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.

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.

Structure of the HLTF HIRAN domain and its functional implications in regression of a stalled replication fork.

HLTF (helicase-like transcription factor) is a yeast RAD5 homolog that is found in mammals. HLTF has E3 ubiquitin ligase and DNA helicase activities, and is a pivotal protein in template-switched DNA synthesis that allows DNA replication to continue even in the presence of DNA damage by utilizing a newly synthesized undamaged strand as a template. In addition, HLTF has a DNA-binding domain termed HIRAN (HIP116 and RAD5 N-terminal). HIRAN has been hypothesized to play a role in DNA binding; however, the structural basis of its role in DNA binding has remained unclear. In the past five years, several crystal structures of HIRAN have been reported. These structures revealed new insights into the molecular mechanism underlying DNA binding by HIRAN. Here, the structural information on HIRAN is summarized and the function of HIRAN in recognizing the 3'-terminus of the daughter strand at a stalled replication fork and the implications for its involvement in fork regression are discussed.

Structure of HIRAN domain of human HLTF bound to duplex DNA provides structural basis for DNA unwinding to initiate replication fork regression.

Replication fork regression is a mechanism to rescue a stalled fork by various replication stresses, such as DNA lesions. Helicase-like transcription factor, a SNF2 translocase, plays a central role in the fork regression and its N-terminal domain, HIRAN (HIP116 and Rad5 N-terminal), binds the 3'-hydroxy group of single-stranded DNA. Furthermore, HIRAN is supposed to bind double-stranded DNA (dsDNA) and involved in strand separation in the fork regression, whereas structural basis for mechanisms underlying dsDNA binding and strand separation by HIRAN are still unclear. Here, we report the crystal structure of HIRAN bound to duplex DNA. The structure reveals that HIRAN binds the 3'-hydroxy group of DNA and unexpectedly unwinds three nucleobases of the duplex. Phe-142 is involved in the dsDNA binding and the strand separation. In addition, the structure unravels the mechanism underlying sequence-independent recognition for purine bases by HIRAN, where the N-glycosidic bond adopts syn conformation. Our findings indicate direct involvement of HIRAN in the fork regression by separating of the daughter strand from the parental template.

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.

Regulation of HLTF-mediated PCNA polyubiquitination by RFC and PCNA monoubiquitination levels determines choice of damage tolerance pathway.

DNA-damage tolerance protects cells via at least two sub-pathways regulated by proliferating cell nuclear antigen (PCNA) ubiquitination in eukaryotes: translesion DNA synthesis (TLS) and template switching (TS), which are stimulated by mono- and polyubiquitination, respectively. However, how cells choose between the two pathways remains unclear. The regulation of ubiquitin ligases catalyzing polyubiquitination, such as helicase-like transcription factor (HLTF), could play a role in the choice of pathway. Here, we demonstrate that the ligase activity of HLTF is stimulated by double-stranded DNA via HIRAN domain-dependent recruitment to stalled primer ends. Replication factor C (RFC) and PCNA located at primer ends, however, suppress en bloc polyubiquitination in the complex, redirecting toward sequential chain elongation. When PCNA in the complex is monoubiquitinated by RAD6-RAD18, the resulting ubiquitin moiety is immediately polyubiquitinated by coexisting HLTF, indicating a coupling reaction between mono- and polyubiquitination. By contrast, when PCNA was monoubiquitinated in the absence of HLTF, it was not polyubiquitinated by subsequently recruited HLTF unless all three-subunits of PCNA were monoubiquitinated, indicating that the uncoupling reaction specifically occurs on three-subunit-monoubiquitinated PCNA. We discuss the physiological relevance of the different modes of the polyubiquitination to the choice of cells between TLS and TS under different conditions.

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.

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.

Lipid Nanoparticle-mediated siRNA Transfer Against PCTAIRE1/PCTK1/Cdk16 Inhibits In Vivo Cancer Growth.

PCTAIRE1/CDK16/PCTK1 plays critical roles in cancer cell proliferation and antiapoptosis. To advance our previously published in vitro results with PCTAIRE1 silencing, we examined the in vivo therapeutic potential of this approach by using small interfering RNA (siRNA) encapsulated by lipid nanoparticles. Therapy experiments of PCTAIRE1 siRNA were performed using human HCT116 colorectal cancer cells and human A2058 melanoma cells. A single dose of PCTAIRE1 siRNA-lipid nanoparticles was found to be highly effective in reducing in vivo PCTAIRE1 expression for up to 4 days as assayed by immunoblotting. Therapy experiments were started 4 days after subcutaneous injection of cancer cells. Treatment with PCTAIRE1 siRNA-lipid nanoparticles (0.5 mg/kg RNA, twice a week) reduced tumor volume and weight significantly compared with the scramble-control group. Histopathological analysis (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) showed increased apoptosis of tumor cells treated with PCTAIRE1-siRNA. Overall, our results demonstrate that siRNA treatment targeting PCTAIRE1 is effective in vivo, suggesting that PCTAIRE1 siRNA-lipid nanoparticles might be a novel therapeutic approach against cancer cells.

Orphan Nuclear Receptor NR4A1 Binds a Novel Protein Interaction Site on Anti-apoptotic B Cell Lymphoma Gene 2 Family Proteins.

B cell lymphoma gene 2 (Bcl-2) family proteins are key regulators of programmed cell death and important targets for drug discovery. Pro-apoptotic and anti-apoptotic Bcl-2 family proteins reciprocally modulate their activities in large part through protein interactions involving a motif known as BH3 (Bcl-2 homology 3). Nur77 is an orphan member of the nuclear receptor family that lacks a BH3 domain but nevertheless binds certain anti-apoptotic Bcl-2 family proteins (Bcl-2, Bfl-1, and Bcl-B), modulating their effects on apoptosis and autophagy. We used a combination of NMR spectroscopy-based methods, mutagenesis, and functional studies to define the interaction site of a Nur77 peptide on anti-apoptotic Bcl-2 family proteins and reveal a novel interaction surface. Nur77 binds adjacent to the BH3 peptide-binding crevice, suggesting the possibility of cross-talk between these discrete binding sites. Mutagenesis of residues lining the identified interaction site on Bcl-B negated the interaction with Nur77 protein in cells and prevented Nur77-mediated modulation of apoptosis and autophagy. The findings establish a new protein interaction site with the potential to modulate the apoptosis and autophagy mechanisms governed by Bcl-2 family proteins.

Crystallographic study of a novel DNA-binding domain of human HLTF involved in the template-switching pathway to avoid the replication arrest caused by DNA damage.

HLTF is a pivotal protein in the template-switching pathway that allows DNA synthesis to continue even in the presence of DNA damage by utilizing a newly synthesized undamaged strand as a template. HLTF has a novel DNA-binding domain termed HIRAN that has been recently found in various proteins, although its detailed function remains unclear. In this study, the HIRAN domain of human HLTF was successfully crystallized. The crystals belonged to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 130.0, c = 150.1 Å.

Structure of a Novel DNA-binding Domain of Helicase-like Transcription Factor (HLTF) and Its Functional Implication in DNA Damage Tolerance.

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 ß-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.

A small molecule inhibitor of monoubiquitinated Proliferating Cell Nuclear Antigen (PCNA) inhibits repair of interstrand DNA cross-link, enhances DNA double strand break, and sensitizes cancer cells to cisplatin.

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.

En bloc transfer of polyubiquitin chains to PCNA in vitro is mediated by two different human E2-E3 pairs.

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.

Identification of small molecule proliferating cell nuclear antigen (PCNA) inhibitor that disrupts interactions with PIP-box proteins and inhibits DNA replication.

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.

Crystallization and X-ray diffraction studies of DNA-free and DNA-bound forms of EcoO109I DNA methyltransferase.

EcoO109I DNA methyltransferase (M.EcoO109I) is a type II modification enzyme from the EcoO109I restriction-modification system identified in Escherichia coli strain H709c. M.EcoO109I recognizes double-stranded RGGNCCY (where R = A or G, Y = T or C and N is any base) and transfers a methyl group to the C5 of the inner cytosines from S-adenosylmethionine. To reveal the mechanism of substrate recognition by M.EcoO109I, DNA-free and DNA-bound forms of M.EcoO109I were successfully crystallized. Crystals of the DNA-free and DNA-bound forms belonged to space groups P4(2)2(1)2, with unit-cell parameters a = b = 120.5, c = 79.8 Å, and P2(1), with unit-cell parameters a = 55.8, b = 77.4, c = 117.4 Å, ß = 93.5°, respectively.