria. However, DnaN from C. difficile has structural differences in the potential binding pocket for partner proteins, implying a non-conventional interaction with its binding partners. Our findings will provide insight into the development of new therapies for C. difficile infection.

Structural basis for molecular interactions on the eukaryotic DNA sliding clamps PCNA and RAD9-RAD1-HUS1.

DNA sliding clamps are widely conserved in all living organisms and play crucial roles in DNA replication and repair. Each DNA sliding clamp is a doughnut-shaped protein with a quaternary structure that encircles the DNA strand and recruits various factors involved in DNA replication and repair, thereby stimulating their biological functions. Eukaryotes have two types of DNA sliding clamp, proliferating cell nuclear antigen (PCNA) and RAD9-RAD1-HUS1 (9-1-1). The homo-trimer PCNA physically interacts with multiple proteins containing a PCNA-interacting protein box and/or AlkB homologue 2 PCNA-interacting motif. The two motifs bind to PCNA by a similar mechanism; in addition, the bound PCNA structure is similar, implying a universality of PCNA interactions. In contrast to PCNA, 9-1-1 is a hetero-trimer composed of RAD9, RAD1 and HUS1 subunits. Although 9-1-1 forms a trimeric ring structure similar to PCNA, the C-terminal extension of the RAD9 is intrinsically unstructured. Based on the structural similarity between PCNA and 9-1-1, the mechanism underlying the interaction of 9-1-1 with its partners was thought to be analogous to that of PCNA. Unexpectedly, however, the recent structure of the 9-1-1 ring bound to a partner has revealed a novel interaction distinct from that of PCNA, potentially providing a new principle for molecular interactions on DNA sliding clamps.

Development of a DNA aptamer that binds to the complementarity-determining region of therapeutic monoclonal antibody and affinity improvement induced by pH-change for sensitive detection.

Therapeutic monoclonal antibodies (mAbs) are successful biomedicines; however, evaluation of their pharmacokinetics and pharmacodynamics demands highly specific discrimination from human immunoglobulin G naturally present in the blood. Here, we developed a novel anti-idiotype aptamer (termed A14#1) with extraordinary specificity against the anti-vascular endothelial growth factor therapeutic mAb, bevacizumab. Structural analysis of the antibody-aptamer complex showed that several bases of A14#1 recognized only the complementarity determining region (CDR) of bevacizumab, thereby contributing to its extraordinary specificity. As the CDR of bevacizumab is predicted to be highly positively charged under mildly acidic conditions and that DNA is negatively charged, the affinity of A14#1 to bevacizumab markedly increased at pH 4.7 (KD = 44 pM) than at pH 7.4 (KD = 12 nM). A14#1-based electrochemical detection method capable of detecting 31 pM of bevacizumab at pH 4.7 was thus developed. A14#1 could be potentially useful for therapeutic drug measurement as a novel ligand of bevacizumab.

Structural basis for peptide recognition by archaeal oligopeptide permease A.

Oligopeptide permease A (OppA) plays an important role in the nutrition of cells and various signaling processes. In archaea, OppA is a major protein present in membrane vesicles of Thermococcales. Because there being no crystal structures of archaeal OppAs determined to date, we report the crystal structure of archaeal OppA from Thermococcus kodakaraensis (TkOppA) at 2.3 Å resolution by the single-wavelength anomalous dispersion method. TkOppA consists of three domains similarly to bacterial OppAs, and the inserted regions not present in bacterial OppAs are at the periphery of the core region. An endogenous pentapeptide was bound in the pocket of domains I and III of TkOppA by hydrogen bonds of main-chain atoms of the peptide and hydrophobic interactions. No hydrogen bonds of side-chain atoms of the peptide were observed; thus, TkOppA may have low peptide selectivity but some preference for residues 2 and 3. TkOppA has a relatively large pocket and can bind a nonapeptide; therefore, it is suitable for the binding of large peptides similarly to OppAs of Gram-positive bacteria.

Catalytic mechanism and endo-to-exo selectivity reversion of an octalin-forming natural Diels-Alderase.

We have previously reported the identification of CghA, a proposed Diels-Alderase responsible for the formation of the bicyclic octalin core of the fungal secondary metabolite Sch210972. Here we show the crystal structure of the CghA-product complex at a resolution of 2.0 Å. Our result provides the second structural determination of eukaryotic Diels-Alderases and adds yet another fold to the family of proteins reported to catalyse [4 + 2] cycloaddition reactions. Site-directed mutagenesis-coupled kinetic characterization and computational analyses allowed us to identify key catalytic residues and propose a possible catalytic mechanism. Most interestingly, we were able to rationally engineer CghA such that the mutant was able to catalyse preferentially the formation of the energetically disfavoured exo adduct. This work expands our knowledge and understanding of the emerging and potentially widespread class of natural enzymes capable of catalysing stereoselective Diels-Alder reactions and paves the way towards developing enzymes potentially useful in various bio/synthetic applications.

Specialized Flavoprotein Promotes Sulfur Migration and Spiroaminal Formation in Aspirochlorine Biosynthesis.

Epidithiodiketopiperazines (ETPs) are a class of ecologically and medicinally important cyclodipeptides bearing a reactive transannular disulfide bridge. Aspirochlorine, an antifungal and toxic ETP isolated from Aspergillus oryzae used in sake brewing, deviates from the common ETP scaffold owing to its unusual ring-enlarged disulfide bridge linked to a spiroaminal ring system. Although this disulfide ring system is implicated in the biological activity of ETPs the biochemical basis for this derailment has remained a mystery. Here we report the discovery of a novel oxidoreductase (AclR) that represents the first-in-class enzyme catalyzing both a carbon-sulfur bond migration and spiro-ring formation, and that the acl pathway involves a cryptic acetylation as a prerequisite for the rearrangement. Genetic screening in A. oryzae identified aclR as the candidate for the complex biotransformation, and the aclR-deficient mutant provided the biosynthetic intermediate, unexpectedly harboring an acetyl group. In vitro assays showed that AclR alone promotes 1,2-sulfamyl migration, elimination of the acetoxy group, and spiroaminal formation. AclR features a thioredoxin oxidoreductase fold with a noncanonical CXXH motif that is distinct from the CXXC in the disulfide forming oxidase for the ETP biosynthesis. Crystallographic and mutational analyses of AclR revealed that the CXXH motif is crucial for catalysis, whereas the flavin-adenine dinucleotide is required as a support of the protein fold, and not as a redox cofactor. AclR proved to be a suitable bioinformatics handle to discover a number of related fungal gene clusters that potentially code for the biosynthesis of derailed ETP compounds. Our results highlight a specialized role of the thioredoxin oxidoreductase family enzyme in the ETP pathway and expand the chemical diversity of small molecules bearing an aberrant disulfide pharmacophore.

Structural and Functional Analyses of a Spiro-Carbon-Forming, Highly Promiscuous Epoxidase from Fungal Natural Product Biosynthesis.

Biosynthesis of fungal nonribosomal peptides frequently involves redox enzymes such as flavin-containing monooxygenase (FMO) to introduce complexity into the core chemical structure. One such example is the formation of spiro-carbons catalyzed by various oxidases. Because many chemically complex spiro-carbon-bearing natural products exhibit useful biological activities, understanding the mechanism of spiro-carbon biosynthesis is of great interest. We previously identified FqzB, an FMO from the fumiquinazoline biosynthetic pathway responsible for epoxidation of fumiquinazoline F that crosstalks with the fumitremorgin biosynthetic pathway to form spirotryprostatin A via epoxidation of the precursor fumitremorgin C. What makes FqzB more interesting is its relaxed substrate specificity, where it can accept a range of other substrates, including tryprostatins A and B along with its original substrate fumiquinazoline F. Here, we characterized FqzB crystallographically and examined FqzB and its site-specific mutants kinetically to understand how this promiscuous epoxidase works. Furthermore, the mutagenesis studies as well as computational docking experiments between the FqzB crystal structure and its known substrates spirotryprostatin A and B, as well as fumitremorgin C and fumiquinazoline F, provided insight into potential modes of substrate recognition and the source of broad substrate tolerance exhibited by this epoxidase. This study serves as a foundation for further characterization and engineering of this redox enzyme, which has potential utility as a valuable catalyst with broad substrate tolerance and an ability to introduce chemical complexity into carbon frameworks for chemoenzymatic and biosynthetic applications.

Inactive dimeric structure of the protease domain of stomatin operon partner protein.

The N-terminal region of the stomatin operon partner protein (STOPP) PH1510 (1510-N) from the hyperthermophilic archaeon Pyrococcus horikoshii is a serine protease with a catalytic Ser-Lys dyad (Ser97 and Lys138) and specifically cleaves the C-terminal hydrophobic region of the p-stomatin PH1511. In a form of human hemolytic anemia known as hereditary stomatocytosis, stomatin is deficient in the erythrocyte membrane owing to mis-trafficking. Stomatin is thought to act as an oligomeric scaffolding protein to support cell membranes. The cleavage of stomatin by STOPP might be involved in a regulatory system. Several crystal structures of 1510-N have previously been determined: the wild type, the K138A mutant and its complex with a substrate peptide. Here, the crystal structure of the S97A mutant of 1510-N (1510-N S97A) was determined at 2.25 Šresolution. The structure contained two 1510-N S97A molecules in the asymmetric unit. On the superposition of one monomer of the 1510-N S97A and wild-type dimers, the S97A Cα atom of the other monomer of 1510-N S97A deviated by 23 Šfrom that of the wild type. This result indicates that 1510-N can greatly change the form of its dimer. Because of crystallographic symmetry in space group P65, a sixfold helical structure is constructed using the 1510-N dimer as a basic unit. This helical structure may be common to STOPP structures.

Structure of the RAD9-RAD1-HUS1 checkpoint clamp bound to RHINO sheds light on the other side of the DNA clamp.

DNA clamp, a highly conserved ring-shaped protein, binds dsDNA within its central pore. Also, DNA clamp interacts with various nuclear proteins on its front, thereby stimulating their enzymatic activities and biological functions. It has been assumed that the DNA clamp is a functionally single-faced ring from bacteria to humans. Here, we report the crystal structure of the heterotrimeric RAD9-RAD1-HUS1 (9-1-1) checkpoint clamp bound to a peptide of RHINO, a recently identified cancer-related protein that interacts with 9-1-1 and promotes activation of the DNA damage checkpoint. This is the first structure of 9-1-1 bound to its partner. The structure reveals that RHINO is unexpectedly bound to the edge and around the back of the 9-1-1 ring through specific interactions with the RAD1 subunit of 9-1-1. Our finding indicates that 9-1-1 is a functionally double-faced DNA clamp.

Functional and Structural Analyses of trans C-Methyltransferase in Fungal Polyketide Biosynthesis.

Biosynthesis of certain fungal polyketide-peptide synthetases involves C-methyltransferase activity that adds one or more S-adenosyl-l-methionine-derived methyl groups to the carbon framework. The previously reported PsoF-MT, the stand-alone C-methyltransferase (MT) from the pseurotin biosynthetic pathway that exists as a domain within a trifunctional didomain enzyme PsoF, was characterized crystallographically and kinetically using mutants with substrate analogs to understand how a trans-acting C-MT works and compare it to known polyketide synthase-associated C-MTs. This study identified key active-site residues involved in catalysis and substrate recognition, which led us to propose the mechanism of C-methylation and substrate specificity determinants in PsoF-MT.

[Structural Basis of the Multifunctional Hub Protein and Identification of a Small-molecule Compound for Drug Discovery].

Translesion DNA synthesis (TLS) is an emergency system activated to inhibit cell death caused by DNA damage-induced replication arrest. Thus, TLS enables cancer cells to acquire resistance to alkylate anticancer drugs. REV7 functions as the hub protein that interacts with both the inserter DNA polymerase REV1 and the extender DNA polymerase REV3 in TLS. REV7-mediated protein-protein interactions (PPIs) are essential for the activation of TLS, and are therefore attractive targets for anticancer drug development. To clarify the REV7-REV3 and REV7-REV1 PPIs, we determined the structures of REV7-REV3 and REV7-REV3-REV1 complexes. In the structures of REV7-REV3 and REV7-REV3-REV1 complexes, REV7 wraps around the REV3 fragment, and the REV1-binding interface is distinct from the REV3-binding site of REV7. We also identified a novel REV7 binding protein, transcription factor II-I (TFII-I), which is required for TLS. Of note, TFII-I binds the REV7-REV3-REV1 complex, suggesting that REV7-TFII-I PPIs are independent of other REV7-mediated PPIs. Furthermore, we found a small-molecule compound that inhibits TLS by targeting the REV7-REV3 PPIs. Lastly, we determined the structure of REV7 in complex with chromosome alignment maintaining phosphoprotein (CAMP), a known kinetochore-microtubule attachment protein. The overall structure of the REV7-CAMP complex is similar to that of the REV7-REV3 complex, but the REV7-CAMP PPIs are markedly different from the REV7-REV3 PPIs. These findings improve our understanding of multifunctional hub proteins, and are helpful for designing small-molecule compounds for novel anticancer drug development.

Structural basis of HEAT-kleisin interactions in the human condensin I subcomplex.

Condensin I is a multi-protein complex that plays an essential role in mitotic chromosome assembly and segregation in eukaryotes. It is composed of five subunits: two SMC (SMC2 and SMC4), a kleisin (CAP-H), and two HEAT-repeat (CAP-D2 and CAP-G) subunits. Although balancing acts of the two HEAT-repeat subunits have been demonstrated to enable this complex to support the dynamic assembly of chromosomal axes in vertebrate cells, its underlying mechanisms remain poorly understood. Here, we report the crystal structure of a human condensin I subcomplex comprising hCAP-G and hCAP-H. hCAP-H binds to the concave surfaces of a harp-shaped HEAT-repeat domain of hCAP-G. Physical interaction between hCAP-G and hCAP-H is indeed essential for mitotic chromosome assembly recapitulated in Xenopus egg cell-free extracts. Furthermore, this study reveals that the human CAP-G-H subcomplex has the ability to interact with not only double-stranded DNA, but also single-stranded DNA, suggesting functional divergence of the vertebrate condensin I complex in proper mitotic chromosome assembly.

Enzymatic one-step ring contraction for quinolone biosynthesis.

The 6,6-quinolone scaffolds on which viridicatin-type fungal alkaloids are built are frequently found in metabolites that display useful biological activities. Here we report in vitro and computational analyses leading to the discovery of a hemocyanin-like protein AsqI from the Aspergillus nidulans aspoquinolone biosynthetic pathway that forms viridicatins via a conversion of the cyclopenin-type 6,7-bicyclic system into the viridicatin-type 6,6-bicyclic core through elimination of carbon dioxide and methylamine through methyl isocyanate.

Structure of proliferating cell nuclear antigen (PCNA) bound to an APIM peptide reveals the universality of PCNA interaction.

Proliferating cell nuclear antigen (PCNA) provides a molecular platform for numerous protein-protein interactions in DNA metabolism. A large number of proteins associated with PCNA have a well characterized sequence termed the PCNA-interacting protein box motif (PIPM). Another PCNA-interacting sequence termed the AlkB homologue 2 PCNA-interacting motif (APIM), comprising the five consensus residues (K/R)-(F/Y/W)-(L/I/V/A)-(L/I/V/A)-(K/R), has also been identified in various proteins. In contrast to that with PIPM, the PCNA-APIM interaction is less well understood. Here, the crystal structure of PCNA bound to a peptide carrying an APIM consensus sequence, RFLVK, was determined and structure-based interaction analysis was performed. The APIM peptide binds to the PIPM-binding pocket on PCNA in a similar way to PIPM. The phenylalanine and leucine residues within the APIM consensus sequence and a hydrophobic residue that precedes the APIM consensus sequence are crucially involved in interactions with the hydrophobic pocket of PCNA. This interaction is essential for overall binding. These results provide a structural basis for regulation of the PCNA interaction and might aid in the development of specific inhibitors of this interaction.

Structural and Thermodynamic Basis of the Enhanced Interaction between Kinesin Spindle Protein Eg5 and STLC-type Inhibitors.

For a better understanding of protein-inhibitor interactions, we report structural, thermodynamic, and biological analyses of the interactions between S-trityl-l-cysteine (STLC) derivatives and the motor domain of kinesin spindle protein Eg5. Binding of STLC-type inhibitors to Eg5 was enthalpically driven and entropically unfavorable. The introduction of a para-methoxy substituent in one phenyl ring of STLC enhances its inhibitory activity resulting from a larger enthalpy gain possibly due to the increased shape complementarity. The substituent fits to a recess in the binding pocket. To avoid steric hindrance, the substituted STLC is nudged toward the side opposite to the recess, which enhances the interaction of Eg5 with the remaining part of the inhibitor. Further introduction of an ethylene linkage between two phenyl rings enhances Eg5 inhibitory activity by reducing the loss of entropy in forming the complex. This study provides valuable examples of enhancing protein-inhibitor interactions without forming additional hydrogen bonds.

Dynamic feature of mitotic arrest deficient 2-like protein 2 (MAD2L2) and structural basis for its interaction with chromosome alignment-maintaining phosphoprotein (CAMP).

Mitotic arrest deficient 2-like protein 2 (MAD2L2), also termed MAD2B or REV7, is involved in multiple cellular functions including translesion DNA synthesis (TLS), signal transduction, transcription, and mitotic events. MAD2L2 interacts with chromosome alignment-maintaining phosphoprotein (CAMP), a kinetochore-microtubule attachment protein in mitotic cells, presumably through a novel "WK" motif in CAMP. Structures of MAD2L2 in complex with binding regions of the TLS proteins REV3 and REV1 have revealed that MAD2L2 has two faces for protein-protein interactions that are regulated by its C-terminal region; however, the mechanisms underlying the MAD2L2-CAMP interaction and the mitotic role of MAD2L2 remain unknown. Here we have determined the structures of human MAD2L2 in complex with a CAMP fragment in two crystal forms. The overall structure of the MAD2L2-CAMP complex in both crystal forms was essentially similar to that of the MAD2L2-REV3 complex. However, the residue interactions between MAD2L2 and CAMP were strikingly different from those in the MAD2L2-REV3 complex. Furthermore, structure-based interaction analyses revealed an unprecedented mechanism involving CAMP's WK motif. Surprisingly, in one of the crystal forms, the MAD2L2-CAMP complex formed a dimeric structure in which the C-terminal region of MAD2L2 was swapped and adopted an immature structure. The structure provides direct evidence for the dynamic nature of MAD2L2 structure, which in turn may have implications for the protein-protein interaction mechanism and the multiple functions of this protein. This work is the first structural study of MAD2L2 aside from its role in TLS and might pave the way to clarify MAD2L2's function in mitosis.

Structural basis for the molecular interactions in DNA damage tolerances.

DNA damage tolerance (DDT) is a cell function to avoid replication arrest by DNA damage during DNA replication. DDT includes two pathways, translesion DNA synthesis (TLS) and template-switched DNA synthesis (TS). DDT is regulated by ubiquitination of proliferating cell nuclear antigen that binds to double-stranded DNA and functions as scaffold protein for DNA metabolism. TLS is transient DNA synthesis using damaged DNA as a template by error-prone DNA polymerases termed TLS polymerases specialized for DNA damage. TS, in which one newly synthesized strand is utilized as an undamaged template for replication by replicative polymerases, is error-free process. Thus, DDT is not inherently a repair pathway. DDT is a mechanism to tolerate DNA damage, giving priority to DNA synthesis and enabling finish of DNA replication for cell survival and genome stability. DDT is associated with cancer development and thus is of great interest in drug discovery for cancer therapy. This review article describes recent progress in structural studies on protein-protein and protein-DNA complexes involved in TLS and TS, providing the molecular mechanisms of interactions in DDT.