Coli DNA Polymerase Research Articles

Involvement of Y-Family DNA Polymerases in Mutagenesis Caused by Oxidized Nucleotides in Escherichia coli

Escherichia coli DNA polymerase IV incorporated 2-hydroxy-dATP opposite template guanine or thymine and 8-hydroxy-dGTP exclusively opposite adenine in vitro. Mutator phenotypes in sod/fur strains were substantially diminished by deletion of dinB and/or umuDC. DNA polymerases IV and V may be involved in mutagenesis caused by incorporation of the oxidized deoxynucleoside triphosphates.

Dideoxy-mediated Sequencing Reactions Using the Klenow Fragment ofE. coliDNA Polymerase I and Single-stranded DNA Templates

Dideoxy-mediated Sequencing Reactions Using the Klenow Fragment of<i>E. coli</i>DNA Polymerase I and Single-stranded DNA Templates

Structure of the θ Subunit ofEscherichia coliDNA Polymerase III in Complex with the ε Subunit

The catalytic core of Escherichia coli DNA polymerase III contains three tightly associated subunits, the alpha, epsilon, and theta subunits. The theta subunit is the smallest and least understood subunit. The three-dimensional structure of theta in a complex with the unlabeled N-terminal domain of the epsilon subunit, epsilon186, was determined by multidimensional nuclear magnetic resonance spectroscopy. The structure was refined using pseudocontact shifts that resulted from inserting a lanthanide ion (Dy3+, Er3+, or Ho3+) at the active site of epsilon186. The structure determination revealed a three-helix bundle fold that is similar to the solution structures of theta in a methanol-water buffer and of the bacteriophage P1 homolog, HOT, in aqueous buffer. Conserved nuclear Overhauser enhancement (NOE) patterns obtained for free and complexed theta show that most of the structure changes little upon complex formation. Discrepancies with respect to a previously published structure of free theta (Keniry et al., Protein Sci. 9:721-733, 2000) were attributed to errors in the latter structure. The present structure satisfies the pseudocontact shifts better than either the structure of theta in methanol-water buffer or the structure of HOT. satisfies these shifts. The epitope of epsilon186 on theta was mapped by NOE difference spectroscopy and was found to involve helix 1 and the C-terminal part of helix 3. The pseudocontact shifts indicated that the helices of theta are located about 15 A or farther from the lanthanide ion in the active site of epsilon186, in agreement with the extensive biochemical data for the theta-epsilon system.

Nuclear Magnetic Resonance Solution Structure of theEscherichia coliDNA Polymerase III θ Subunit

The catalytic core of Escherichia coli DNA polymerase III holoenzyme contains three subunits: alpha, epsilon, and theta. The alpha subunit contains the polymerase, and the epsilon subunit contains the exonucleolytic proofreading function. The small (8-kDa) theta subunit binds only to epsilon. Its function is not well understood, although it was shown to exert a small stabilizing effect on the epsilon proofreading function. In order to help elucidate its function, we undertook a determination of its solution structure. In aqueous solution, theta yielded poor-quality nuclear magnetic resonance spectra, presumably due to conformational exchange and/or protein aggregation. Based on our recently determined structure of the theta homolog from bacteriophage P1, named HOT, we constructed a homology model of theta. This model suggested that the unfavorable behavior of theta might arise from exposed hydrophobic residues, particularly toward the end of alpha-helix 3. In gel filtration studies, theta elutes later than expected, indicating that aggregation is potentially responsible for these problems. To address this issue, we recorded 1H-15N heteronuclear single quantum correlation (HSQC) spectra in water-alcohol mixed solvents and observed substantially improved dispersion and uniformity of peak intensities, facilitating a structural determination under these conditions. The structure of theta in 60/40 (vol/vol) water-methanol is similar to that of HOT but differs significantly from a previously reported theta structure. The new theta structure is expected to provide additional insight into its physiological role and its effect on the epsilon proofreading subunit.

Mutator Phenotype Resulting from DNA Polymerase IV Overproduction in Escherichia coli : Preferential Mutagenesis on the Lagging Strand

We investigated the mutator effect resulting from overproduction of Escherichia coli DNA polymerase IV. Using lac mutational targets in the two possible orientations on the chromosome, we observed preferential mutagenesis during lagging strand synthesis. The mutator activity likely results from extension of mismatches produced by polymerase III holoenzyme.

3′-End Polishing of the Kinetoplastid Spliced Leader RNA Is Performed by SNIP, a 3′→5′ Exonuclease with a Motley Assortment of Small RNA Substrates

In all trypanosomatids, trans splicing of the spliced leader (SL) RNA is a required step in the maturation of all nucleus-derived mRNAs. The SL RNA is transcribed with an oligo-U 3' extension that is removed prior to trans splicing. Here we report the identification and characterization of a nonexosomal, 3'-->5' exonuclease required for SL RNA 3'-end formation in Trypanosoma brucei. We named this enzyme SNIP (for snRNA incomplete 3' processing). The central 158-amino-acid domain of SNIP is related to the exonuclease III (ExoIII) domain of the 3'-->5' proofreading epsilon subunit of Escherichia coli DNA polymerase III holoenzyme. SNIP had a preference for oligo(U) 3' extensions in vitro. RNA interference-mediated knockdown of SNIP resulted in a growth defect and correlated with the accumulation of one- to two- nucleotide 3' extensions of SL RNA, U2 and U4 snRNAs, a five-nucleotide extension of 5S rRNA, and the destabilization of U3 snoRNA and U2 snRNA. SNIP-green fluorescent protein localized to the nucleoplasm, and substrate SL RNA derived from SNIP knockdown cells showed wild-type cap 4 modification, indicating that SNIP acts on SL RNA after cytosolic trafficking. Since the primary SL RNA transcript was not the accumulating species in SNIP knockdown cells, SL RNA 3'-end formation is a multistep process in which SNIP provides the ultimate 3'-end polishing. We speculate that SNIP is part of an organized nucleoplasmic machinery responsible for processing of SL RNA.

Side‐Chain Conformational Restriction in Template‐Competitive Inhibitors of E. coliDNA Polymerase I Klenow Fragment: Synthesis, Structural Characterization and Inhibition Activity #

Nucleotide triphosphate α‐(4‐azidophenyl)‐1,N6‐etheno‐dATP 3 and its monophosphate 3m were synthesized by condensation of 2‐halo‐2‐(4‐azidophenyl)acetaldehyes with dATP and dAMP, respectively. Structure analysis shows that the azidophenyl side chain is attached to the α‐position of the etheno ring (i.e., the carbon attached to N1 of the purine), and conformation calculations show minima in the etheno‐phenyl bond rotation at 50 and 130° where the bulk of the phenyl ring projects out from the plane of the etheno group. Like DNA Pol inhibitor 2‐(4‐azidophenacyl)thio‐2′‐deoxyadenosine 5′‐triphosphate 1, nucleotide 3 is a template‐competitive DNA polymerase inhibitor (TCPI), with a competitive Ki for Pol I KF of 3.41 µM, but has only weak activity as an HIV RT inhibitor relative to the template‐competitive reverse transcriptase inhibitor 2‐(4‐azidophenacyl)thio‐1,N6‐etheno‐2′‐deoxyadenosine 5′‐triphosphate 2. Additionally, 3 photoinactivates KF in a time‐dependent manner, confirming the kinetic data that 3 binds to the free form of KF. The TCPI activity of 3 provides evidence for an extended side chain conformational preference in the combined substrate polymerase inhibitors. #This work was funded in part by the Louisiana Board of Regents 044UG‐02 and the National Institutes of Health GM067686.

Roles of YqjH and YqjW, homologs of the Escherichia coli UmuC/DinB or Y superfamily of DNA polymerases, in stationary-phase mutagenesis and UV-induced mutagenesis of Bacillus subtilis.

YqjH and YqjW are Bacillus subtilis homologs of the UmuC/DinB or Y superfamily of DNA polymerases that are involved in SOS-induced mutagenesis in Escherichia coli. While the functions of YqjH and YqjW in B. subtilis are still unclear, the comparisons of protein structures demonstrate that YqjH has 36% identity to E. coli DNA polymerase IV (DinB protein), and YqjW has 26% identity to E. coli DNA polymerase V (UmuC protein). In this report, we demonstrate that both YqjH and the products of the yqjW operon are involved in UV-induced mutagenesis in this bacterium. Furthermore, resistance to UV-induced damage is significantly reduced in cells lacking a functional YqjH protein. Analysis of stationary-phase mutagenesis indicates that absences of YqjH, but not that of YqjW, decreases the ability of B. subtilis to generate revertants at the hisC952 allele via this system. These data suggest a role for YqjH in the generation of at least some types of stationary-phase-induced mutagenesis.

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.

Baculovirus replication factor LEF-1 is a DNA primase.

The baculovirus replication factors LEF-1 and LEF-2 of the Autographa californica multinucleocapsid nucleopolyhedrovirus were overexpressed as fusions containing a hemagglutinin (HA) epitope and a HIS(6) tag using recombinant baculoviruses. LEF-1 was purified to near homogeneity and found to have primase activity in an indirect assay employing Escherichia coli DNA polymerase I (Klenow enzyme) and poly(dT) template. The LEF-1 primase products were also directly characterized by electrophoresis in 20% polyacrylamide-8 M urea gels and agarose gels. Primer synthesis was time dependent, and products of several hundred nucleotides or more were observed from the M13 single-stranded DNA (ssDNA) template. The LEF-1 primase was absolutely dependent on divalent cations (Mg(2+)), and optimal activity was supported by 10 mM MgCl(2). An alkaline pH (8.8 to 9.4) was optimal, whereas monovalent salt (KCl) was inhibitory. Mutation of an invariant aspartic acid in a putative primase domain caused LEF-1 activity to be abolished. Upon ultracentrifugation in glycerol gradients, LEF-1 was found to have a sedimentation coefficient of 3S that is consistent with its being present as a monomer. Elution profiles of LEF-1 and LEF-2 from ssDNA-cellulose and DEAE resin suggested that LEF-2 may bind to both DNA and LEF-1.

Stimulation of DNA Polymerase α by Hypergravity Generated by Centrifugal Acceleration

Gravity alteration is known to influence cell proliferation. Here we tested the effects of hypergravity on the action of DNA polymerase α, one of the DNA replication enzymes in eukaryotes. Hypergravity was produced by horizontal centrifugal acceleration with a hand-made rotator. The reaction rate of DNA polymerase α in centrifuge tubes increased along with the acceleration up to 4g, when a plateau was reached. In contrast, no stimulation was observed with primase, DNA polymerase ϵ, and the E. coli DNA polymerase I Klenow fragment. Kinetic analysis of DNA polymerase α reactions revealed that, under high gravity conditions, the Km value for template DNA decreased while the Vmax stayed constant. In contrast, the centrifugal acceleration did not affect the Km values for deoxyribonucleoside triphosphates. These results suggest that the hypergravity enhances the activity of DNA polymerase α by increasing the affinity of the enzyme for template DNA. Such enhancement was more prominent with a low concentration of DNA polymerase α under low ionic conditions.

A universal protein-protein interaction motif in the eubacterial DNA replication and repair systems.

The interaction between DNA polymerases and sliding clamp proteins confers processivity in DNA synthesis. This interaction is critical for most DNA replication machines from viruses and prokaryotes to higher eukaryotes. The clamp proteins also participate in a variety of dynamic and competing protein-protein interactions. However, clamp-protein binding sequences have not so far been identified in the eubacteria. Here we show from three lines of evidence, bioinformatics, yeast two-hybrid analysis, and inhibition of protein-protein interaction by modified peptides, that variants of a pentapeptide motif (consensus QL[SD]LF) are sufficient to enable interaction of a number of proteins with an archetypal eubacterial sliding clamp (the beta subunit of Escherichia coli DNA polymerase III holoenzyme). Representatives of this motif are present in most sequenced members of the eubacterial DnaE, PolC, PolB, DinB, and UmuC families of DNA polymerases and the MutS1 mismatch repair protein family. The component tripeptide DLF inhibits the binding of the alpha (DnaE) subunit of E. coli DNA polymerase III to beta at microM concentration, identifying key residues. Comparison of the eubacterial, eukaryotic, and archaeal sliding clamp binding motifs suggests that the basic interactions have been conserved across the evolutionary landscape.

Mechanism of Processivity Clamp Opening by the Delta Subunit Wrench of the Clamp Loader Complex of E. coli DNA Polymerase III

The dimeric ring-shaped sliding clamp of E. coli DNA polymerase III (β subunit, homolog of eukaryotic PCNA) is loaded onto DNA by the clamp loader γ complex (homolog of eukaryotic Replication Factor C, RFC). The δ subunit of the γ complex binds to the β ring and opens it. The crystal structure of a β:δ complex shows that δ, which is structurally related to the δ′ and γ subunits of the γ complex, is a molecular wrench that induces or traps a conformational change in β such that one of its dimer interfaces is destabilized. Structural comparisons and molecular dynamics simulations suggest a spring-loaded mechanism in which the β ring opens spontaneously once a dimer interface is perturbed by the δ wrench.

Mechanism of β Clamp Opening by the δ Subunit ofEscherichia coli DNA Polymerase III Holoenzyme

The β sliding clamp encircles the primer-template and tethers DNA polymerase III holoenzyme to DNA for processive replication of the Escherichia coli genome. The clamp is formed via hydrophobic and ionic interactions between two semicircular β monomers. This report demonstrates that the β dimer is a stable closed ring and is not monomerized when the γ complex clamp loader (γ3δ1δ1χ1ψ1) assembles the β ring around DNA. δ is the subunit of the γ complex that binds β and opens the ring; it also does not appear to monomerize β. Point mutations were introduced at the β dimer interface to test its structural integrity and gain insight into its interaction with δ. Mutation of two residues at the dimer interface of β, I272A/L273A, yields a stable β monomer. We find that δ binds the β monomer mutant at least 50-fold tighter than the β dimer. These findings suggest that when δ interacts with the β clamp, it binds one β subunit with high affinity and utilizes some of that binding energy to perform work on the dimeric clamp, probably cracking one dimer interface open.

Cross-utilization of the β Sliding Clamp by Replicative Polymerases of Evolutionary Divergent Organisms

Chromosomal replicases are multiprotein machines comprised of a DNA polymerase, a sliding clamp, and a clamp loader. This study examines replicase components for their ability to be switched between Gram-positive and Gram-negative organisms. These two cell types diverged over 1 billion years ago, and their sequences have diverged widely. Yet the Escherichia coli beta clamp binds directly to Staphylococcus aureus PolC and makes it highly processive, confirming and extending earlier results (Low, R. L., Rashbaum, S. A. , and Cozzarelli, N. R. (1976) J. Biol. Chem. 251, 1311-1325). We have also examined the S. aureus beta clamp. The results show that it functions with S. aureus PolC, but not with E. coli polymerase III core. PolC is a rather potent polymerase by itself and can extend a primer with an intrinsic speed of 80-120 nucleotides per s. Both E. coli beta and S. aureus beta converted PolC to a highly processive polymerase, but surprisingly, beta also increased the intrinsic rate of DNA synthesis to 240-580 nucleotides per s. This finding expands the scope of beta function beyond a simple mechanical tether for processivity to include that of an effector that increases the intrinsic rate of nucleotide incorporation by the polymerase.

The Mutagenesis Protein UmuC Is a DNA Polymerase Activated by UmuD′, RecA, and SSB and Is Specialized for Translesion Replication

Replication of DNA lesions leads to the formation of mutations. In Escherichia coli this process is regulated by the SOS stress response, and requires the mutagenesis proteins UmuC and UmuD'. Analysis of translesion replication using a recently reconstituted in vitro system (Reuven, N. B., Tomer, G., and Livneh, Z. (1998) Mol. Cell 2, 191-199) revealed that lesion bypass occurred with a UmuC fusion protein, UmuD', RecA, and SSB in the absence of added DNA polymerase. Further analysis revealed that UmuC was a DNA polymerase (E. coli DNA polymerase V), with a weak polymerizing activity. Upon addition of UmuD', RecA, and SSB, the UmuC DNA polymerase was greatly activated, and replicated a synthetic abasic site with great efficiency (45% bypass in 6 min), 10-100-fold higher than E. coli DNA polymerases I, II, or III holoenzyme. Analysis of bypass products revealed insertion of primarily dAMP (69%), and to a lesser degree dGMP (31%) opposite the abasic site. The UmuC104 mutant protein was defective both in lesion bypass and in DNA synthesis. These results indicate that UmuC is a UmuD'-, RecA-, and SSB-activated DNA polymerase, which is specialized for lesion bypass. UmuC is a member of a new family of DNA polymerases which are specialized for lesion bypass, and include the yeast RAD30 and the human XP-V genes, encoding DNA polymerase eta.

Construction of nonnatural DNA library.

Nonnatural DNA polymerase substrates which contain many kinds of modified functional groups are synthesized. The C-5 position of dUTP was modified by amino acid, saccharide. These compounds were incorporated on a DNA double strand using E. Coli DNA polymerase. This nonnatural DNA library contains a larger diversity than native DNA or RNA and has a higher chemical stability than RNA. This library will be useful for in vitro selection study, combinatorial chemistry, and the preparation of supramolecular structures.

The accessory subunit of mtDNA polymerase shares structural homology with aminoacyl-tRNA synthetases: implications for a dual role as a primer recognition factor and processivity clamp.

The accessory subunit of the heterodimeric mtDNA polymerase (polgamma) from Drosophila embryos is required to maintain the structural integrity or catalytic efficiency of the holoenzyme. cDNAs for the accessory subunit from Drosophila, man, mouse, and rat have been identified, and comparative sequence alignment reveals that the C-terminal region of about 120 aa is the most conserved. Furthermore, we demonstrate that the accessory subunit of animal polgamma has both sequence and structural similarity with class IIa aminoacyl-tRNA synthetases. Based on sequence similarity and fold recognition followed by homology modeling, we have developed a model of the three-dimensional structure of the C-terminal region of the accessory subunit of polgamma. The model reveals a rare five-stranded beta-sheet surrounded by four alpha-helices with structural homology to the anticodon-binding domain of class IIa aminoacyl-tRNA synthetases. We postulate that the accessory subunit plays a role in the recognition of RNA primers in mtDNA replication, to recruit polgamma to the template-primer junction. A similar role is served by the gamma-complex in Escherichia coli DNA polymerase III, and indeed our accessory subunit model shows structural similarity with the N-terminal domain of the delta' subunit of the gamma-complex. Structural similarity is also found with E. coli thioredoxin, the accessory subunit and processivity factor in bacteriophage T7 DNA polymerase. Thus, we propose that the accessory subunit of polgamma is involved both in primer recognition and in processive DNA strand elongation.

Simultaneous Determination of Pyrimidine or Purine Deoxyribonucleoside Triphosphates Using a Polymerase Assay

In this paper, we describe an improved enzymatic assay for the determination of deoxyribonucleoside triphosphates (dNTPs). This is based on the elongation of32P 5′-end-labeled oligonucleotide primers annealed to complementary oligonucleotide templates. Incorporation within the primer/template (p/t) was catalyzed by the Klenow fragment ofEscherichia coliDNA polymerase I under conditions where the concentration of the dNTP to be analyzed is limiting. Using a combination of two different sized p/t pairs, dCTP and dTTP (or dATP and dGTP) were assayed together. Since the elongated products were clearly separated after electrophoresis on a denaturing 10% polyacrylamide gel, the two dNTPs could be quantified in a single lane. This method allows for the first time the simultaneous determination of two pyrimidine or two purine deoxyribonucleoside triphosphates. Consequently, a large number of biological samples can be tested in a single experiment. The high sensitivity of this method enables the quantification of low concentrations of dNTPs, such as those found in resting nondividing cells. Furthermore, this new protocol is well suited for the determination of dNTPs in cells treated with the antiretroviral ddI, since the Klenow fragment has a low affinity for ddATP, the active form of ddI.

AAA+: A Class of Chaperone-Like ATPases Associated with the Assembly, Operation, and Disassembly of Protein Complexes

Using a combination of computer methods for iterative database searches and multiple sequence alignment, we show that protein sequences related to the AAA family of ATPases are far more prevalent than reported previously. Among these are regulatory components of Lon and Clp proteases, proteins involved in DNA replication, recombination, and restriction (including subunits of the origin recognition complex, replication factor C proteins, MCM DNA-licensing factors and the bacterial DnaA, RuvB, and McrB proteins), prokaryotic NtrC-related transcription regulators, the Bacillus sporulation protein SpoVJ, Mg2+, and Co2+ chelatases, theHalobacterium GvpN gas vesicle synthesis protein, dynein motor proteins, TorsinA, and Rubisco activase. Alignment of these sequences, in light of the structures of the clamp loader δ′ subunit ofEscherichia coli DNA polymerase III and the hexamerization component of N-ethylmaleimide-sensitive fusion protein, provides structural and mechanistic insights into these proteins, collectively designated the AAA+ class. Whole-genome analysis indicates that this class is ancient and has undergone considerable functional divergence prior to the emergence of the major divisions of life. These proteins often perform chaperone-like functions that assist in the assembly, operation, or disassembly of protein complexes. The hexameric architecture often associated with this class can provide a hole through which DNA or RNA can be thread; this may be important for assembly or remodeling of DNA–protein complexes.