Structure-guided mutational analysis of the nucleotidyltransferase domain of Escherichia coli NAD+-dependent DNA ligase (LigA)

NAD+-dependent DNA ligase (LigA) is essential for bacterial growth and a potential target for antimicrobial drug discovery. Here we queried the role of 14 conserved amino acids of Escherichia coli LigA by alanine scanning and thereby identified five new residues within the nucleotidyltransferase domain as being essential for LigA function in vitro and in vivo. Structure activity relationships were determined by conservative mutagenesis for the Glu-173, Arg-200, Arg-208, and Arg-277 side chains, as well as four other essential side chains that had been identified previously (Lys-115, Asp-117, Asp-285, and Lys-314). In addition, we identified Lys-290 as important for LigA activity. Reference to the structure of Enterococcus faecalis LigA allowed us to discriminate three classes of essential/important side chains that: (i) contact NAD+ directly (Lys-115, Glu-173, Lys-290, and Lys-314); (ii) comprise the interface between the NMN-binding domain (domain Ia) and the nucleotidyltransferase domain or comprise part of a nick-binding site on the surface of the nucleotidyltransferase domain (Arg-200 and Arg-208); or (iii) stabilize the active site fold of the nucleotidyltransferase domain (Arg-277). Analysis of mutational effects on the isolated ligase adenylylation and phosphodiester formation reactions revealed different functions for essential side chains at different steps of the DNA ligase pathway, consistent with the proposal that the active site is serially remodeled as the reaction proceeds.     

Structure-guided mutational analysis of T4 RNA ligase 1

T4 RNA ligase 1 (Rnl1) is a tRNA repair enzyme that circumvents an RNA-damaging host antiviral response. Whereas the three-step reaction scheme of Rnl1 is well established, the structural basis for catalysis has only recently been appreciated as mutational and crystallographic approaches have converged. Here we performed a structure-guided alanine scan of nine conserved residues, including side chains that either contact the ATP substrate via adenine (Leu179, Val230), the 2'-OH (Glu159), or the gamma phosphate (Tyr37) or coordinate divalent metal ions at the ATP alpha phosphate (Glu159, Tyr246) or beta phosphate (Asp272, Asp273). We thereby identified Glu159 and Tyr246 as essential for RNA sealing activity in vitro and for tRNA repair in vivo. Structure-activity relationships at Glu159 and Tyr246 were clarified by conservative substitutions. Eliminating the phosphate-binding Tyr37, and the magnesium-binding Asp272 and Asp273 side chains had little impact on sealing activity in vitro or in vivo, signifying that not all atomic interactions in the active site are critical for function. Analysis of mutational effects on individual steps of the ligation pathway underscored how different functional groups come into play during the ligase-adenylylation reaction versus the subsequent steps of RNA-adenylylation and phosphodiester formation. Moreover, the requirements for sealing exogenous preformed RNA-adenylate are more stringent than are those for sealing the RNA-adenylate intermediate formed in situ during ligation of a 5'-PO4 RNA.     

Structure-function analysis of the OB and latch domains of chlorella virus DNA ligase

Chlorella virus DNA ligase (ChVLig) is a minimized eukaryal ATP-dependent DNA sealing enzyme with an intrinsic nick-sensing function. ChVLig consists of three structural domains, nucleotidyltransferase (NTase), OB-fold, and latch, that envelop the nicked DNA as a C-shaped protein clamp. The OB domain engages the DNA minor groove on the face of the duplex behind the nick, and it makes contacts to amino acids in the NTase domain surrounding the ligase active site. The latch module occupies the DNA major groove flanking the nick. Residues at the tip of the latch contact the NTase domain to close the ligase clamp. Here we performed a structure-guided mutational analysis of the OB and latch domains. Alanine scanning defined seven individual amino acids as essential in vivo (Lys-274, Arg-285, Phe-286, and Val-288 in the OB domain; Asn-214, Phe-215, and Tyr-217 in the latch), after which structure-activity relations were clarified by conservative substitutions. Biochemical tests of the composite nick sealing reaction and of each of the three chemical steps of the ligation pathway highlighted the importance of Arg-285 and Phe-286 in the catalysis of the DNA adenylylation and phosphodiester synthesis reactions. Phe-286 interacts with the nick 5'-phosphate nucleotide and the 3'-OH base pair and distorts the DNA helical conformation at the nick. Arg-285 is a key component of the OB-NTase interface, where it forms a salt bridge to the essential Asp-29 side chain, which is imputed to coordinate divalent metal catalysts during the nick sealing steps.     

Functional domains of the Escherichia coli dnaK heat shock protein as revealed by mutational analysis

The employment of a set of truncated dnaK peptides produced by deletion and insertion mutations in the Escherichia coli dnaK gene allowed us to define regions of the dnaK protein which are involved in particular enzymatic functions. The results obtained suggest that the dnaK polypeptide is organized into at least two distinct functional domains. The highly conserved amino-terminal portion is required for the ATPase activity. The carboxyl-terminal portion, characterized by relatively low similarity among species, is responsible for the autophosphorylating activity. The mutant dnaK protein C[74], which lacks amino acid sequences at the extreme carboxyl-terminal portion of the protein, retains both the ATPase and the autophosphorylating activities. The results obtained with the full-length (70-kDa) dnaK756 protein suggest that the thermolabile defect of the dnaK756 mutation affects directly or indirectly the ATPase active site of the enzyme. The autophosphorylating activity of the dnaK+, dnaK756, and C[74] polypeptides was activated at least 10-fold by the addition of CaCl2.     

Genetics and function of DNA ligase in Escherichia coli

The characterization of two classes of DNA ligase mutants in Escherichia coli is described. The first class consists of three mutations coding for a temperature-sensitive ligase and defines the structural gene for DNA ligase (lig). The second class of mutants (lop) overproduces an apparently wild-type enzyme; a genetic diploid analysis implies that these are promoter or operator mutations, lig and lop are cotransduced by phage P1 and map at 46 minutes on the E. coli map. Detailed studies of two lig mutants (lig4 and lig ts7) are reported, lig ts7 is a conditionally lethal mutation, proving the essential nature of the ligase gene product. Neither mutant has a major defect in recombination or ultraviolet-repair, but both show retarded sealing of 10 S pulse-labeled DNA (Okazaki fragments).     

Metagenomic DNA fragments that affect Escherichia coli mutational pathways

A multicopy cloning approach was used to search for metagenomic DNA fragments that affect Escherichia coli mutational pathways. Soil metagenomic expression libraries were constructed with DNA samples prepared directly from soil samples collected from the UCLA Botanical Garden. Using frameshift mutator screening, we obtained a total of 26 unique metagenomic fragments that stimulate frameshift rates in an E. coli wild-type host. Mutational enhancer strains such as an ndk-deficient strain and a temperature sensitive mutS strain (mutS60) were used to further verify the mutator phenotype. We found that the presence of multiple copies of certain types of metagenomic DNA sequence repeats cause general genome instability in the wild-type E. coli host and the effect can be suppressed by overproducing a DNA mismatch component MutL. In addition, we identified nine metagenomic mutator genes (designated as smu genes) that encode proteins that have not been linked to mutator phenotypes prior to this study including a putative RNA methyltransferase Smu10A. The strain overproducing Smu10A displays one prominent base substitution hotspot in the rpoB gene, which coincides with the base substitution hotspot we have observed in cells that are partially deficient in the proofreading function carried out by the DNA polymerase III epsilon subunit. Based on the structural conservation of DNA replication/recombination/repair machineries among microorganisms, this approach would allow us to both identify new mutational pathways in E. coli and to find genes involved in DNA replication, recombination or DNA repair from vast unculturable microbes.     

Chlorella virus DNA ligase: nick recognition and mutational analysis.

Chlorella virus PBCV-1 DNA ligase seals nicked DNA substrates consisting of a 5'-phosphate-terminated strand and a 3'-hydroxyl-terminated strand annealed to a bridging DNA template strand. The enzyme discriminates at the DNA binding step between substrates containing a 5'-phosphate versus a 5'-hydroxyl at the nick. Mutational analysis of the active site motif KxDGxR (residues 27-32) illuminates essential roles for the conserved Lys, Asp and Arg moieties at different steps of the ligase reaction. Mutant K27A is unable to form the covalent ligase-(Lys-straightepsilonN-P)-adenylate intermediate and hence cannot activate a nicked DNA substrate via formation of the DNA-adenylate intermediate. Nonetheless, K27A catalyzes phosphodiester bond formation at a pre-adenylated nick. This shows that the active site lysine is not required for the strand closure reaction. K27A binds to nicked DNA-adenylate, but not to a standard DNA nick. This suggests that occupancy of the AMP binding pocket of DNA ligase is important for nick recognition. Mutant D29A is active in enzyme-adenylate formation and binds readily to nicked DNA, but is inert in DNA-adenylate formation. R32A is unable to catalyze any of the three reactions of the ligation pathway and does not bind to nicked DNA.     

Sequencing, mutational analysis, and transcriptional regulation of the Escherichia coli htrB gene

The Escherichia coli htrB gene was originally discovered because its insertional inactivation led to an exquisitely temperature-sensitive phenotype in rich media, i.e. the ability to form colonies at temperatures below 32 degrees C, but not above 33 degrees C. The htrB gene has been sequenced. It can potentially code for two proteins, with Mr values of 35,407 Da and 8669 Da, that are encoded by overlapping, divergent open reading frames. Our data are consistent with the 35,407 Da protein being HtrB. Northern blot analysis clearly shows that the monocistronic htrB message is not under heat-shock regulation. We have also sequenced the flanking DNA and have discovered a new gene, designated orf39.9, located immediately adjacent to htrB, but divergently transcribed.     

Expression, purification, and biophysical characterization of the BRCT domain of human DNA ligase IIIalpha

The C-terminal regions of several DNA repair and cell cycle checkpoint proteins are homologous to the breast-cancer-associated BRCA-1 protein C-terminal region. These regions, known as BRCT domains, have been found to mediate important protein-protein interactions. We produced the BRCT domain of DNA ligase IIIalpha (L3[86]) for biophysical and structural characterization. A glutathione S-transferase (GST) fusion with the L3[86] domain (residues 837-922 of ligase IIIalpha) was expressed in Escherichia coli and purified by glutathione affinity chromatography. The GST fusion protein was removed by thrombin digestion and further purification steps. Using this method, (15)N-labeled and (13)C/(15)N-double-labeled L3[86] proteins were prepared to enable a full determination of structure and dynamics using heteronuclear NMR spectroscopy. To obtain evidence of binding activity to the distal BRCT of the repair protein XRCC1 (X1BRCTb), as well as to provide insight into the interaction between these two BRCT binding partners, the corresponding BRCT heterocomplexes were also prepared and studied. Changes in the secondary structures (amount of helix and sheet components) of the two constituents were not observed upon complex formation. However, the melting temperature of the complex was significantly higher relative to the values obtained for the L3[86] or X1BRCTb proteins alone. This increased thermostability imparted by the interaction between the two BRCT domains may explain why cells require XRCC1 to maintain ligase IIIalpha activity.     

Cloning, nucleotide sequence, and engineered expression of Thermus thermophilus DNA ligase, a homolog of Escherichia coli DNA ligase.

We have cloned and sequenced the gene for DNA ligase from Thermus thermophilus. A comparison of this sequence and those of other ligases reveals significant homology only with that of Escherichia coli. The overall amino acid composition of the thermophilic ligase and the pattern of amino acid substitutions between the two proteins are consistent with compositional biases in other thermophilic enzymes. We have engineered the expression of the T. thermophilus gene in Escherichia coli, and we show that E. coli proteins may be substantially removed from the thermostable ligase by a simple heat precipitation step.     

Functional domains of Escherichia coli recA protein deduced from the mutational sites in the gene

Summary The sites of recA mutations of Escherichia coli, recA441 (tif-1), recA1, recA430 (lexB30) and recA44, were determined by analyses of the nucleotide sequences. All mutations are single point missense mutations within the coding region of the recA gene. In the recA441, recA1, recA430 and recA44 proteins, the 38th, 160th, 204th, and 246th amino acids, respectively, from the amino terminal ends are altered. Based on the properties of these mutant proteins and modified forms of recA protein, the locations of various regions of the recA protein that are involved in binding with ATP, binding with single-stranded DNA, hydrolysis of ATP, interaction between the recA protein molecules and interaction with the cI or lexA repressors are mapped on the primary structure of the protein.     

Crystal structure and mutational analysis of the DaaE adhesin of Escherichia coli

DaaE is a member of the Dr adhesin family of Escherichia coli, members of which are associated with diarrhea and urinary tract infections. A receptor for Dr adhesins is the cell surface protein, decay-accelerating factor (DAF). We have carried out a functional analysis of Dr adhesins, as well as mutagenesis and crystallographic studies of DaaE, to obtain detailed molecular information about interactions of Dr adhesins with their receptors. The crystal structure of DaaE has been solved at 1.48 A resolution. Trimers of the protein are found in the crystal, as has been the case for other Dr adhesins. Naturally occurring variants and directed mutations in DaaE have been generated and analyzed for their ability to bind DAF. Mapping of the mutation sites onto the DaaE molecular structure shows that several of them contribute to a contiguous surface that is likely the primary DAF-binding site. The DAF-binding properties of purified fimbriae and adhesin proteins from mutants and variants correlated with the ability of bacteria expressing these proteins to bind to human epithelial cells in culture. DaaE, DraE, AfaE-III, and AfaE-V interact with complement control protein (CCP) domains 2-4 of DAF, and analysis of the ionic strength dependence of their binding indicates that the intermolecular interactions are highly electrostatic in nature. The adhesins AfaE-I and NfaE-2 bind to CCP-3 and CCP-4 of DAF, and electrostatic interactions contribute significantly less to these interactions. These observations are consistent with structural predictions for these Dr variants and also suggest a role for the positively charged region linking CCP-2 and CCP-3 of DAF in electrostatic Dr adhesin-DAF interactions.     

Structure-function analysis of Escherichia coli DNA helicase I reveals non-overlapping transesterase and helicase domains

TraI (DNA helicase I) is an Escherichia coli F plasmid-encoded protein required for bacterial conjugative DNA transfer. The protein is a sequence-specific DNA transesterase that provides the site- and strand-specific nick required to initiate DNA strand transfer and a 5' to 3' DNA helicase that unwinds the F plasmid to provide the single-stranded DNA that is transferred from donor to recipient. Sequence comparisons with other transesterases and helicases suggest that these activities reside in the N- and C-terminal regions of TraI, respectively. Computer-assisted secondary structure probability analysis identified a potential interdomain region spanning residues 304-309. Proteins encoded by segments of traI, whose N or C terminus either flanked or coincided with this region, were purified and assessed for catalytic activity. Amino acids 1-306 contain the transesterase activity, whereas amino acids 309-1504 contain the helicase activity. The C-terminal 252 amino acids of the 1756-amino acid TraI protein are not required for either helicase or transesterase activity. Protein and nucleic acid sequence similarity searches indicate that the occurrence of both transesterase- and helicase-associated motifs in a conjugative DNA transfer initiator protein is rare. Only two examples (other than R100 plasmid TraI) were found: R388 plasmid TrwC and R46 plasmid (pKM101) TraH, belonging to the IncW and IncN groups of broad host range conjugative plasmids, respectively. The most significant structural difference between these proteins and TraI is that TraI contains an additional region of approximately 650 residues between the transesterase domain and the helicase-associated motifs. This region is required for helicase activity.     

Analysis of ligation and DNA binding by Escherichia coli DNA ligase (LigA)

NAD(+)-dependent DNA ligases are essential enzymes in bacteria, with the most widely studied of this class of enzymes being LigA from Escherichia coli. NAD(+)-dependent DNA ligases comprise several discrete structural domains, including a BRCT domain at the C-terminus that is highly-conserved in this group of proteins. The over-expression and purification of various fragments of E. coli LigA allowed the investigation of the different domains in DNA-binding and ligation by this enzyme. Compared to the full-length protein, the deletion of the BRCT domain from LigA reduced in vitro ligation activity by 3-fold and also reduced DNA binding. Using an E. coli strain harbouring a temperature-sensitive mutation of ligA, the over-expression of protein with its BRCT domain deleted enabled growth at the non-permissive temperature. In gel-mobility shift experiments, the isolated BRCT domain bound DNA in a stable manner and to a wider range of DNA molecules compared to full LigA. Thus, the BRCT domain of E. coli LigA can bind DNA, but it is not essential for DNA nick-joining activity in vitro or in vivo.     

14-3-3 proteins, FHA domains and BRCT domains in the DNA damage response

The DNA damage response depends on the concerted activity of protein serine/threonine kinases and modular phosphoserine/threonine-binding domains to relay the damage signal and recruit repair proteins. The PIKK family of protein kinases, which includes ATM/ATR/DNA-PK, preferentially phosphorylate Ser-Gln sites, while their basophilic downstream effecter kinases, Chk1/Chk2/MK2 preferentially phosphorylate hydrophobic-X-Arg-X-X-Ser/Thr-hydrophobic sites. A subset of tandem BRCT domains act as phosphopeptide binding modules that bind to ATM/ATR/DNA-PK substrates after DNA damage. Conversely, 14-3-3 proteins interact with substrates of Chk1/Chk2/MK2. FHA domains have been shown to interact with substrates of ATM/ATR/DNA-PK and CK2. In this review we consider how substrate phsophorylation together with BRCT domains, FHA domains and 14-3-3 proteins function to regulate ionizing radiation-induced nuclear foci and help to establish the G₂/M checkpoint. We discuss the role of MDC1 a molecular scaffold that recruits early proteins to foci, such as NBS1 and RNF8, through distinct phosphodependent interactions. In addition, we consider the role of 14-3-3 proteins and the Chk2 FHA domain in initiating and maintaining cell cycle arrest.     

Modification of Escherichia coli DNA ligase by cleavage with trypsin.

Limited treatment of Escherichia coli DNA ligase with trypsin results in rapid loss of DNA joining activity. However, the ability to react with DPN to form the covalent enzyme-AMP intermediate is unaffected. The cleaved enzyme is also unable to catalyze the formation of DNA-adenylate, the second covalent intermediate in the ligase-catalyzed reaction. These findings demonstrate that portions of the DNA ligase molecule that are required for phosphodiester bond formation are not required for at least one of the partial reactions catalyzed by this enzyme.     

Solution structure and backbone dynamics of the human DNA ligase IIIalpha BRCT domain.

BRCT (BRCA1 carboxyl terminus) domains are found in a number of DNA repair enzymes and cell cycle regulators and are believed to mediate important protein-protein interactions. The DNA ligase IIIalpha BRCT domain partners with the distal BRCT domain of the DNA repair protein XRCC1 (X1BRCTb) in the DNA base excision repair (BER) pathway. To elucidate the mechanisms by which these two domains can interact, we have determined the solution structure of human ligase IIIalpha BRCT (L3[86], residues 837-922). The structure of L3[86] consists of a beta2beta1beta3beta4 parallel sheet with a two-alpha-helix bundle packed against one face of the sheet. This fold is conserved in several proteins having a wide range of activities, including X1BRCTb [Zhang, X. D., et al. (1998) EMBO J. 17, 6404-6411]. L3[86] exists as a dimer in solution, but an insufficient number of NOE restraints precluded the determination of the homodimer structure. However, 13C isotope-filtered and hydrogen-deuterium exchange experiments indicate that the N-terminus, alpha1, the alpha1-beta2 loop, and the three residues following alpha2 are involved in forming the dimer interface, as similarly observed in the structure of X1BRCTb. NOE and dynamic data indicate that several residues (837-844) in the N-terminal region appear to interconvert between helix and random coil conformations. Further studies of other BRCT domains and of their complexes are needed to address how these proteins interact with one another, and to shed light on how mutations can lead to disruption of function and ultimately disease.     

Cloning, sequencing, and mutational analysis of the hyb operon encoding Escherichia coli hydrogenase 2.

The genes encoding the two structural subunits of Escherichia coli hydrogenase 2 (HYD2) have been cloned and sequenced. They occur in an operon (hyb) which contains seven open reading frames. An hyb deletion mutant (strain AP3) failed to grown on dihydrogen-fumarate medium and also produced very low levels of HYD1. All seven open reading frames are required for restoration of wild-type levels of active HYD2 in AP3. The hyb operon was mapped at 65 min on the E. coli chromosome.     

An amber dna mutant of Escherichia coli K12 affecting DNA ligase

We have isolated an amber mutant (dnaL321) of Escherichia coli K12, which affects DNA ligase and which is lethal unless it is suppressed. DNA is degraded under the restrictive conditions. The mutation also affects the sensitivity of the cell to ultraviolet light irradiation, and the capacity to support the growth of phage λ that is deficient in general recombination. This pleiotropy is considered to be due to a single mutation, and is suppressed by supD^?_Isu+ and by supF^?su_III⁺). The mutation is cotransducible with dapE(2%), and with ptsI(85%), by phage Plvir.     

Escherichia coli DNA polymerase IV mutator activity: genetic requirements and mutational specificity

The dinB gene of Escherichia coli is known to be involved in the untargeted mutagenesis of lambda phage. Recently, we have demonstrated that this damage-inducible and SOS-controlled gene encodes a novel DNA polymerase, DNA Pol IV, which is able to dramatically increase the untargeted mutagenesis of F' plasmid. At the amino acid level, DNA Pol IV shares sequence homologies with E. coli UmuC (DNA Pol V), Rev1p, and Rad30p (DNA polymerase eta) of Saccharomyces cerevisiae and human Rad30A (XPV) proteins, all of which are involved in translesion DNA synthesis. To better characterize the Pol IV-dependent untargeted mutagenesis, i.e., the DNA Pol IV mutator activity, we analyzed the genetic requirements of this activity and determined the forward mutation spectrum generated by this protein within the cII gene of lambda phage. The results indicated that the DNA Pol IV mutator activity is independent of polA, polB, recA, umuDC, uvrA, and mutS functions. The analysis of more than 300 independent mutations obtained in the wild-type or mutS background revealed that the mutator activity clearly promotes single-nucleotide substitutions as well as one-base deletions in the ratio of about 1:2. The base changes were strikingly biased for substitutions toward G:C base pairs, and about 70% of them occurred in 5'-GX-3' sequences, where X represents the base (T, A, or C) that is mutated to G. These results are discussed with respect to the recently described biochemical characteristics of DNA Pol IV.