<Emphasis Type="Italic">Escherichia coli</Emphasis> topoisomerase I is an iron and zinc binding protein

Escherichia coli topoisomerase I (TopA) cleaves and rejoins one strand of double-stranded DNA to relax the negatively supercoiled DNA. Structurally, TopA contains an N-terminal catalytic fragment and a C-terminal zinc-binding region that is required for relaxation of the negatively supercoiled DNA. Here we report that E. coli TopA is an iron and zinc binding protein. The UV–Vis absorption measurements and metal content analyses reveal that TopA purified from E. coli cells grown in the rich LB medium contains both iron and zinc. However, TopA purified from E. coli cells grown in the M9 minimal medium has negligible amounts of zinc or iron and no topoisomerase activity. Nevertheless, supplement of exogenous zinc or iron in E. coli cells grown in the M9 minimal medium produces the zinc- or iron-bound TopA, respectively. Whereas the zinc-bound TopA is fully active to relax the negatively supercoiled DNA, the iron-bound TopA has little or no enzyme activity. Furthermore, excess iron in the M9 minimal medium is able to compete with the zinc binding in TopA in E. coli cells and attenuate the topoisomerase activity, suggesting that E. coli TopA may be modulated by iron and zinc binding in vivo.     

氧化还原对铁结合的大肠杆菌拓扑异构酶Ⅰ活性的调控

大肠杆菌拓扑异构酶 I（E. coli TopA）属于 I 型拓扑异构酶,在DNA复制、转录、重组和基因表达调控等过程中发挥关键作用。E. coli TopA 不仅能结合锌,还可以结合铁。细胞内过量铁可与锌竞争,通过与锌指结构域结合减弱其 DNA 结合能力和改变蛋白质空间构象,从而抑制TopA拓扑异构酶活性。然而,铁结合形式TopA的氧化还原特性以及氧化还原条件对其活性的影响仍不清楚。本研究通过紫外分光光谱和体外DNA拓扑异构酶活性分析,发现体外纯化得到的铁结合形式的 TopA 呈氧化状态,能够被二硫苏糖醇和连二亚硫酸钠还原,原本氧化状态下无活性的TopA在还原条件下,可恢复其拓扑异构酶活性。当还原剂被去除后,铁结合的TopA在空气中能够重新被氧化,且其活性重新受到抑制。这说明,氧化还原条件对铁结合的 TopA 功能具有可逆调节作用。通过金属 蛋白体外结合实验进一步发现,无金属结合的TopA蛋白(apo-TopA)在无氧条件下,与 Fe²⁺ 和 Fe³⁺ 均能结合,但与Fe²⁺ 结合能力较弱,并且TopA结合的Fe³⁺ 被还原成Fe²⁺ 后,结合力显著下降,能够被铁螯合指示剂菲咯嗪快速捕获。此外,蛋白质内源性荧光光谱分析实验表明,铁结合的TopA在氧化还原的不同状态时,其在330 nm左右的荧光值有显著差异。这提示,氧化还原条件可能通过影响铁离子与TopA的结合状态,引起蛋白质空间构象改变,从而对TopA的拓扑异构酶活性进行调节。此研究表明,铁结合TopA的拓扑异构酶活性会受到细胞内氧化还原信号的可逆调控,也提示I型拓扑异构酶可能是细胞铁超载通过氧化损伤引起细胞功能障碍（或铁死亡）的靶点之一。     

大肠杆菌拓扑异构酶I结合重金属的特性及功能影响

【背景】大肠杆菌拓扑异构酶Ⅰ(Escherichia coli topoisomerase I,E.coli TopA)在DNA复制、转录、重组和基因表达调控等过程发挥关键作用。研究表明E.coli TopA只有结合锌离子才具有活性,然而E.coli TopA能否结合其他金属离子尤其是重金属离子,以及结合其他金属后是否具有活性,目前仍不清楚。【目的】探究大肠杆菌拓扑异构酶Ⅰ是否结合环境中常见重金属离子,研究重金属离子结合E.coli TopA蛋白后对其活性的影响。【方法】在分别添加有锌、钴、镍、镉、铁、汞、砷、铬、铅、铜离子的M9基础培养中表达、纯化出E.coli TopA蛋白,并对纯化得到的蛋白用电感耦合等离子体质谱仪进行相应金属离子含量的测定;利用表达E.coli TopA锌指结构的突变体蛋白鉴定重金属离子的结合位点;通过体外超螺旋DNA松弛实验测定不同金属结合E.coli TopA的拓扑异构酶活性;通过测定蛋白内源性荧光推测不同金属结合E.coli TopA的空间构象差异。【结果】E.coli TopA在体内除了能结合锌和铁之外,还能够结合钴、镍、镉3种离子,但是不能结合汞、砷、铬、铅、铜离子。钴、镍、镉结合形式的E.coli TopA,每个蛋白分子最多可以结合3个相应的金属离子,他们与TopA蛋白的结合位点也是位于3个锌指结构域,而且每个锌指结构域结合1个金属离子。此外,E.coli TopA结合钴、镍、镉离子后,其DNA拓扑异构酶活性并未受到影响,可能是由于钴、镍、镉离子结合形式的E.coli TopA蛋白,其空间构象与锌结合形式相比并未发生显著变化。【结论】由于DNA拓扑异构酶在维持细胞正常生理功能中发挥关键作用,研究表明E.coli TopA的功能不会受到常见重金属的干扰(不结合或者结合后活性无影响),这也有可能是大肠杆菌在进化过程中产生的对抗环境中重金属离子毒害作用的一种自我保护和耐受机制,具有重要的生理意义。     

Structural basis for the MukB‐topoisomerase IV interaction and its functional implications in vivo

Chromosome partitioning in Escherichia coli is assisted by two interacting proteins, topoisomerase (topo) IV and MukB. MukB stimulates the relaxation of negative supercoils by topo IV; to understand the mechanism of their action and to define this functional interplay, we determined the crystal structure of a minimal MukB–topo IV complex to 2.3 Å resolution. The structure shows that the so‐called ‘hinge’ region of MukB forms a heterotetrameric assembly with a C‐terminal DNA binding domain (CTD) on topo IV's ParC subunit. Biochemical studies show that the hinge stimulates topo IV by competing for a site on the CTD that normally represses activity on negatively supercoiled DNA, while complementation tests using mutants implicated in the interaction reveal that the cellular dependency on topo IV derives from a joint need for both strand passage and MukB binding. Interestingly, the configuration of the MukB·topo IV complex sterically disfavours intradimeric interactions, indicating that the proteins may form oligomeric arrays with one another, and suggesting a framework by which MukB and topo IV may collaborate during daughter chromosome disentanglement.     

A highly processive topoisomerase I: studies at the single-molecule level

Amongst enzymes which relieve torsional strain and maintain chromosome supercoiling, type IA topoisomerases share a strand-passage mechanism that involves transient nicking and re-joining of a single deoxyribonucleic acid (DNA) strand. In contrast to many bacterial species that possess two type IA topoisomerases (TopA and TopB), Actinobacteria possess only TopA, and unlike its homologues this topoisomerase has a unique C-terminal domain that lacks the Zn-finger motifs characteristic of type IA enzymes. To better understand how this unique C-terminal domain affects the enzyme''s activity, we have examined DNA relaxation by actinobacterial TopA from Streptomyces coelicolor (ScTopA) using real-time single-molecule experiments. These studies reveal extremely high processivity of ScTopA not described previously for any other topoisomerase of type I. Moreover, we also demonstrate that enzyme processivity varies in a torque-dependent manner. Based on the analysis of the C-terminally truncated ScTopA mutants, we propose that high processivity of the enzyme is associated with the presence of a stretch of positively charged amino acids in its C-terminal region.     

Localization of a DNA topoisomerase II to mitochondria inDictyostelium discoideum: Deletion mutant analysis and mitochondrial targeting signal presequence

DNA topoisomerase II ofDictyostelium discoideum (TopA), the gene (topA) encoding which we cloned, was shown to have an additional N-terminal region which contains a putative mitochondrial targeting signal presequence. We constructed overexpression mutants which expressed the wild-type or the N-terminally deleted enzyme, and examined its localization by immunofluorescence microscopy and proteinase K digestion experiment. These experiments revealed that the enzyme is located in the mitochondria by virtue of the additional N-terminal region. Furthermore, in the cell extract depleted the enzyme by immunoprecipitation, nuclear DNA topoisomerase II activity was not decreased. These results confirmed that TopA is located in the mitochondria, even through its amino acid sequence is highly similar to those of nuclear type topoisomerase II of other organisms. Thus, this report is the first to establish the location of the mitochondrial targeting signal presequence in DNA topoisomerase II and in proteins ofD. discoideum directly by analyzing deletion mutants. Tsukuba Advanced Research Alliance (TARA researcher for the Sakabe project)     

Structure of the zinc‐bound amino‐terminal domain of the NMDA receptor NR2B subunit

N‐methyl‐D ‐aspartate (NMDA) receptors belong to the family of ionotropic glutamate receptors (iGluRs) that mediate the majority of fast excitatory synaptic transmission in the mammalian brain. One of the hallmarks for the function of NMDA receptors is that their ion channel activity is allosterically regulated by binding of modulator compounds to the extracellular amino‐terminal domain (ATD) distinct from the L ‐glutamate‐binding domain. The molecular basis for the ATD‐mediated allosteric regulation has been enigmatic because of a complete lack of structural information on NMDA receptor ATDs. Here, we report the crystal structures of ATD from the NR2B NMDA receptor subunit in the zinc‐free and zinc‐bound states. The structures reveal the overall clamshell‐like architecture distinct from the non‐NMDA receptor ATDs and molecular determinants for the zinc‐binding site, ion‐binding sites, and the architecture of the putative phenylethanolamine‐binding site.     

Molecular cloning and characterization of a C‐type lectin in yellow catfish Tachysurus fulvidraco

This study represents the first report of a C‐type lectin (ctl) in yellow catfish Tachysurus fulvidraco. The complete sequence of ctl complementary (c)DNA consisted of 685 nucleotides. The open reading frame potentially encoded a protein of 177 amino acids with a calculated molecular mass of c.y 20.204 kDa. The deduced amino‐acid sequence contained a signal peptide and a single carbohydrate recognition domain with four cysteine residues and GlnProAsp (QPD) and TrpAsnAsp (WND) motifs. Ctl showed the highest identity (56.0%) to the predicted lactose binding lectin from channel catfish Ictalurus punctatus. Quantitative real‐time (qrt)‐PCR analysis showed that ctl messenger (m)RNA was constitutively expressed in all examined tissues in normal fish, with high expression in trunk kidney and head kidney, which was increased following Aeromonas hydrophila challenge in a duration‐dependent manner. Purified recombinant Ctl (rCtl) from Escherichia coli BL21 was able to bind and agglutinate Gram‐positive and Gram‐negative bacteria in a calcium‐dependent manner. These results suggested that Ctl might be a C‐type lectin of T. fulvidraco involved in innate immune responses as receptors (PRR).     

The mechanism of Single strand binding protein–RecG binding: Implications for SSB interactome function

The Escherichia coli single‐strand DNA binding protein (SSB) is essential to viability where it functions to regulate SSB interactome function. Here it binds to single‐stranded DNA and to target proteins that comprise the interactome. The region of SSB that links these two essential protein functions is the intrinsically disordered linker. Key to linker function is the presence of three, conserved PXXP motifs that mediate binding to oligosaccharide‐oligonucleotide binding folds (OB‐fold) present in SSB and its interactome partners. Not surprisingly, partner OB‐fold deletions eliminate SSB binding. Furthermore, single point mutations in either the PXXP motifs or, in the RecG OB‐fold, obliterate SSB binding. The data also demonstrate that, and in contrast to the view currently held in the field, the C‐terminal acidic tip of SSB is not required for interactome partner binding. Instead, we propose the tip has two roles. First, and consistent with the proposal of Dixon, to regulate the structure of the C‐terminal domain in a biologically active conformation that prevents linkers from binding to SSB OB‐folds until this interaction is required. Second, as a secondary binding domain. Finally, as OB‐folds are present in SSB and many of its partners, we present the SSB interactome as the first family of OB‐fold genome guardians identified in prokaryotes.     

Cloning and biochemical characterization of Staphylococcus aureus type IA DNA topoisomerase comprised of distinct five domains

DNA topoisomerases play critical roles in regulating DNA topology and are essential enzymes for cell survival. In this study, a gene encoding type IA DNA topoisomerase was cloned from Staphylococcus aureus (S. aureus) sp. strain C-66, and the biochemical properties of recombinant enzyme was characterized. The nucleotide sequence analysis showed that the cloned gene contained an open reading frame (2070 bp) that could encode a polypeptide of 689 amino acids. The cloned gene actually produced 79.1 kDa functional enzyme (named Sau-TopoI) in Escherichia coli (E. coli). Sau-TopoI enzyme purified from E. coli showed ATP-independent and Mg²⁺-dependent manners for relaxing negatively supercoiled DNA. The relaxation activity of Sau-TopoI was inhibited by camptothecin, but not by nalidixic acid and etoposide. Cleavage site mapping showed that the enzyme could preferentially bind to and cleave the sequence GGNN↓CAT (N and ↓ represent any nucleotide and cleavage site, respectively). All these results suggest that the purified enzyme is type IA DNA topoisomerase. In addition, domain mapping analysis showed that the enzyme was composed of conserved four domains (I through IV), together with a variable C-terminal region containing a unique domain V.     

Nuclear‐encoded DnaJ homologue of Plasmodium falciparum interacts with replication ori of the apicoplast genome

The apicoplast of Plasmodium falciparum carries a 35 kb circular genome (plDNA) that replicates at the late trophozoite stage of the parasite intraerythocytic cycle. plDNA replication proceeds predominantly via a d ‐loop/bi‐directional ori mechanism with replication ori localized within inverted repeat region. Although replication of the apicoplast genome is a validated drug target, the proteins involved in the replication process are only partially characterized. We analysed DNA–protein interactions at a plDNA replication ori region and report the identification of a nuclear‐encoded DnaJ homologue that binds directly to ori elements of the plDNA molecule. PfDnaJ_A interacted with the minor groove of the DNA double‐helix and recognized a 13 bp sequence within the ori. Inhibition of binding with anti‐PfDnaJ_A antibodies confirmed identity of the protein in DNA‐binding experiments with organellar protein fractions. The DNA‐binding domain of the ～69 kDa PfDnaJ_A lay within the N‐terminal 38 kDa region that carries DnaJ signature motifs. In contrast to PfDnaJ_A in parasite organellar fractions, the recombinant protein interacted with DNA in a sequence non‐specific manner. Our results suggest a role for PfDnaJ_A in replication/repair of the apicoplast genome.     

Critical roles of tyrosyl‐DNA phosphodiesterases in cell tolerance to carnosol‐induced DNA damage

Carnosol is a natural compound with pharmacological action due to its anti‐cancer properties. However, the precise mechanism for its anti‐carcinogenic effect remains elusive. In this study, we used lymphoblastoid TK6 cell lines to identify the DNA damage and repair mechanisms of carnosol. Our results showed that carnosol induced DNA double‐strand breaks (DSBs). We also found that cells lacking tyrosyl‐DNA phosphodiesterase 1 (TDP1), an enzyme related to topoisomerase 1 (TOP1), and tyrosyl‐DNA phosphodiesterase 2 (TDP2), an enzyme related to topoisomerase 2 (TOP2), were supersensitive to carnosol. Carnosol was found to induce the formation of the TOP1‐DNA cleavage complex (TOP1cc) and TOP2‐DNA cleavage complex (TOP2cc). When comparing the accumulation of γ‐H2AX foci and the number of chromosomal aberrations (CAs) with wild‐type (WT) cells, the susceptivity of the TDP1^?/? and TDP2^?/? cells were associated with an increased DNA damage. Our results provided evidence of carnosol inducing DNA lesions in TK6 cells and demonstrated that the damage induced by carnosol was associated with abnormal topoisomerase activity. We conclude that TDP1 and TDP2 play important roles in the anti‐cancer effect of carnosol.     

Iron and zinc binding activity of Escherichia coli topoisomerase I homolog YrdD

YrdD, a homolog of the C-terminal zinc-binding region of Escherichia coli topoisomerase I, is highly conserved among proteobacteria and enterobacteria. However, the function of YrdD remains elusive. Here we report that YrdD purified from E. coli cells grown in LB media contains both zinc and iron. Supplement of exogenous zinc in the medium abolishes the iron binding of YrdD in E. coli cells, indicating that iron and zinc may compete for the same metal binding sites in the protein. While the zinc-bound YrdD is able to bind single-stranded (ss) DNA and protect ssDNA from the DNase I digestion in vitro, the iron-bound YrdD has very little or no binding activity for ssDNA, suggesting that the zinc-bound YrdD may have an important role in DNA repair by interacting with ssDNA in cells.     

Designed zinc finger protein interacting with the HIV‐1 integrase recognition sequence at 2‐LTR‐circle junctions

Integration of HIV‐1 cDNA into the host genome is a crucial step for viral propagation. Two nucleotides, cytosine and adenine (CA), conserved at the 3′ end of the viral cDNA genome, are cleaved by the viral integrase (IN) enzyme. As IN plays a crucial role in the early stages of the HIV‐1 life cycle, substrate blockage of IN is an attractive strategy for therapeutic interference. In this study, we used the 2‐LTR‐circle junctions of HIV‐1 DNA as a model to design zinc finger protein (ZFP) targeting at the end terminal portion of HIV‐1 LTR. A six‐contiguous ZFP, namely 2LTRZFP was designed using zinc finger tools. The designed motif was expressed and purified from E. coli to determine its binding properties. Surface plasmon resonance (SPR) was used to determine the binding affinity of 2LTRZFP to its target DNA. The level of dissociation constant (K_d) was 12.0 nM. The competitive SPR confirmed that 2LTRZFP specifically interacted with its target DNA. The qualitative binding activity was subsequently determined by EMSA and demonstrated the aforementioned correlation. In addition, molecular modeling and binding energy analyses were carried out to provide structural insight into the binding of 2LTRZFP to the specific and nonspecific DNA target. It is suggested that hydrogen‐bonding interactions play a key role in the DNA recognition mechanisms of the designed ZFP. Our study suggested an alternative HIV therapeutic strategy using ZFP interference of the HIV integration process.     

Mass spectrometric characterization of the Campylobacter jejuni adherence factor CadF reveals post‐translational processing that removes immunogenicity while retaining fibronectin binding

Campylobacter jejuni is a major gastrointestinal pathogen that colonizes host mucosa via interactions with extracellular matrix proteins, such as fibronectin (Fn). Fn‐binding is mediated by a 37 kDa outer membrane protein termed Campylobacter adherence Factor (CadF). The outer membrane protein profile of a recent gastrointestinal C. jejuni clinical isolate (JHH1) was analysed using 2‐DE and MS. Several spots were identified as products of the cadF gene. These included mass and pI variants of 34 and 30 kDa, as well as 24 kDa (CadF₂₄) and 22 kDa (CadF₂₂) mass variants. CadF variants were fully characterized by MALDI‐TOF MS and MALDI‐MS/MS. These data confirmed that CadF forms re‐folding variants resulting in spots with lower mass and varying pI that are identical at the amino acid sequence level and are not modified post‐translationally. CadF₂₂ and CadF₂₄, however, were characterized as N‐terminal, membrane‐associated polypeptides resulting from cleavage between serine¹⁹⁵ and leucine¹⁹⁶, and glycine²⁰¹ and phenylalanine²⁰², respectively. These variants were more abundant in the virulent (O) isolate of C. jejuni NCTC11168 when compared with the avirulent (genome sequenced) isolate. Hexahistidine fusion constructs of full‐length CadF (34 kDa), CadF₂₄, and the deleted C‐terminal OmpA domain (14 kDa; CadF₁₄) were created in Escherichia coli. Recombinant CadF variants were probed against patient sera and revealed that only full‐length CadF retained reactivity. Binding assays showed that CadF₂₄ retained Fn‐binding capability, while CadF₁₄ did not bind Fn. These data suggest that the immunogenic epitope of CadF is cleaved to generate smaller Fn‐binding polypeptides, which are not recognized by the host humoral response. CadF cleavage therefore may be associated with virulence in C. jejuni.     

Nucleotides sequestered at different subsite loci within DNA‐binding pockets of two OB‐fold single‐stranded DNA‐binding proteins are unstacked to different extents

The gene 5 protein (g5p) encoded by the Ff strains of Escherichia coli bacteriophages is a dimeric single‐stranded DNA‐binding protein (SSB) that consists of two identical OB‐fold (oligonucleotide/oligosaccharide‐binding) motifs. Ultrafast time‐resolved fluorescence measurements were carried out to investigate the effect of g5p binding on the conformation of 2‐aminopurine (2AP) labels positioned between adenines or cytosines in the 16‐nucleotide antiparallel tails of DNA hairpins. The measurements revealed significant changes in the conformational heterogeneity of the 2AP labels caused by g5p binding. The extent of the changes was dependent on sub‐binding‐site location, but generally resulted in base unstacking. When bound by g5p, the unstacked 2AP population increased from ～22% to 59–67% in C‐2AP‐C segments and from 39% to 77% in an A‐2AP‐A segment. The OB‐fold RPA70A domain of the human replication protein A also caused a significant amount of base unstacking at various locations within the DNA binding site as evidenced by steady‐state fluorescence titration measurements using 2AP‐labeled 5‐mer DNAs. These solution studies support the concept that base unstacking at most of a protein's multiple sub‐binding‐site loci may be a feature that allows non‐sequence specific OB‐fold proteins to bind to single‐stranded DNAs (ssDNAs) with minimal preference for particular sequences. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 484–496, 2013.     

The cleidocranial dysplasia‐related R131G mutation in the Runt‐related transcription factor RUNX2 disrupts binding to DNA but not CBF‐β

Cleidocranial dysplasia (CCD) is caused by haploinsufficiency in RUNX2 function. We have previously identified a series of RUNX2 mutations in Korean CCD patients, including a novel R131G missense mutation in the Runt‐homology domain. Here, we examine the functional consequences of the RUNX2^R131G mutation, which could potentially affect DNA binding, nuclear localization signal, and/or heterodimerization with core‐binding factor‐β (CBF‐β). Immunofluorescence microscopy and western blot analysis with subcellular fractions show that RUNX2^R131G is localized in the nucleus. Immunoprecipitation analysis reveals that heterodimerization with CBF‐β is retained. However, precipitation assays with biotinylated oligonucleotides and reporter gene assays with RUNX2 responsive promoters together reveal that DNA‐binding activity and consequently the transactivation of potential of RUNX2^R131G is abrogated. We conclude that loss of DNA binding, but not nuclear localization or CBF‐β heterodimerization, causes RUNX2 haploinsufficiency in patients with the RUNX2^R131G mutation. Retention of specific functions including nuclear localization and binding to CBF‐β of the RUNX2^R131G mutation may render the mutant protein an effective competitor that interferes with wild‐type function. J. Cell. Biochem. 110: 97–103, 2010. © 2010 Wiley‐Liss, Inc.     

Structure and DNA binding characteristics of the three‐Cys2His2 domain of mouse testis zinc finger protein

The C‐terminal three‐Cys₂His₂ zinc‐finger domain (TZD) of mouse testis zinc‐finger protein binds to the 5′‐TGTACAGTGT‐3′ at the Aie1 (aurora‐C) promoter with high specificity. Interestingly, the primary sequence of TZD is unique, possessing two distinct linkers, TGEKP and GAAP, and distinct residues at presumed DNA binding sites at each finger, especially finger 3. A K_d value of ～10^?8 M was obtained from surface plasmon resonance analysis for the TZD‐DNA complex. NMR structure of the free TZD showed that each zinc finger forms a typical ββα fold. On binding to DNA, chemical shift perturbations and the R₂ transverse relaxation rate in finger 3 are significantly smaller than those in fingers 1 and 2, which indicates that the DNA binding affinity in finger 3 is weaker. Furthermore, the shift perturbations between TZD in complex with the cognate DNA and its serial mutants revealed that both ADE7 and CYT8, underlined in 5′‐ATATGTACAGTGTTAT‐3′, are critical in specific binding, and the DNA binding in finger 3 is sequence independent. Remarkably, the shift perturbations in finger 3 on the linker mutation of TZD (GAAP mutated to TGEKP) were barely detected, which further indicates that finger 3 does not play a critical role in DNA sequence‐specific recognition. The complex model showed that residues important for DNA binding are mainly located on positions ?1, 2, 3, and 6 of α‐helices in fingers 1 and 2. The DNA sequence and nonsequence‐specific bindings occurring simultaneously in TZD provide valuable information for better understanding of protein–DNA recognition. Proteins 2010. © 2010 Wiley‐Liss, Inc.