Role In DNA Replication Research Articles

Pold4 is dispensable for mouse development, DNA replication and DNA repair

The DNA polymerase delta (Pol δ), a heterotetramer of four subunits (Pol δ4), plays a pivotal role in DNA replication, as well as in DNA damage repair. Pold4, as the smallest subunit of Pol δ, is degraded in response to DNA damage or when entering into S-phase. This leads to the conversion of Pol δ4 to the trimeric complex Pol δ3. However, the contribution of Pold4 has not been fully elucidated in mammals. Cdm1, the Pold4 ortholog in Schizosaccharomyces pombe, is dispensable for cell growth and DNA damage repair, and there are no Pold4 orthologs in Saccharomyces cerevisiae. We previously generated a knockout mouse model of Pold3 and revealed its essential role in genome stability. Unexpectedly, we here found that Pold4 knockout mice are viable and fertile. In addition, Pold4 knockout mice do not exhibit any pathologic changes in the lung and spleen, tissues with the most abundant expression of Pold4. Moreover, Pold4 knockout mouse tail tip fibroblasts (TTF) exhibited normal cell growth, cell cycle, DNA replication, DNA damage and DNA repair capacity. These results suggested that Pol δ3 but not Pol δ4 may be responsible for these processes in normal cells. Interestingly, 19-month-old wild-type (WT) mice had tumors in the liver, while Pold4 knockout mice did not, and Pold4 knockout mice showed increased longevity. In further, this provided evidence suggested that Pold4 could be a potential novel target for lung carcinoma because its depletion does not affect normal cells but does affect cancer cells.

DNA polymerase epsilon binds histone H3.1-H4 and recruits MORC1 to mediate meiotic heterochromatin condensation

Heterochromatin is essential for genomic integrity and stability in eukaryotes. The mechanisms that regulate meiotic heterochromatin formation remain largely undefined. Here, we show that the catalytic subunit (POL2A) of Arabidopsis DNA polymerase epsilon (POL ε) is required for proper formation of meiotic heterochromatin. The POL2A N terminus interacts with the GHKL adenosine triphosphatase (ATPase) MORC1 (Microrchidia 1), and POL2A is required for MORC1's localization on meiotic heterochromatin. Mutations affecting the POL2A N terminus cause aberrant morphology of meiotic heterochromatin, which is also observed in morc1. Moreover, the POL2A C-terminal zinc finger domain (ZF1) specifically binds to histone H3.1-H4 dimer or tetramer and is important for meiotic heterochromatin condensation. Interestingly, we also found similar H3.1-binding specificity for the mouse counterpart. Together, our results show that two distinct domains of POL2A, ZF1 and N terminus bind H3.1-H4 and recruit MORC1, respectively, to induce a continuous process of meiotic heterochromatin organization. These activities expand the functional repertoire of POL εbeyond its classic role in DNA replication and appear to be conserved in animals and plants.

Go-Ichi-Ni-San 2: A potential biomarker and therapeutic target in human cancers.

Cancer incidence and mortality are increasing globally, leading to its rising status as a leading cause of death. The Go-Ichi-Ni-San (GINS) complex plays a crucial role in DNA replication and the cell cycle. The GINS complex consists of four subunits encoded by the GINS1, GINS2, GINS3, and GINS4 genes. Recent findings have shown that GINS2 expression is upregulated in many diseases, particularly tumors. For example, increased GINS2 expression has been found in cervical cancer, gastric adenocarcinoma, glioma, non-small cell lung cancer, and pancreatic cancer. It correlates with the clinicopathological characteristics of the tumors. In addition, high GINS2 expression plays a pro-carcinogenic role in tumor development by promoting tumor cell proliferation and migration, inhibiting tumor cell apoptosis, and blocking the cell cycle. This review describes the upregulation of GINS2 expression in most human tumors and the pathway of GINS2 in tumor development. GINS2 may serve as a new marker for tumor diagnosis and a new biological target for therapy.

283 (PB063) - Replication protein A targeted therapy: In vivo anticancer activity and cellular target engagement

Background: Replication protein A (RPA) plays essential roles in DNA replication, repair, recombination and the DNA-damage response (DDR). Retrospective analysis of lung cancer patient data demonstrates high RPA expression as a negative prognostic biomarker for overall survival in smoking-related lung cancers. These observations are consistent with the increase in RPA expression serving as an adaptive mechanism that allows tolerance of the genotoxic stress resulting from carcinogen exposure.

ASPM promotes ATR-CHK1 activation and stabilizes stalled replication forks in response to replication stress.

ASPM is a protein encoded by primary microcephaly 5 (MCPH5) and is responsible for ensuring spindle position during mitosis and the symmetrical division of neural stem cells. We recently reported that ASPM promotes homologous recombination (HR) repair of DNA double strand breaks. However, its potential role in DNA replication and replication stress response remains elusive. Interestingly, we found that ASPM is dispensable for DNA replication under unperturbed conditions. However, ASPM is enriched at stalled replication forks in a RAD17-dependent manner in response to replication stress and promotes RAD9 and TopBP1 loading onto chromatin, facilitating ATR-CHK1 activation. ASPM depletion results in failed fork restart and nuclease MRE11-mediated nascent DNA degradation at the stalled replication fork. The overall consequence is chromosome instability and the sensitization of cancer cells to replication stressors. These data support a role for ASPM in loading RAD17-RAD9/TopBP1 onto chromatin to activate the ATR-CHK1 checkpoint and ultimately ensure genome stability.

Comparative single-cell transcriptional atlases of Babesia species reveal conserved and species-specific expression profiles.

Babesia is a genus of apicomplexan parasites that infect red blood cells in vertebrate hosts. Pathology occurs during rapid replication cycles in the asexual blood stage of infection. Current knowledge of Babesia replication cycle progression and regulation is limited and relies mostly on comparative studies with related parasites. Due to limitations in synchronizing Babesia parasites, fine-scale time-course transcriptomic resources are not readily available. Single-cell transcriptomics provides a powerful unbiased alternative for profiling asynchronous cell populations. Here, we applied single-cell RNA sequencing to 3 Babesia species (B. divergens, B. bovis, and B. bigemina). We used analytical approaches and algorithms to map the replication cycle and construct pseudo-synchronized time-course gene expression profiles. We identify clusters of co-expressed genes showing “just-in-time” expression profiles, with gradually cascading peaks throughout asexual development. Moreover, clustering analysis of reconstructed gene curves reveals coordinated timing of peak expression in epigenetic markers and transcription factors. Using a regularized Gaussian graphical model, we reconstructed co-expression networks and identified conserved and species-specific nodes. Motif analysis of a co-expression interactome of AP2 transcription factors identified specific motifs previously reported to play a role in DNA replication in Plasmodium species. Finally, we present an interactive web application to visualize and interactively explore the datasets.

Bombyx mori Flap endonuclease 1 correlates with the repair of ultraviolet-induced DNA damage

Solar ultraviolet radiation (UV) can cause DNA damage in microorganisms. Flap endonuclease 1 (FEN1) is a structure-specific nuclease and plays important roles in DNA replication and repair. At present, the properties and functions of FEN1 have not been characterized in detail in invertebrates such as Bombyx mori. In this study, Bombyx mori FEN1 (BmFEN1) was expressed in E. coli, and was shown to have nuclease activity that nonspecifically cleaved DNA in vitro. However, inside the cell, BmFEN1 did not cleave DNA randomly. Truncated BmFEN1 missing the nuclear localization signal (346–380 aa) still had the nuclease activity, but was no longer precisely localized to the sites of UV-induced DNA damage. It was further found that BmFEN1 favored the faster repair of UV-damaged DNA. The present study will provide a reference for further understanding the functions of BmFEN1 and UV-induced DNA damage repair mechanisms in insects.

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.

Abstract 802: Development of replication protein A (RPA) targeted PROTACs for the treatment of lung and ovarian cancers

Abstract Despite remarkable advances within the field of molecular cancer therapeutics over the preceding decades, DNA-damaging agents retain a central role in the pharmacotherapy of neoplastic diseases. The clinical response to DNA-damaging therapies is varied within and across cancer subtypes where a robust DNA damage response (DDR) predicts recalcitrance. Replication protein A (RPA) is the predominant human single-stranded DNA (ssDNA)-binding protein, playing essential roles in DNA replication, repair, recombination, and the DNA-damage response (DDR). Inhibition of the RPA−DNA interaction is an approach to cancer drug discovery that holds potential to provide single-agent activity and/or synergy with existing therapeutics. Proteolysis targeting chimera (PROTAC) based drug design presages a paradigm shift within drug discovery as PROTACs begin to carve out a space in the clinical landscape. The PROTAC platform is particularly suited for drugging overexpressed proteins such as RPA because iterative target degradation improves drug:target stoichiometry and precludes RPA’s role in protein scaffolding. Herein, we describe the synthesis and biological evaluation of an RPA targeted PROTAC library conceived by conjugation of the diaryl pyrazole RPA inhibitor TDRL-551 and an E3-ligand via a linker of variable length and composition. Residing within the beyond rule of five (bRo5) chemical space, careful consideration was given to minimize the liability to cell permeability imposed by the high molecular weight of PROTACs. Excitingly, cellular uptake across the compound library was broadly improved relative to the unconjugated warhead TDRL-551. Similarly, RPA engagement by the PROTAC series was widely unhindered and, in many cases, improved relative to the warhead TDRL-551. A subset of the compound library unified by flexible linkers and alkyne attachment of the E3-ligand (typified by GL-3311) display low single-digit micromolar IC50 values in viability assays across H460, A549, and A2780 cell lines. Citation Format: Jeremy M. Kelm, Jitender Dev Gaddameedi, Pamela S. VanderVere-Carozza, Amirreza Samarbakhsh, Nivisa Vakeesan, Hussein W. Kansou, Sara Serafimovski, Katherine S. Pawelczak, John J. Turchi, Navnath S. Gavande. Development of replication protein A (RPA) targeted PROTACs for the treatment of lung and ovarian cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 802.

Abstract 808: In vivo targeting of replication protein A for cancer therapy

Abstract Replication protein A (RPA) plays essential roles in DNA replication, repair, recombination and the DNA-damage response (DDR). Retrospective analysis of lung cancer patient data demonstrates high RPA expression as a negative prognostic biomarker for overall survival in smoking-related lung cancers. Similarly, relative expression of RPA is a predictive marker for response to chemotherapy. These observations are consistent with the increase in RPA expression serving as an adaptive mechanism that allows tolerance of the genotoxic stress resulting from carcinogen exposure. We have developed second generation RPA inhibitors (RPAi’s) that block the RPA-DNA interaction and optimized formulation for in vivo analyses. Data demonstrate that unlike first generation RPAi’s, second generation molecules with increased cellular permeability, induce cell death via apoptosis. Second gen RPAi’s elicit single agent in vitro anticancer activity across a broad spectrum of cancers and the cellular response supports a threshold exists before chemical RPA exhaustion induces cell death. Chemical RPA inhibition potentiates the anticancer activity of a series of DDR inhibitors as well as traditional DNA damaging cancer therapeutics. Consistent with the chemical RPA exhaustion model, we demonstrate that the effects of RPAi on replication fork dynamics. An optimized formulation was developed that resulted in single agent anticancer activity in two non-small cell lung cancer models. These data demonstrate a unique mechanism of action of RPAi’s eliciting a state of chemical RPA exhaustion can provide an effective therapeutic option for difficult to treat lung cancers. Citation Format: Pamela VanderVere-Carozza, Navnath Gavande, Shadia Jalal, Karen Pollok, Elmira Ekinci, Joshua Heyza, Steve Patrick, Andi Masters, John Turchi, Katherine Pawelczak. In vivo targeting of replication protein A for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 808.

CDC7 as a novel biomarker and druggable target in cancer

Due to the bottlenecks encountered in traditional treatment for tumor, more effective drug targets need to bedeveloped. Cell division cycle 7 kinase plays an important role in DNA replication, DNA repair and recombination signaling pathways. In this review, we first describe recent studies on the role of CDC7 in DNA replication in normal human tissues, and then we integrate new evidence focusing on the important role of CDC7 in replication stress tolerance of tumor cells and its impact on the prognosis of clinical oncology patients. Finally, we comb through the CDC7 inhibitors identified in recent studies as a reference for further research in clinical practice.

Comprehensive Analysis of the Expression and Prognosis for MCM4 in Uterine Corpus Endometrial Carcinoma.

Background: Mini chromosome maintenance protein 4 (MCM4) belongs to the family of mini chromosome maintenance proteins (MCMs) that plays a crucial role in DNA replication and cell cycle regulation. Given that MCM4 has been reported to be aberrantly expressed in a variety of tumor tissues, and is strongly associated with poor patient prognosis, it has rarely been reported in uterine corpus endometrial carcinoma (UCEC). Methods: We explored the role of MCM4 in UCEC through multi-omics analysis, including gene expression levels, survival prognosis, the biological function of interacting proteins, immune infiltration, and diagnostic value. Finally, these results were confirmed by biological experiments. Results: MCM4 was highly expressed in various malignancies including UCEC compared to normal samples and was associated with poor prognosis in patients with UCEC [including OS (HR = 1.74, p = 0.009), PFI (HR = 1.73, p = 0.002), PFI (HR = 2.23, p = 0.003)]. In the Cox regression analysis, MCM4 was an independent prognostic biomarker. Further studies showed those interacting proteins of MCM4 were enriched in DNA repair and cell cycle. Moreover, high expression of MCM4 was accompanied by lower infiltration of immune cells such as Treg cells and B cells. The distribution of MCM4 expression in molecular and immune subtypes was significantly different (p < 0.05), with high expression in the copynumber high (CN_HIGH) molecular subtype and the IFN-gamma dominant (C2) immune subtype. RT-qPCR and immunohistochemistry results also showed that MCM4 expression was significantly upregulated in endometrial cancer tissues and negatively correlated with patient prognosis (p < 0.05). Subsequent biological experiments confirmed that MCM4 promoted cell growth and invasion and inhibited apoptosis in vitro. Conclusion: Therefore, MCM4 could be a new potential biomarker for UCEC.

19F Electron-Nuclear Double Resonance Reveals Interaction between Redox-Active Tyrosines across the α/β Interface of E. coli Ribonucleotide Reductase.

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, thereby playing a key role in DNA replication and repair. Escherichia coli class Ia RNR is an α2β2 enzyme complex that uses a reversible multistep radical transfer (RT) over 32 Å across its two subunits, α and β, to initiate, using its metallo-cofactor in β2, nucleotide reduction in α2. Each step is proposed to involve a distinct proton-coupled electron-transfer (PCET) process. An unresolved step is the RT involving Y356(β) and Y731(α) across the α/β interface. Using 2,3,5-F3Y122-β2 with 3,5-F2Y731-α2, GDP (substrate) and TTP (allosteric effector), a Y356• intermediate was trapped and its identity was verified by 263 GHz electron paramagnetic resonance (EPR) and 34 GHz pulse electron–electron double resonance spectroscopies. 94 GHz 19F electron-nuclear double resonance spectroscopy allowed measuring the interspin distances between Y356• and the 19F nuclei of 3,5-F2Y731 in this RNR mutant. Similar experiments with the double mutant E52Q/F3Y122-β2 were carried out for comparison to the recently published cryo-EM structure of a holo RNR complex. For both mutant combinations, the distance measurements reveal two conformations of 3,5-F2Y731. Remarkably, one conformation is consistent with 3,5-F2Y731 within the H-bond distance to Y356•, whereas the second one is consistent with the conformation observed in the cryo-EM structure. The observations unexpectedly suggest the possibility of a colinear PCET, in which electron and proton are transferred from the same donor to the same acceptor between Y356 and Y731. The results highlight the important role of state-of-the-art EPR spectroscopy to decipher this mechanism.

Studies on the antibacterial activities and molecular mechanism of GyrB inhibitors by 3D-QSAR, molecular docking and molecular dynamics simulation

DNA gyrase B (GyrB) plays a critical role in DNA replication, repair, recombination, and transcription and has become an attractive target for a number of antibacterial agents. In the present work, to better comprehend the structure–activity relationship and mechanism of action of novel series of GyrB inhibitors, a theoretical study of three-dimensional-quantitative structure–activity relationship (3D-QSAR), molecular docking, molecular dynamics (MD) simulations, and binding free energy analysis were performed. The results showed that CoMFA (R 2 cv = 0.591, R 2 pred = 0.7698) and CoMSIA (R 2 cv = 0.629, R 2 pred = 0.6848) models possessed robust stability and predictability. The derived contour maps of steric, electrostatic, and hydrogen bond donor field further displayed the modified information of these inhibitors. Molecular docking was further conducted to provide the initial binding conformation for MD simulations. Subsequently, MD simulations were applied to explore the key amino acids and binding modes at the active site. The binding free energy decomposition analysis further indicates that the residues Ile54, Glu55, Arg83, Ala85, Val86 and Thr128 are essential for the high selectivity of inhibitors. Overall, these results would serve as a significant guideline for the discovery and design of novel GyrB inhibitors.

ADRV 12L: A Ranaviral Putative Rad2 Family Protein Involved in DNA Recombination and Repair.

The Andrias davidianus ranavirus (ADRV) is a member of the family Iridoviridae and belongs to the nucleocytoplasmic large DNA viruses. Based on genomic analysis, an ADRV-encoding protein, ADRV 12L, and its homologs from other iridoviruses were predicted as Rad2 family proteins based on the conserved amino acids, domains, and secondary structures. Expression analysis showed that the transcription of ADRV 12L started at 4 h post infection, and its expression was not inhibited by a DNA-replication inhibitor. Meanwhile, immunofluorescence localization showed that ADRV 12L mainly localized in viral factories and colocalized with the viral nascent DNA, which hinted at a possible role in DNA replication. Furthermore, a mutant ADRV lacking 12L (ADRV-Δ12L) was constructed. In both luciferase assays based on homologous recombination (HR) and double-strand break repair (DSBR) that followed, ADRV-Δ12L induced less luciferase activity than the wild-type ADRV, indicating that HR and DSBR were impaired in ADRV-Δ12L infected cells. In addition, infection with ADRV-Δ12L resulted in smaller plaque sizes and lower viral titers than that with wild-type ADRV, indicating an important role for 12L in efficient virus infection. Therefore, the results suggest that Rad2 homologs encoded by iridovirus have important roles in HR- and DSBR-process of the viral DNA and, thus, affect virus replication and the production of progeny virions.

Novel Benzotriazole Acetamide Derivatives as Benzo-Fused Five- Membered Nitrogen-Containing Heterocycles - In silico Screening, Molecular Docking, and Synthesis

Background: DNA gyrase subunit B (1KZN) is an attractive target for antibacterial drug development because of its role in DNA replication. The fast development of antimicrobial medication resistance necessitates the quick discovery of new antimicrobial medicines. Objective: The goal of this research was to design, synthesize, and discover benzo-fused five-membered nitrogen-containing heterocycles that bind to DNA gyrase subunit B via molecular docking (1KZN). Methods: Based on literature research, 2-(1H-1,2,3-Benzotriazol-1-yl)-N-substituted acetamide was synthesized using an efficient method. All synthesized compounds were evaluated for antibacterial activity against three distinct organisms: E. coli, Pseudomonas aeruginosa, Staphylococcus aureus. In a docking investigation, the chemical interacts with the active site of DNA gyrase subunit B (1KZN), indicating that it might have antibacterial action. Conclusion: According to the findings of this research, the compounds 3d and 3f showed antibacterial properties. For Staphylococcus aureus, 3c has the potential to be an antibacterial agent.

Structural basis of R-loop recognition by the S9.6 monoclonal antibody

R-loops are ubiquitous, dynamic nucleic-acid structures that play fundamental roles in DNA replication and repair, chromatin and transcription regulation, as well as telomere maintenance. The DNA-RNA hybrid–specific S9.6 monoclonal antibody is widely used to map R-loops. Here, we report crystal structures of a S9.6 antigen-binding fragment (Fab) free and bound to a 13-bp hybrid duplex. We demonstrate that S9.6 exhibits robust selectivity in binding hybrids over double-stranded (ds) RNA and in categorically rejecting dsDNA. S9.6 asymmetrically recognizes a compact epitope of two consecutive RNA nucleotides via their 2′-hydroxyl groups and six consecutive DNA nucleotides via their backbone phosphate and deoxyribose groups. Recognition is mediated principally by aromatic and basic residues of the S9.6 heavy chain, which closely track the curvature of the hybrid minor groove. These findings reveal the molecular basis for S9.6 recognition of R-loops, detail its binding specificity, identify a new hybrid-recognition strategy, and provide a framework for S9.6 protein engineering.

Systematic Analysis and Accurate Identification of DNA N4-Methylcytosine Sites by Deep Learning.

DNA N4-methylcytosine (4mC) is a pivotal epigenetic modification that plays an essential role in DNA replication, repair, expression and differentiation. To gain insight into the biological functions of 4mC, it is critical to identify their modification sites in the genomics. Recently, deep learning has become increasingly popular in recent years and frequently employed for the 4mC site identification. However, a systematic analysis of how to build predictive models using deep learning techniques is still lacking. In this work, we first summarized all existing deep learning-based predictors and systematically analyzed their models, features and datasets, etc. Then, using a typical standard dataset with three species (A. thaliana, C. elegans, and D. melanogaster), we assessed the contribution of different model architectures, encoding methods and the attention mechanism in establishing a deep learning-based model for the 4mC site prediction. After a series of optimizations, convolutional-recurrent neural network architecture using the one-hot encoding and attention mechanism achieved the best overall prediction performance. Extensive comparison experiments were conducted based on the same dataset. This work will be helpful for researchers who would like to build the 4mC prediction models using deep learning in the future.

BERT6mA: prediction of DNA N6-methyladenine site using deep learning-based approaches.

N6-methyladenine (6mA) is associated with important roles in DNA replication, DNA repair, transcription, regulation of gene expression. Several experimental methods were used to identify DNA modifications. However, these experimental methods are costly and time-consuming. To detect the 6mA and complement these shortcomings of experimental methods, we proposed a novel, deep leaning approach called BERT6mA. To compare the BERT6mA with other deep learning approaches, we used the benchmark datasets including 11 species. The BERT6mA presented the highest AUCs in eight species in independent tests. Furthermore, BERT6mA showed higher and comparable performance with the state-of-the-art models while the BERT6mA showed poor performances in a few species with a small sample size. To overcome this issue, pretraining and fine-tuning between two species were applied to the BERT6mA. The pretrained and fine-tuned models on specific species presented higher performances than other models even for the species with a small sample size. In addition to the prediction, we analyzed the attention weights generated by BERT6mA to reveal how the BERT6mA model extracts critical features responsible for the 6mA prediction. To facilitate biological sciences, the BERT6mA online web server and its source codes are freely accessible at https://github.com/kuratahiroyuki/BERT6mA.git, respectively.

Pivotal role of a conserved histidine in Escherichia coli ribonuclease HI as proposed by X-ray crystallography.

The ribonuclease (RNase) H family of enzymes catalyze the specific cleavage of RNA strands of RNA/DNA hybrid duplexes and play an important role in DNA replication and repair. Since the first report of the crystal structure of RNase HI, its catalytic mechanisms, which require metal ions, have been discussed based on numerous structural and functional analyses, including X-ray crystallography. In contrast, the function of the conserved histidine residue (His124 in Escherichia coli) in the flexible loop around the active site remains poorly understood, although an important role was suggested by NMR analyses. Here, novel high-resolution X-ray crystal structures of E. coli RNase HI are described, with a particular focus on the interactions of divalent cations with His124 oriented towards the active site. The enzyme-Mg2+ complex contains two metal ions in the active site, one of which has previously been observed. The second ion lies alongside the first and binds to His124 in an octahedral coordination scheme. In the enzyme-Zn2+ complex a single metal ionwas found to bind to the active site, showing a tetrahedral coordination geometry with the surrounding atoms, including His124. These results provide structural evidence that His124 plays a crucial role in the catalytic activity of RNase HI by interacting weakly and transiently with metal ions in the catalytic center.