DNA Rejoining Research Articles

Prediction of DNA rejoining kinetics and cell survival after proton irradiation for V79 cells using Geant4-DNA

Purpose:Track structure Monte Carlo (MC) codes have achieved successful outcomes in the quantitative investigation of radiation-induced initial DNA damage. The aim of the present study is to extend a Geant4-DNA radiobiological application by incorporating a feature allowing for the prediction of DNA rejoining kinetics and corresponding cell surviving fraction along time after irradiation, for a Chinese hamster V79 cell line, which is one of the most popular and widely investigated cell lines in radiobiology. Methods:We implemented the Two-Lesion Kinetics (TLK) model, originally proposed by Stewart, which allows for simulations to calculate residual DNA damage and surviving fraction along time via the number of initial DNA damage and its complexity as inputs. Results:By optimizing the model parameters of the TLK model in accordance to the experimental data on V79, we were able to predict both DNA rejoining kinetics at low linear energy transfers (LET) and cell surviving fraction. Conclusion:This is the first study to demonstrate the implementation of both the cell surviving fraction and the DNA rejoining kinetics with the estimated initial DNA damage, in a realistic cell geometrical model simulated by full track structure MC simulations at DNA level and for various LET. These simulation and model make the link between mechanistic physical/chemical damage processes and these two specific biological endpoints.

Performance Evaluation for Repair of HSGc-C5 Carcinoma Cell Using Geant4-DNA.

Simple SummaryTo evaluate the repair performance of HSGc-C5 carcinoma cell against radiation-induced DNA damage, a Geant4-DNA application for radiobiological research was extended by using newly measured experimental data acquired in this study. Concerning fast- and slow-DNA rejoining, the two-lesion kinetics (TLK) model parameters were adequately optimized (the repair speeds of each process were reasonably close to the DNA rejoining speed of the nonhomologous end-joining and homologous recombination pathways). The lethality probabilities of the DNA damage induced by complex double strand breaks (DSBs) and binary repair were approximately and , respectively. Using the optimized repair parameters, the Geant4-DNA simulation was able to predict the cell surviving fraction (SF) and the DNA repair kinetics.Track-structure Monte Carlo simulations are useful tools to evaluate initial DNA damage induced by irradiation. In the previous study, we have developed a Gean4-DNA-based application to estimate the cell surviving fraction of V79 cells after irradiation, bridging the gap between the initial DNA damage and the DNA rejoining kinetics by means of the two-lesion kinetics (TLK) model. However, since the DNA repair performance depends on cell line, the same model parameters cannot be used for different cell lines. Thus, we extended the Geant4-DNA application with a TLK model for the evaluation of DNA damage repair performance in HSGc-C5 carcinoma cells which are typically used for evaluating proton/carbon radiation treatment effects. For this evaluation, we also performed experimental measurements for cell surviving fractions and DNA rejoining kinetics of the HSGc-C5 cells irradiated by 70 MeV protons at the cyclotron facility at the National Institutes for Quantum and Radiological Science and Technology (QST). Concerning fast- and slow-DNA rejoining, the TLK model parameters were adequately optimized with the simulated initial DNA damage. The optimized DNA rejoining speeds were reasonably agreed with the experimental DNA rejoining speeds. Using the optimized TLK model, the Geant4-DNA simulation is now able to predict cell survival and DNA-rejoining kinetics for HSGc-C5 cells.

Chromothripsis in High-Risk Myelodysplastic Syndromes: Incidence, Genetic Features, Clinical Implications, and Impact on Survival of Patients Treated with Azacytidine (Data from Czech MDS Group)

Introduction: Chromothripsis is a recently identified genomic instability phenomenon that plays a role in the genesis and progression of cancer. It is a one-step catastrophic genomic event involving multiple chromosomal breakages and random DNA rejoining. This genetic abnormality can affect an entire chromosome, a chromosomal arm, or a single chromosomal region. Chromothripsis is associated with highly complex karyotypes and a very poor prognosis, and has been detected in a wide range of tumor entities, including hematological malignancies. However, this complex genomic abnormality has not been comprehensively studied in patients with myelodysplastic syndromes (MDS). The aim of the study was to assess the incidence, associated genetic features, and clinical significance of chromothripsis in a large homogeneous cohort of patients newly diagnosed with high-risk MDS and complex karyotypes.Methods: A detailed genome-wide analysis of fixed bone-marrow cells from adults with complex karyotypes (≥ 3 aberrations), identified with conventional G-banding at the diagnosis of MDS, was performed. The complex rearrangements were studied with integrative genetic methodologies: fluorescence in situ hybridization (FISH) with Vysis DNA probes (Abbott, Des Plaines, IL), multicolor FISH (mFISH) and/or multicolor banding (mBAND) methods with the 24XCyte and the XCyte color kits (MetaSystems, Altlussheim, Germany), and array-based comparative genomic hybridization with CytoChip Cancer SNP 180K (Illumina, San Diego, CA) or the SurePrint G3 Cancer CGH+SNP 4x180K Microarray (Agilent, Santa Clara, CA). A mutational analysis of the TP53 gene was also performed in selected cases using amplicon-based deep sequencing on a 454 GS Junior System (Roche, Basel, Switzerland) or the TruSight Myeloid Sequencing Panel on MiSeq sequencing instruments (Illumina).Results: In total, 265 patients with complex karyotypes and newly diagnosed high-risk MDS were included (131 females, 134 males; median age, 70 years). The hallmarks of chromothripsis were detected in 67.7% of cases, in both the main clones and one or more subclones. At the cytogenetic level, chromothripsis was apparent as multiple deletions, insertions, ring chromosomes, amplification of individual genes or chromosomal regions, and/or the formation of chaotically reassembled chromosomes. Chromothripsis affected almost all the chromosomes, except the Y chromosome. The most frequently involved were chromosomes 5 (33.3% of events), 7 (28.1% of events), 17 (18.9% of events), 11 and 12 (15.6% of events each). In samples with signs of chromothripsis, the higher frequency of aberrations on chromosome 17p was observed (loss of heterozygosity [LOH], copy-number neutral LOH [cnLOH] and/or homozygous mutation of TP53) (p = 0.05). Patients with chromothripsis had significantly worse overall survival (OS; median, 3 months). We also investigated the effect of chromothripsis on the survival of 183 patients treated with azacytidine. In this cohort, the median OS of chromothripsis-positive patients was 10.1 months (mean, 12.2 months), whereas the median OS of chromothripsis-negative patients was 17.3 months (mean, 31.0 months).Conclusions: Our results demonstrate that chromothripsis is a frequent genomic abnormality in patients with high-risk MDS, which influences the patient's prognosis and disease biology. Chromothripsis was associated with a higher frequency of LOH/cnLOH 17p, rapid disease progression, and short survival. The adverse outcomes may be attributable to the effects on the functions of many important genes. The OS of patients with high-risk MDS with chromotripsis may be slightly improved by azacytidine treatment, but the prognosis of these patients remains very poor. Therefore, a better understanding of the mechanistic basis of chromothripsis is extremely important and could lead to the development of new treatment strategies based on drugs that target the genes present in amplified or deleted regions and/or the DNA damage response pathways.Thisstudy was supported by research projects RVO-VFN64165, GACR P302/12/G157,AZV 16-27790A,Progres Q26 and Q28/LF1, GACR 18-01687S, MHCR 00023736 and UNCE/MED/016. DisclosuresNo relevant conflicts of interest to declare.

Computational screening of chalcones acting against topoisomerase IIα and their cytotoxicity towards cancer cell lines

Targeted cancer therapy has become one of the high potential cancer treatments. Human topoisomerase II (hTopoII), which catalyzes the cleavage and rejoining of double-stranded DNA, is an important molecular target for the development of novel cancer therapeutics. In order to diversify the pharmacological activity of chalcones and to extend the scaffold of topoisomerase inhibitors, a series of chalcones was screened against hTopoIIα by computational techniques, and subsequently tested for their in vitro cytotoxicity. From the experimental IC50 values, chalcone 3d showed a high cytotoxicity with IC50 values of 10.8, 3.2 and 21.1 µM against the HT-1376, HeLa and MCF-7 cancer-derived cell lines, respectively, and also exhibited an inhibitory activity against hTopoIIα-ATPase that was better than the known inhibitor, salvicine. The observed ligand–protein interactions from a molecular dynamics study affirmed that 3d strongly interacts with the ATP-binding pocket residues. Altogether, the newly synthesised chalcone 3d has a high potential to serve as a lead compound for topoisomerase inhibitors.

Investigating mycobacterial topoisomerase I mechanism from the analysis of metal and DNA substrate interactions at the active site.

We have obtained new crystal structures of Mycobacterium tuberculosis topoisomerase I, including structures with ssDNA substrate bound to the active site, with and without Mg2+ ion present. Significant enzyme conformational changes upon DNA binding place the catalytic tyrosine in a pre-transition state position for cleavage of a specific phosphodiester linkage. Meanwhile, the enzyme/DNA complex with bound Mg2+ ion may represent the post-transition state for religation in the enzyme's multiple-step DNA relaxation catalytic cycle. The first observation of Mg2+ ion coordinated with the TOPRIM residues and DNA phosphate in a type IA topoisomerase active site allows assignment of likely catalytic role for the metal and draws a comparison to the proposed mechanism for type IIA topoisomerases. The critical function of a strictly conserved glutamic acid in the DNA cleavage step was assessed through site-directed mutagenesis. The functions assigned to the observed Mg2+ ion can account for the metal requirement for DNA rejoining but not DNA cleavage by type IA topoisomerases. This work provides new structural insights into a more stringent requirement for DNA rejoining versus cleavage in the catalytic cycle of this essential enzyme, and further establishes the potential for selective interference of DNA rejoining by this validated TB drug target.

Structural snapshots of Xer recombination reveal activation by synaptic complex remodeling and DNA bending.

Bacterial Xer site-specific recombinases play an essential genome maintenance role by unlinking chromosome multimers, but their mechanism of action has remained structurally uncharacterized. Here, we present two high-resolution structures of Helicobacter pylori XerH with its recombination site DNA difH, representing pre-cleavage and post-cleavage synaptic intermediates in the recombination pathway. The structures reveal that activation of DNA strand cleavage and rejoining involves large conformational changes and DNA bending, suggesting how interaction with the cell division protein FtsK may license recombination at the septum. Together with biochemical and in vivo analysis, our structures also reveal how a small sequence asymmetry in difH defines protein conformation in the synaptic complex and orchestrates the order of DNA strand exchanges. Our results provide insights into the catalytic mechanism of Xer recombination and a model for regulation of recombination activity during cell division.

DNA Polymerase θ: Duct Tape and Zip Ties for a Fragile Genome

Using a combination of genetics and cellular DNA rejoining assays, in this issue of Molecular Cell, Wyatt etal. (2016) demonstrate a critical role for mammalian DNA polymerase θ in the rejoining of DNA ends that are poor substrates for classical non-homologous end joining.

Mechanisms of human lymphoid chromosomal translocations.

Analysis of chromosomal translocation sequence locations in human lymphomas has provided valuable clues about the mechanism of the translocations and when they occur. Biochemical analyses on the mechanisms of DNA breakage and rejoining permit formulation of detailed models of the human chromosomal translocation process in lymphoid neoplasms. Most human lymphomas are derived from B cells in which a DNA break at an oncogene is initiated by activation-induced deaminase (AID). The partner locus in many cases is located at one of the antigen receptor loci, and this break is generated by the recombination activating gene (RAG) complex or by AID. After breakage, the joining process typically occurs by non-homologous DNA end-joining (NHEJ). Some of the insights into this mechanism also apply to translocations that occur in non-lymphoid neoplasms.

Recovery of the poisoned topoisomerase II for DNA religation: coordinated motion of the cleavage core revealed with the microsecond atomistic simulation.

Type II topoisomerases resolve topological problems of DNA double helices by passing one duplex through the reversible double-stranded break they generated on another duplex. Despite the wealth of information in the cleaving operation, molecular understanding of the enzymatic DNA ligation remains elusive. Topoisomerase poisons are widely used in anti-cancer and anti-bacterial therapy and have been employed to entrap the intermediates of topoisomerase IIβ with religatable DNA substrate. We removed drug molecules from the structure and conducted molecular dynamics simulations to investigate the enzyme-mediated DNA religation. The drug-unbound intermediate displayed transitions toward the resealing-compliant configuration: closing distance between the cleaved DNA termini, B-to-A transformation of the double helix, and restoration of the metal-binding motif. By mapping the contact configurations and the correlated motions between enzyme and DNA, we identified the indispensable role of the linker preceding winged helix domain (WHD) in coordinating the movements of TOPRIM, the nucleotide-binding motifs, and the bound DNA substrate during gate closure. We observed a nearly vectorial transition in the recovery of the enzyme and identified the previously uncharacterized roles of Asn508 and Arg677 in DNA rejoining. Our findings delineate the dynamic mechanism of the DNA religation conducted by type II topoisomerases.

Improvement of metabolic parameters in healthy older adult men following a fasting calorie restriction intervention

Objective: Calorie restriction and intermittent fasting are two dietary interventions that can improve aging. Religious fasting also suggested having similar benefit; however, such studies are still scarce. Thus, this study aimed to determine the effect of fasting calorie restriction (FCR) on metabolic parameters and DNA damage among healthy older adult men.Methods: A randomized controlled study was done on men, aged 50–70 years in Klang Valley, Malaysia. Subjects were divided into two groups; FCR (reduction of 300–500 kcal/d combined with 2 days/week of Muslim Sunnah Fasting) and control. Assessment was ascertained at three time point; baseline, weeks 6 and 12. Blood samples were analyzed for lipid profile, DNA damage and malondialdehyde (MDA).Results: The FCR group reduced their energy intake for approximately 18% upon completion of the study. A significant interaction effect was found in body weight, body mass index, fat percentage, fat mass, blood pressure, total cholesterol, low-density lipoprotein cholesterol and the ratio of total cholesterol/high-density lipoprotein cholesterol (p < 0.05). A significant improvement (p < 0.001) in total DNA rejoining cells and MDA (p < 0.05) was also observed in the FCR group.Conclusion: FCR improved metabolic parameters and DNA damage in healthy older adult men. Therefore, there is a need to further examine the mechanism of FCR.

Breaking and joining single-stranded DNA: the HUH endonuclease superfamily

HUH endonucleases are numerous and widespread in all three domains of life. The major function of these enzymes is processing a range of mobile genetic elements by catalysing cleavage and rejoining of single-stranded DNA using an active-site Tyr residue to make a transient 5'-phosphotyrosine bond with the DNA substrate. These enzymes have a key role in rolling-circle replication of plasmids and bacteriophages, in plasmid transfer, in the replication of several eukaryotic viruses and in various types of transposition. They have also been appropriated for cellular processes such as intron homing and the processing of bacterial repeated extragenic palindromes. Here, we provide an overview of these fascinating enzymes and their functions, using well-characterized examples of Rep proteins, relaxases and transposases, and we explore the molecular mechanisms used in their diverse activities.

A High-Throughput Scintillation Proximity-Based Assay for Human DNA Ligase IV

Ionizing radiation (IR) and certain chemotherapeutic drugs are designed to generate cytotoxic DNA double-strand breaks (DSBs) in cancer cells. Inhibition of the major DSB repair pathway, nonhomologous end joining (NHEJ), will enhance the cytotoxicity of these agents. Screening for inhibitors of the DNA ligase IV (Lig4), which mediates the final ligation step in NHEJ, offers a novel target-based drug discovery opportunity. For this purpose, we have developed an enzymatic assay to identify chemicals that block the transfer of [α-(33)P]-AMP from the complex Lig4-[α-(33)P]-AMP onto the 5' end of a double-stranded DNA substrate and adapted it to a scintillation proximity assay (SPA). A screen was performed against a collection of 5,280 compounds. Assay statistics show an average Z' value of 0.73, indicative of a robust assay in this SPA format. Using a threshold of >20% inhibition, 10 compounds were initially scored as positive hits. A follow-up screen confirmed four compounds with IC(50) values ranging from 1 to 30 μM. Rabeprazole and U73122 were found to specifically block the adenylate transfer step and DNA rejoining; in whole live cell assays, these compounds were found to inhibit the repair of DSBs generated by IR. The ability to screen and identify Lig4 inhibitors suggests that they may have utility as chemo- and radio-sensitizers in combination therapy and provides a rationale for using this screening strategy to identify additional inhibitors.

Enhancement of non-homologous end joining DNA repair capacity confers cancer cells resistance to the novel selenophene compound, D-501036

D-501036 is a promising anti-cancer compound that exhibits potent anti-proliferative activity against various types of human cancers through the induction of double strand DNA breaks. To determine drug resistance mechanism related to this class of DNA-damaging agents, a KB-derived D-501036-resistant cell line (S4) was established. Results showed that S4 cells exhibit enhanced DNA rejoining ability as compare to KB cells, through up-regulation of the non-homologous end joining activity. In conclusion, enhancement of NHEJ activity plays important role in the development of D-501036-resistance and targeting NHEJ-related molecules maybe able to overcome drug resistance to DNA damaging agents.

The Strictly Conserved Arg-321 Residue in the Active Site of Escherichia coli Topoisomerase I Plays a Critical Role in DNA Rejoining

The strictly conserved arginine residue proximal to the active site tyrosine of type IA topoisomerases is required for the relaxation of supercoiled DNA and was hypothesized to be required for positioning of the scissile phosphate for DNA cleavage to take place. Mutants of recombinant Yersinia pestis topoisomerase I with hydrophobic substitutions at this position were found in genetic screening to exhibit a dominant lethal phenotype, resulting in drastic loss in Escherichia coli viability when overexpressed. In depth biochemical analysis of E. coli topoisomerase I with the corresponding Arg-321 mutation showed that DNA cleavage can still take place in the absence of this arginine function if Mg(2+) is present to enhance the interaction of the enzyme with the scissile phosphate. However, DNA rejoining is inhibited in the absence of this conserved arginine, resulting in accumulation of the cleaved covalent intermediate and loss of relaxation activity. These new experimental results demonstrate that catalysis of DNA rejoining by type IA topoisomerases has a more stringent requirement than DNA cleavage. In addition to the divalent metal ions, the side chain of this arginine residue is required for the precise positioning of the phosphotyrosine linkage for nucleophilic attack by the 3'-OH end to result in DNA rejoining. Small molecules that can interfere or distort the enzyme-DNA interactions required for DNA rejoining by bacterial type IA topoisomerases could be developed into novel antibacterial drugs.

Mechanistic Investigation of ROS-Induced DNA Damage by Oestrogenic Compounds in Lymphocytes and Sperm Using the Comet Assay

Past research has demonstrated that oestrogenic compounds produce strand breaks in the DNA of sperm and lymphocytes via reactive oxygen species (ROS). In the current investigation, sperm and lymphocytes were treated in vitro with oestrogenic compounds (diethylstilboestrol, progesterone, 17β-oestradiol, noradrenaline and triiodotyronine) and several aspects of DNA damage were investigated. Firstly, mediation of DNA damage by lipid peroxidation was investigated in the presence of BHA (a lipid peroxidation blocker). BHA reduced the DNA damage generated by 17β-oestradiol and diethylstilboestrol in a statistically significant manner. No effects were observed for sperm. Secondly, the presence of oxidized bases employing FPG and EndoIII were detected for lymphocytes and sperm in the negative control and after 24 h recovery in lymphocytes but not immediately after treatment for both cell types. The successful detection of oxidized bases in the negative control (untreated) of sperm provides an opportunity for its application in biomonitoring studies. DNA repair at 24 h after exposure was also studied. A nearly complete recovery to negative control levels was shown in lymphocytes 24 h recovery after oestrogenic exposure and this was statistically significant in all cases. Rapid rejoining of DNA, in a matter of hours, is a characteristic of DNA damaged by ROS.

Crystal structure of a covalent intermediate in DNA cleavage and rejoining by Escherichia coli DNA topoisomerase I

DNA topoisomerases control DNA topology by breaking and rejoining DNA strands via covalent complexes with cleaved DNA substrate as catalytic intermediates. Here we report the structure of Escherichia coli topoisomerase I catalytic domain (residues 2-695) in covalent complex with a cleaved single-stranded oligonucleotide substrate, refined to 2.3-Å resolution. The enzyme-substrate intermediate formed after strand cleavage was captured due to the presence of the D111N mutation. This structure of the covalent topoisomerase-DNA intermediate, previously elusive for type IA topoisomerases, shows distinct conformational changes from the structure of the enzyme without bound DNA and provides detailed understanding of the covalent catalysis required for strand cleavage to take place. The portion of cleaved DNA 5' to the site of cleavage is anchored tightly with extensive noncovalent protein-DNA interactions as predicted by the "enzyme-bridged" model. Distortion of the scissile strand at the -4 position 5' to the cleavage site allows specific selectivity of a cytosine base in the binding pocket. Many antibacterial and anticancer drugs initiate cell killing by trapping the covalent complexes formed by topoisomerases. We have demonstrated in previous mutagenesis studies that accumulation of the covalent complex of bacterial topoisomerase I is bactericidal. This structure of the covalent intermediate provides the basis for the design of novel antibiotics that can trap the enzyme after formation of the covalent complex.

A specific inhibitor of protein kinase CK2 delays gamma-H2Ax foci removal and reduces clonogenic survival of irradiated mammalian cells

BackgroundThe protein kinase CK2 sustains multiple pro-survival functions in cellular DNA damage response and its level is tightly regulated in normal cells but elevated in cancers. Because CK2 is thus considered as potential therapeutic target, DNA double-strand break (DSB) formation and rejoining, apoptosis induction and clonogenic survival was assessed in irradiated mammalian cells upon chemical inhibition of CK2.MethodsMRC5 human fibroblasts and WIDR human colon carcinoma cells were incubated with highly specific CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB), or mock-treated, 2 hours prior to irradiation. DSB was measured by pulsed-field electrophoresis (PFGE) as well as gamma-H2AX foci formation and removal. Apoptosis induction was tested by DAPI staining and sub-G1 flow cytometry, survival was quantified by clonogenic assay.ResultsTBB treatment did not affect initial DNA fragmention (PFGE; up to 80 Gy) or foci formation (1 Gy). While DNA fragment rejoining (PFGE) was not inhibited by the drug, TBB clearly delayed gamma-H2AX foci disappearence during postirradiation incubation. No apoptosis induction could be detected for up to 38 hours for both cell lines and exposure conditions (monotherapies or combination), but TBB treatment at this moderately toxic concentration of 20 μM (about 40% survival) enhanced radiation-induced cell killing in the clonogenic assay.ConclusionsThe data imply a role of CK2 in gamma-H2AX dephosporylation, most likely through its known ability to stimulate PP2A phosphatase, rather than DSB rejoining. The slight but definite clonogenic radiosensitization by TBB does apparently not result from interference with an apoptosis suppression function of CK2 in these cells but could reflect inhibitor-induced uncoupling of DNA damage response decay from break ligation.

Control of directionality in the DNA strand-exchange reaction catalysed by the tyrosine recombinase TnpI

In DNA site-specific recombination catalysed by tyrosine recombinases, two pairs of DNA strands are sequentially exchanged between separate duplexes and the mechanisms that confer directionality to this theoretically reversible reaction remain unclear. The tyrosine recombinase TnpI acts at the internal resolution site (IRS) of the transposon Tn4430 to resolve intermolecular transposition products. Recombination is catalysed at the IRS core sites (IR1–IR2) and is regulated by adjacent TnpI-binding motifs (DR1 and DR2). These are dispensable accessory sequences that confer resolution selectivity to the reaction by stimulating synapsis between directly repeated IRSs. Here, we show that formation of the DR1–DR2-containing synapse imposes a specific order of activation of the TnpI catalytic subunits in the complex so that the IR1-bound subunits catalyse the first strand exchange and the IR2-bound subunits the second strand exchange. This ordered pathway was demonstrated for a complete recombination reaction using a TnpI catalytic mutant (TnpI-H234L) partially defective in DNA rejoining. The presence of the DR1- and DR2-bound TnpI subunits was also found to stabilize transient recombination intermediates, further displacing the reaction equilibrium towards product formation. Implication of TnpI/IRS accessory elements in the initial architecture of the synapse and subsequent conformational changes taking place during strand exchange is discussed.

Untangling the double helix: DNA entanglement and the action of the DNA topoisomerases

Preface 1. An Insuperable Problem 2. Nature's Solutions: The DNA Topoisomerases 3. Breaking a DNA Strand and Holding on to the Broken Ends 4. A Magic Swivel 5. Moving One DNA Double Helix through Another 6. Real-Time Viewing of the DNA Topoisomerases in Action 7. Manifestations of DNA Entanglement: Replication and Recombination 8. Manifestations of DNA Entanglement: Gene Expression 9. Beyond DNA Entanglement: New Twists on an Old Theme 10. From Nature's Battlefields to the Clinical Wards Appendices 1. Identification of the Covalent Intermediates in Topoisomerase-Catalyzed Reactions 2. Catalysis of DNA Breakage and Rejoining by DNA Topoisomerases Index

The catalytic residues of Tn3 resolvase.

To characterize the residues that participate in the catalysis of DNA cleavage and rejoining by the site-specific recombinase Tn3 resolvase, we mutated conserved polar or charged residues in the catalytic domain of an activated resolvase variant. We analysed the effects of mutations at 14 residues on proficiency in binding to the recombination site (‘site I’), formation of a synaptic complex between two site Is, DNA cleavage and recombination. Mutations of Y6, R8, S10, D36, R68 and R71 resulted in greatly reduced cleavage and recombination activity, suggesting crucial roles of these six residues in catalysis, whereas mutations of the other residues had less dramatic effects. No mutations strongly inhibited binding of resolvase to site I, but several caused conspicuous changes in the yield or stability of the synapse of two site Is observed by non-denaturing gel electrophoresis. The involvement of some residues in both synapsis and catalysis suggests that they contribute to a regulatory mechanism, in which engagement of catalytic residues with the substrate is coupled to correct assembly of the synapse.