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regions of bacteriophage t4 and rb69 rega translational repressor proteins that determine rna-binding specificity.rega protein of t4 and related bacteriophages is a highly conserved rna-binding protein that represses the translation of many phage mrnas that encode enzymes involved in dna metabolism. rb69, a t4-related bacteriophage, has a unique rega gene, which we have cloned, sequenced, and expressed. the predicted amino acid sequence of rb69 rega is 78% identical to that of t4 rega. plasmid-encoded rb69 rega expressed in vivo represses the translation of t4 early mrnas, including those of riia, riib, 44, ...19921594613
sequence analysis of conserved rega and variable orf43.1 genes in t4-like bacteriophages.bacteriophage t4 rega protein is a translational repressor of several phage mrnas. in the t4-related phages examined, rega nucleotide sequences are highly conserved and the inferred amino acid sequences are identical. the exceptional phage, rb69, did not produce a rega protein reproducibly identifiable by western blots (immunoblots) nor did it produce mrna that hybridized to t4 rega primers. nucleotide sequences of either 223 or 250 base pairs were identified immediately 3' to rega in rb18 and r ...19902168375
rna-protein interactions of the bacteriophage rb69 rega translational repressor protein.we report the rna-protein interactions of phage rb69 rega and rega691 proteins. in rnase protection assays, rb69 rega protects from 10 (44 mrna) to >70 (rpba) nucleotides; for rpba in particular, the length of the protected region is rega-concentration dependent. quantitative fluorescence quenching measurements with rb69 wildtype rega and rega691 show that both proteins bind poly(u) similarly, but rega691 binds with lower affinity to both 44 and rpba rna. the results indicate that both rb69 rega ...19958643388
structural and functional insights provided by crystal structures of dna polymerases and their substrate complexes.new levels in the understanding of dna replication have been achieved from recent crystal structure determinations of several dna polymerases and their substrate complexes. the structure of an alpha family dna polymerase from bacteriophage rb69 shows some similarities, but also considerable differences in structure and organization from the pol i family dna polymerases. also, the functions of three polymerase domains and their conserved residues have been clarified by studying structures of pol ...19989519297
sensitivity of bacteriophage rb69 dna polymerase to n2-(p-n-butylphenyl)-2'-deoxyguanosine nucleotides.the response of bacteriophage rb69 dna polymerase to n2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (bupdgtp), related nucleotides and non-nucleoside inhibitors was measured and compared to values obtained for the closely related dna polymerase from bacteriophage t4. both enzymes showed similar responses to inhibitors in terms of ki values and the ability to utilize bupdgtp as a substrate. these results provide the relevance of using the recent crystal structure of rb69 dna polymerase fo ...199910616725
identification of conserved residues contributing to the activities of adenovirus dna polymerase.adenovirus codes for a dna polymerase that is a member of the dna polymerase alpha family and uses a protein primer for initiation of dna synthesis. it contains motifs characteristic of a proofreading 3'-5'-exonuclease domain located in the n-terminal region and several polymerase motifs located in the c-terminal region. to determine the role of adenovirus dna polymerase in dna replication, 22 site-directed mutations were introduced into the conserved dna polymerase motifs in the c-terminal regi ...200011090167
rega proteins from phage t4 and rb69 have conserved helix-loop groove rna binding motifs but different rna binding specificities.the rega proteins from the bacteriophage t4 and rb69 are translational repressors that control the expression of multiple phage mrnas. rega proteins from the two phages share 78% sequence identity; however, in vivo expression studies have suggested that the rb69 rega protein binds target rnas with a higher affinity than t4 rega protein. to study the rna binding properties of t4 and rb69 rega proteins more directly, the binding sites of rb69 rega protein on synthetic rnas corresponding to the tra ...200111222767
clamp loaders and sliding clamps.a coherent view of the structure and function of dna polymerase processivity factors (sliding clamps and clamp loaders) is emerging from recent structural studies. crystal structures of sliding clamps from the t4 and rb69 bacteriophages, and from an archaebacterium expand the gallery of ring-shaped processivity factors and clarify how the clamp interacts with the dna polymerase. crystallographic and electron microscopic views of clamp loaders from bacteria, archaebacteria and eukaryotes emphasiz ...200211959500
rna determinants of translational operator recognition by the dna polymerases of bacteriophages t4 and rb69.the dna polymerases (gp43s) of the two related phages t4 and rb69 are dna-binding proteins that also function as mrna-binding autogenous translational repressors. as repressors, t4 gp43 is narrowly specific to its own mrna whereas rb69 gp43 is equally effective against mrna for either protein. we used in vitro rnase-sensitivity and rna footprinting assays to identify features of the non-identical t4 and rb69 mrna targets (translational operators) that allow for their identical binding affinities ...200212140318
a conserved tyr residue is required for sugar selectivity in a pol alpha dna polymerase.many dna polymerases select their natural substrates, deoxy- as opposed to ribonucleoside triphosphates, with a selectivity greater than 10000-fold. the function of a highly conserved residue, tyr416, in the palm domain of the parental enzyme, an exo(-) derivative of rb69 dna polymerase (gp43), a member of the pol alpha dna polymerase family, was examined for its role in helping the polymerase discriminate between ribo-, dideoxyribo-, and deoxyribonucleoside triphosphates. the parental enzyme se ...200212162740
biochemical characterization of interactions between dna polymerase and single-stranded dna-binding protein in bacteriophage rb69.the organization and proper assembly of proteins to the primer-template junction during dna replication is essential for accurate and processive dna synthesis. dna replication in rb69 (a t4-like bacteriophage) is similar to those of eukaryotes and archaea and has been a prototype for studies on dna replication and assembly of the functional replisome. to examine protein-protein interactions at the dna replication fork, we have established solution conditions for the formation of a discrete and h ...200312458197
correlation of the kinetics of finger domain mutants in rb69 dna polymerase with its structure.we have estimated pre-steady-state kinetic parameters for the addition of a single nucleotide residue by a set of rb69 dna polymerase mutants in which four highly conserved residues in the fingers domain have been replaced by ala. the relationship between the kinetic constants exhibited by the mutants and the structure of the ternary complex [franklin, m., wang, j., and steitz t. (2001) cell 105, 657-667] was consistent with the following sets of interactions between the conserved residues and o ...200211851399
crystal structure of dna polymerase from hyperthermophilic archaeon pyrococcus kodakaraensis kod1.the crystal structure of family b dna polymerase from the hyperthermophilic archaeon pyrococcus kodakaraensis kod1 (kod dna polymerase) was determined. kod dna polymerase exhibits the highest known extension rate, processivity and fidelity. we carried out the structural analysis of kod dna polymerase in order to clarify the mechanisms of those enzymatic features. structural comparison of dna polymerases from hyperthermophilic archaea highlighted the conformational difference in thumb domains. th ...200111178906
lesion (in)tolerance reveals insights into dna replication fidelity.the initial encounter of an unrepaired dna lesion is likely to be with a replicative dna polymerase, and the outcome of this event determines whether an error-prone or error-free damage avoidance pathway is taken. to understand the atomic details of this critical encounter, we have determined the crystal structures of the pol alpha family rb69 dna polymerase with dna containing the two most prevalent, spontaneously generated premutagenic lesions, an abasic site and 2'-deoxy-7,8-dihydro-8-oxoguan ...200415057282
crystallographic snapshots of a replicative dna polymerase encountering an abasic site.abasic sites are common dna lesions, which are strong blocks to replicative polymerases and are potentially mutagenic when bypassed. we report here the 2.8 a structure of the bacteriophage rb69 replicative dna polymerase attempting to process an abasic site analog. four different complexes were captured in the crystal asymmetric unit: two have dna in the polymerase active site whereas the other two molecules are in the exonuclease mode. when compared to complexes with undamaged dna, the dna surr ...200415057283
crystal structure of a pol alpha family dna polymerase from the hyperthermophilic archaeon thermococcus sp. 9 degrees n-7.the 2.25 a resolution crystal structure of a pol alpha family (family b) dna polymerase from the hyperthermophilic marine archaeon thermococcus sp. 9 degrees n-7 (9 degrees n-7 pol) provides new insight into the mechanism of pol alpha family polymerases that include essentially all of the eukaryotic replicative and viral dna polymerases. the structure is folded into nh(2)- terminal, editing 3'-5' exonuclease, and polymerase domains that are topologically similar to the two other known pol alpha ...200010860752
bacteriophage-based genetic system for selection of nonsplicing inteins.a genetic selection system that detects splicing and nonsplicing activities of inteins was developed based on the ability to rescue a t4 phage strain with a conditionally inactive dna polymerase. this phage defect can be complemented by expression of plasmid-encoded phage rb69 dna polymerase. insertion of an intein gene into the active site of the rb69 dna polymerase gene renders polymerase activity and phage viability dependent on protein splicing. the effectiveness of the system was tested by ...200415128583
crystal structure of an archaebacterial dna polymerase.members of the pol ii family of dna polymerases are responsible for chromosomal replication in eukaryotes, and carry out highly processive dna replication when attached to ring-shaped processivity clamps. the sequences of pol ii polymerases are distinct from those of members of the well-studied pol i family of dna polymerases. the dna polymerase from the archaebacterium desulfurococcus strain tok (d. tok pol) is a member of the pol ii family that retains catalytic activity at elevated temperatur ...199910545321
rna-binding properties of in vitro expressed histidine-tagged rb69 rega translational repressor protein.to facilitate rna-binding studies of the phage rb69 rega translational repressor protein, rega was configured to add six histidines to the carboxyl end of the protein. in vitro transcription-translation from the t7 promoter on plasmid psa1 yielded a rega69-his6 protein that binds nickel-sepharose and elutes with 0.5 m imidazole. the system was further modified to avoid cloning and the toxic effects of rega on escherichia coli by the polymerase chain reaction (pcr), producing linear templates wit ...199910094772
role of the first aspartate residue of the "yxdtds" motif of phi29 dna polymerase as a metal ligand during both tp-primed and dna-primed dna synthesis.almost all known nucleic acid polymerases require three acidic residues to bind the metal ion during catalysis of nucleotide incorporation. nevertheless, recent crystallographic data on bacteriophage rb69 dna polymerase indicate that the first aspartate residue belonging to the conserved motif "yxdtds" could have a merely structural role. to address this question, a mutant protein at the homologous aspartate residue (asp456) in phi29 dna polymerase was made 3'-5' exonuclease deficient. this allo ...19989784372
aspects of the ultraviolet photobiology of some t-even bacteriophages.bacteriophage t4 dna metabolism is largely insulated from that of its host, although some host functions assist in the repair of t4 dna damage. environmental factors sometimes affect survival and mutagenesis after ultraviolet (uv) irradiation of t4, and can affect mutagenesis in many organisms. we therefore tested the effect of certain environmental factors and host genetic defects upon spontaneous and uv-induced mutagenesis and survival in t4 and some related t-even phages. plating at ph 9 enha ...19989560380
residues at the carboxy terminus of t4 dna polymerase are important determinants for interaction with the polymerase accessory proteins.three t4 dna polymerase accessory proteins (44p/62p and 45p) stimulate the polymerase (pol) activity and the 3'-5' exonuclease (exo) activity of t4 dna polymerase (43p) on long, double-stranded dna substrates. the 44p/62p "clamp loader" facilitates the binding of 45p, the "sliding clamp", to dna that is primed for replication. using a series of truncated 43p mutants, we identified a region at the extreme carboxy terminus of the dna polymerase that is required for its interaction with accessory p ...19979265627
retention of replication fidelity by a dna polymerase functioning in a distantly related environment.the primary structures of the replicative dna polymerases (gp43s) of bacteriophage t4 and its distant phylogenetic relative rb69 are diverged, retaining only 61% identity and 74% similarity. nevertheless, rb69 gp43 substitutes effectively for t4 gp43 in t4 dna replication in vivo. we show here that rb69 gp43 replicates t4 genomes in vivo with a fidelity similar to that achieved by t4 gp43. furthermore, replication by rb69 gp43 in the distantly related environment does not enhance the mutator act ...19979223311
base selectivity is impaired by mutants that perturb hydrogen bonding networks in the rb69 dna polymerase active site.to investigate the molecular basis for the selective utilization of nucleoside triphosphates complementary to templating bases, by rb69 dna polymerase (rb69 pol), we constructed a set of mutants that we predicted would perturb the "floor" of the nascent base-pairing interface in the enzyme. we then determined the pre-steady-state kinetic parameters for the incorporation of complementary and noncomplementary dntps by the exo(-) form of rb69 pol and its mutants. we found that the y567a mutant had ...200515736944
crystal structure of a pol alpha family replication dna polymerase from bacteriophage rb69.the 2.8 a resolution crystal structure of the bacteriophage rb69 gp43, a member of the eukaryotic pol alpha family of replicative dna polymerases, shares some similarities with other polymerases but shows many differences. although its palm domain has the same topology as other polymerases, except rat dna polymerase beta, one of the three carboxylates required for nucleotidyl transfer is located on a different beta strand. the structures of the fingers and thumb domains are unrelated to all othe ...19979215631
clusters of mutations from transient hypermutability.collections of mutants usually contain more mutants bearing multiple mutations than expected from the mutant frequency and a random distribution of mutations. this excess is seen in a variety of organisms and also after dna synthesis in vitro. the excess is unlikely to originate in mutator mutants but rather from transient hypermutability resulting from a perturbation of one of the many transactions that maintain genetic fidelity. the multiple mutations are sometimes clustered and sometimes rand ...200516118275
evolution of rna-binding specificity in t4 dna polymerase.dna polymerase of phage t4 (t4 gp43), an essential component of the t4 dna replicase, is a multifunctional single-chained (898-amino acid) protein that catalyzes the highly accurate synthesis of dna in phage replication. the enzyme functions both as a dna-binding replication protein and as a sequence-specific rna-binding autogenous translational repressor. we have utilized a phylogenetic approach to study the relationships between the two nucleic acid-binding functions of the protein. we found t ...19979211921
experimental examination of bacteriophage latent-period evolution as a response to bacterial availability.for obligately lytic bacteriophage (phage) a trade-off exists between fecundity (burst size) and latent period (a component of generation time). this trade-off occurs because release of phage progeny from infected bacteria coincides with destruction of the machinery necessary to produce more phage progeny. here we employ phage mutants to explore issues of phage latent-period evolution as a function of the density of phage-susceptible bacteria. theory suggests that higher bacterial densities shou ...200314660403
dissecting the fidelity of bacteriophage rb69 dna polymerase: site-specific modulation of fidelity by polymerase accessory proteins.bacteriophage rb69 encodes a replicative b-family dna polymerase (rb69 gp43) with an associated proofreading 3' exonuclease. crystal structures have been determined for this enzyme with and without dna substrates. we previously described the mutation rates and kinds of mutations produced in vivo by the wild-type (pol(+) exo(+)) enzyme, an exonuclease-deficient mutator variant (pol(+) exo(-)), mutator variants with substitutions at tyr(567) in the polymerase active site (pol(m) exo(+)), and the d ...200212454051
the l561a substitution in the nascent base-pair binding pocket of rb69 dna polymerase reduces base discrimination.several variants of rb69 dna polymerase (rb69 pol) with single-site replacements in the nascent base-pair binding pocket are less discriminating with respect to noncomplementary dnmp incorporation than the wild-type enzyme. to quantify the loss in base selectivity, we determined the transient-state kinetic parameters for incorporation of correct and all combinations of incorrect dnmps by the exonuclease-deficient form of one of these rb69 pol variants, l561a, using rapid chemical quench assays. ...200616475809
protein determinants of rna binding by dna polymerase of the t4-related bacteriophage rb69.dna polymerase (gp43) of phage t4 plays two biological roles, one as an essential dna binding replication enzyme and the other as an mrna-specific autogenous translational repressor. binding of t4 gp43 to its mrna target (translational operator rna) interferes with gp43-dna interactions, but it is unclear how the protein determinants for binding dna are affected by the dynamics of gp43-mrna interactions. we have used rb69 gp43, a natural variant of the t4 enzyme whose crystal structure has been ...200212087102
direct interaction of proliferating cell nuclear antigen with the small subunit of dna polymerase delta.the interaction between proliferating cell nuclear antigen (pcna) and dna polymerase delta is essential for processive dna synthesis during dna replication/repair; however, the identity of the subunit of dna polymerase delta that directly interacts with pcna has not been resolved until now. in the present study we have used reciprocal co-immunoprecipitation experiments to determine which of the two subunits of core dna polymerase delta, the 125-kda catalytic subunit or the 50-kda small subunit, ...200211986310
rna aptamers selected against dna polymerase beta inhibit the polymerase activities of dna polymerases beta and kappa.dna polymerase beta (polbeta), a member of the x family of dna polymerases, is the major polymerase in the base excision repair pathway. using in vitro selection, we obtained rna aptamers for polbeta from a variable pool of 8 x 10(12) individual rna sequences containing 30 random nucleotides. a total of 60 individual clones selected after seven rounds were screened for the ability to inhibit polbeta activity. all of the inhibitory aptamers analyzed have a predicted tri-lobed structure. gel mobil ...200616707660
genetic diversity among five t4-like bacteriophages.bacteriophages are an important repository of genetic diversity. as one of the major constituents of terrestrial biomass, they exert profound effects on the earth's ecology and microbial evolution by mediating horizontal gene transfer between bacteria and controlling their growth. only limited genomic sequence data are currently available for phages but even this reveals an overwhelming diversity in their gene sequences and genomes. the contribution of the t4-like phages to this overall phage di ...200616716236
plasticity of the gene functions for dna replication in the t4-like phages.we have completely sequenced and annotated the genomes of several relatives of the bacteriophage t4, including three coliphages (rb43, rb49 and rb69), three aeromonas salmonicida phages (44rr2.8t, 25 and 31) and one aeromonas hydrophila phage (aeh1). in addition, we have partially sequenced and annotated the t4-like genomes of coliphage rb16 (a close relative of rb43), a. salmonicida phage 65, acinetobacter johnsonii phage 133 and vibrio natriegens phage nt-1. each of these phage genomes exhibit ...200616828113
structure of the replicating complex of a pol alpha family dna polymerase.we describe the 2.6 a resolution crystal structure of rb69 dna polymerase with primer-template dna and dttp, capturing the step just before primer extension. this ternary complex structure in the human dna polymerase alpha family shows a 60 degrees rotation of the fingers domain relative to the apo-protein structure, similar to the fingers movement in pol i family polymerases. minor groove interactions near the primer 3' terminus suggest a common fidelity mechanism for pol i and pol alpha family ...200111389835
accuracy, lesion bypass, strand displacement and translocation by dna polymerases.the structures of dna polymerases from different families show common features and significant differences that shed light on the ability of these enzymes to accurately copy dna and translocate. the structure of a b family dna polymerase from phage rb69 exhibits an active-site closing conformational change in the fingers domain upon forming a ternary complex with primer template in deoxynucleoside triphosphate. the rotation of the fingers domain alpha-helices by 60 degrees upon dntp binding is a ...200415065652
interacting fidelity defects in the replicative dna polymerase of bacteriophage rb69.the dna polymerases (gp43s) of the related bacteriophages t4 and rb69 are b family (polymerase alpha class) enzymes that determine the fidelity of phage dna replication. a t4 whose gene 43 has been mutationally inactivated can be replicated by a cognate rb69 gp43 encoded by a recombinant plasmid in t4-infected escherichia coli. we used this phage-plasmid complementation assay to obtain rapid and sensitive measurements of the mutational specificities of mutator derivatives of the rb69 enzyme. rb6 ...200111133987
dna polymerase of the t4-related bacteriophages.the dna polymerase of bacteriophage t4, product of phage gene 43 (gp43), has served as a model replicative dna polymerase in nucleic acids research for nearly 40 years. the base-selection (polymerase, or pol) and editing (3'-exonuclease, or exo) functions of this multifunctional protein, which have counterparts in the replicative polymerases of other organisms, are primary determinants of the high fidelity of dna synthesis in phage dna replication. t4 gp43 is considered to be a member of the "b ...200010697407
building a replisome from interacting pieces: sliding clamp complexed to a peptide from dna polymerase and a polymerase editing complex.we have solved the crystal structures of the bacteriophage rb69 sliding clamp, its complex with a peptide essential for dna polymerase interactions, and the dna polymerase complexed with primer-template dna. the editing complex structure shows a partially melted duplex dna exiting from the exonuclease domain at an unexpected angle and significant changes in the protein structure. the clamp complex shows the c-terminal 11 residues of polymerase bound in a hydrophobic pocket, and it allows docking ...199910535734
steady-state kinetic characterization of rb69 dna polymerase mutants that affect dntp incorporation.the function of six highly conserved residues (arg482, lys483, lys486, lys560, asn564, and tyr567) in the fingers domain of bacteriophage rb69 dna polymerase (rb69 gp43) were analyzed by kinetic studies with mutants in which each of these residues was replaced with ala. our results suggest that arg482, lys486, lys560, and asn564 contact the incoming dntp during the nucleotidyl transfer reaction as judged by variations in apparent km and kcat values for dntp incorporation by these mutants compare ...199910387055
crystal structure of a thermostable type b dna polymerase from thermococcus gorgonarius.most known archaeal dna polymerases belong to the type b family, which also includes the dna replication polymerases of eukaryotes, but maintain high fidelity at extreme conditions. we describe here the 2.5 a resolution crystal structure of a dna polymerase from the archaea thermococcus gorgonarius and identify structural features of the fold and the active site that are likely responsible for its thermostable function. comparison with the mesophilic b type dna polymerase gp43 of the bacteriopha ...199910097083
exonuclease-polymerase active site partitioning of primer-template dna strands and equilibrium mg2+ binding properties of bacteriophage t4 dna polymerase.the binding of bacteriophage t4 dna polymerase (t4 pol) to primer-template dna with 2-aminopurine (2ap) located at the primer terminus results in the formation of a hyperfluorescent 2ap state. changes in this hyperfluorescent state were utilized to investigate the fractional concentration of primer-templates bound at the exonuclease and statically quenched polymerase sites. in the absence of mg2+, a hydrophobic exonuclease site dominates over the polymerase site for possession of the primer term ...19989665720
utilization of a deoxynucleoside diphosphate substrate by hiv reverse transcriptase.deoxynucleoside triphosphates (dntps) are the normal substrates for dna synthesis catalyzed by polymerases such as hiv-1 reverse transcriptase (rt). however, substantial amounts of deoxynucleoside diphosphates (dndps) are also present in the cell. use of dndps in hiv-1 dna synthesis could have significant implications for the efficacy of nucleoside rt inhibitors such as azt which are first line therapeutics for treatment of hiv infection. our earlier work on hiv-1 reverse transcriptase (rt) sugg ...200818446195
divergence of a dna replication gene cluster in the t4-related bacteriophage rb69.the genomes of bacteriophages t4 and rb69 are phylogenetically related but diverge in nucleotide sequence at many loci and are incompatible with each other in vivo. we describe here the biological implications of divergence in a genomic segment that encodes four essential dna replication proteins: gp45 (sliding clamp), gp44/62 complex (clamp loader), and gp46 (a recombination protein). we have cloned, sequenced, and expressed several overlapping segments of the rb69 gene 46-45.2-(rpba)-45-44-62 ...19989555879
modular organization of t4 dna polymerase. evidence from phylogenetics.we describe the use of a phylogenetic approach to analyze the modular organization of the single-chained (898 amino acids) and multifunctional dna polymerase of phage t4. we have identified, cloned in expression vectors, and sequenced the dna polymerase gene (gene 43) of phage rb69, a distant relative of t4. the deduced primary structure of the rb69 protein (rb69 gp43) differs from that of t4 gp43 in discrete clusters of short sequence that are interspersed with clusters of high similarity betwe ...19957592876
autogenous translational operator recognized by bacteriophage t4 dna polymerase.the synthesis of the dna polymerase of bacteriophage t4 is autogenously regulated. this protein (gp43), the product of gene 43, binds to a segment of its mrna that overlaps its ribosome binding site, and thereby blocks translation. we have determined the kd of the gp43-operator interaction to be 1.0 x 10(-9) m. the minimum operator sequence to which gp43 binds consists of 36 nucleotides that include a hairpin (containing a 5 base-pair helix and an 8 nucleotide loop) and a single-stranded segment ...19902359122
role of helix p of the human cytomegalovirus dna polymerase in resistance and hypersusceptibility to the antiviral drug foscarnet.mutations in the human cytomegalovirus dna polymerase (ul54) can not only decrease but also increase susceptibility to the pyrophosphate (pp(i)) analogue foscarnet. the proximity of l802m, which confers resistance, and k805q, which confers hypersusceptibility, suggests a possible unifying mechanism that affects drug susceptibility in one direction or the other. we found that the polymerase activities of l802m- and k805q-containing mutant enzymes were literally indistinguishable from that of wild ...200616415021
kinetics of error generation in homologous b-family dna polymerases.the kinetics of forming a proper watson-crick base pair as well incorporating bases opposite furan, an abasic site analog, have been well characterized for the b family replicative dna polymerase from bacteriophage t4. structural studies of these reactions, however, have only been performed with the homologous enzyme from bacteriophage rb69. in this work, the homologous enzymes from rb69 and t4 were compared in parallel reactions to determine the relative abilities of the two polymerases to inco ...200616687658
different behaviors in vivo of mutations in the beta hairpin loop of the dna polymerases of the closely related phages t4 and rb69.the t4 and rb69 dna replicative polymerases are members of the b family and are highly similar. both replicate dna with high fidelity and employ the same mechanism that allows efficient switching of the primer terminus between the polymerase and exonuclease sites. both polymerases have a beta hairpin loop (hereafter called the beta loop) in their exonuclease domains that plays an important role in active-site switching. the beta loop is involved in strand separation and is needed to stabilize pa ...200919409904
three-dimensional modeling of cytomegalovirus dna polymerase and preliminary analysis of drug resistance.cytomegalovirus (cmv) is the leading cause of congenital infection and a frequent opportunistic agent in immunocompromised hosts such as transplant recipients and aids patients. cmv dna polymerase, a member of the polymerase b family, is the primary target of all available antivirals (ganciclovir, cidofovir, and foscarnet) and certain variations of this enzyme could lead to drug resistance. however, understanding the drug resistance mechanisms at the atomic level is hampered by the lack of its t ...200616705640
structure and enzymatic properties of a chimeric bacteriophage rb69 dna polymerase and single-stranded dna binding protein with increased processivity.in vivo, replicative dna polymerases are made more processive by their interactions with accessory proteins at the replication fork. single-stranded dna binding protein (ssb) is an essential protein that binds tightly and cooperatively to single-stranded dna during replication to remove adventitious secondary structures and protect the exposed dna from endogenous nucleases. using information from high resolution structures and biochemical data, we have engineered a functional chimeric enzyme of ...200616881051
the crystal structure of pf-8, the dna polymerase accessory subunit from kaposi's sarcoma-associated herpesvirus.kaposi's sarcoma-associated herpesvirus is an emerging pathogen whose mechanism of replication is poorly understood. pf-8, the presumed processivity factor of kaposi's sarcoma-associated herpesvirus dna polymerase, acts in combination with the catalytic subunit, pol-8, to synthesize viral dna. we have solved the crystal structure of residues 1 to 304 of pf-8 at a resolution of 2.8 a. this structure reveals that each monomer of pf-8 shares a fold common to processivity factors. like human cytomeg ...200919759157
pre-steady-state kinetics of rb69 dna polymerase and its exo domain mutants: effect of ph and thiophosphoryl linkages on 3'-5' exonuclease activity.dna polymerases from the a and b families with 3'-5' exonucleolytic activity have exonuclease domains with similar three-dimensional structures that require two divalent metal ions for catalysis. b family dna polymerases that are part of a replicase generally have a more potent 3'-5' exonuclease (exo) activity than a family dna polymerases that mainly function in dna repair. to investigate the basis for these differences, we determined ph-activity profiles for the exonuclease reactions of t4, rb ...200415049692
structural and biochemical investigation of the role in proofreading of a beta hairpin loop found in the exonuclease domain of a replicative dna polymerase of the b family.replicative dna polymerases, as exemplified by the b family polymerases from bacteriophages t4 and rb69, not only replicate dna but also have the ability to proofread misincorporated nucleotides. because the two activities reside in separate protein domains, polymerases must employ a mechanism that allows for efficient switching of the primer strand between the two active sites to achieve fast and accurate replication. prior mutational and structural studies suggested that a beta hairpin structu ...200717098747
a structural rationale for stalling of a replicative dna polymerase at the most common oxidative thymine lesion, thymine glycol.thymine glycol (tg) is a common product of oxidation and ionizing radiation, including that used for cancer treatment. although tg is a poor mutagenic lesion, it has been shown to present a strong block to both repair and replicative dna polymerases. the 2.65-a crystal structure of a binary complex of the replicative rb69 dna polymerase with dna shows that the templating tg is intrahelical and forms a regular watson-crick base pair with the incorporated a. the c5 methyl group protrudes axially f ...200717210917
genome properties and the limits of adaptation in bacteriophages.eight bacteriophages were adapted for rapid growth under similar conditions to compare their evolved, endpoint fitnesses. four pairs of related phages were used, including two rna phages with small genomes (ms2 and qbeta) two single-stranded dna phages with small genomes (phix174 and g4), two t-odd phages with medium-sized, double-stranded dna genomes (t7 and t3), and two t-even phages with large, double-stranded dna genomes (t6 and rb69). fitness was measured as absolute growth rate per hour un ...200415154545
two proton transfers in the transition state for nucleotidyl transfer catalyzed by rna- and dna-dependent rna and dna polymerases.the rate-limiting step for nucleotide incorporation in the pre-steady state for most nucleic acid polymerases is thought to be a conformational change. as a result, very little information is available on the role of active-site residues in the chemistry of nucleotidyl transfer. for the poliovirus rna-dependent rna polymerase (3d(pol)), chemistry is partially (mg(2+)) or completely (mn(2+)) rate limiting. here we show that nucleotidyl transfer depends on two ionizable groups with pk(a) values of ...200717360513
the activity of selected rb69 dna polymerase mutants can be restored by manganese ions: the existence of alternative metal ion ligands used during the polymerization cycle.site specific mutants in the pol active center of rb69 dna polymerase have been produced and studied using rapid chemical-quench techniques. pre-steady-state kinetic analysis carried out with mg(2+) and mn(2+) has enabled us to divide the mutants into two groups. one group had greatly reduced k(pols) values in the presence of mg(2+) but responded to mn(2+) which restored the k(pol) values for the nucleotidyl transfer reaction to near wild-type levels. the other group of mutants also had lower k( ...200415157091
fluorescence of 2-aminopurine reveals rapid conformational changes in the rb69 dna polymerase-primer/template complexes upon binding and incorporation of matched deoxynucleoside triphosphates.we have used 2-aminopurine (2ap) as a fluorescent probe in the template strand of a 13/20mer primer/template (d) to detect deoxynucleoside triphosphates (n)-dependent conformational changes exhibited by rb69 dna polymerase (ed) complexes. the rates and amplitudes of fluorescence quenching depend hyperbolically on the [dttp] when a dideoxy-primer/template (ddp/t) with 2ap as the templating base (n position) is used. no detectable fluorescence changes occur when a ddp/t with 2ap positioned 5' to t ...200717766250
the sequences and activities of regb endoribonucleases of t4-related bacteriophages.the regb endoribonuclease encoded by bacteriophage t4 is a unique sequence-specific nuclease that cleaves in the middle of ggag or, in a few cases, ggau tetranucleotides, preferentially those found in the shine-dalgarno regions of early phage mrnas. in this study, we examined the primary structures and functional properties of regb ribonucleases encoded by t4-related bacteriophages. we show that all but one of 36 phages tested harbor the regb gene homologues and the similar signals for transcrip ...200415486207
divergence of the mrna targets for the ssb proteins of bacteriophages t4 and rb69.the single-strand binding (ssb) protein of phage t4 (t4 gp32, product of gene 32) is a mrna-specific autogenous translational repressor, in addition to being a sequence-independent ssdna-binding protein that participates in phage dna replication, repair and recombination. it is not clear how this physiologically essential protein distinguishes between specific rna and nonspecific nucleic acid targets. here, we present phylogenetic evidence suggesting that ssdna and specific rna bind the same gp3 ...200415507125
a family of anti-sigma70 proteins in t4-type phages and bacteria that are similar to asia, a transcription inhibitor and co-activator of bacteriophage t4.anti-sigma70 factors interact with sigma70 proteins, the specificity subunits of prokaryotic rna polymerase. the bacteriophage t4 anti-sigma70 protein, asia, binds tightly to regions 4.1 and 4.2 of the sigma70 subunit of escherichia coli rna polymerase and inhibits transcription from sigma70 promoters that require recognition of the canonical sigma70 -35 dna sequence. in the presence of the t4 transcription activator mota, asia also functions as a co-activator of transcription from t4 middle pro ...200415561138
processivity clamp gp45 and ssdna-binding-protein gp32 modulate the fidelity of bacteriophage rb69 dna polymerase in a sequence-specific manner, sometimes enhancing and sometimes compromising accuracy.numerous studies of the impact of accessory proteins upon the fidelity of dna synthesis have provided a complex and sometimes discordant picture. we previously described such an analysis conducted in vitro using various bacteriophage rb69 gp43 mutator dna polymerases with or without the accessory proteins gp32 (which binds single-stranded dna) plus gp45/44/62 (processivity clamp and its loaders). mutations were scored at many sites in the laczalpha mutation reporter sequence. unexpectedly, the a ...200515695359
in vitro selection of phage rb69 rega rna binding sites yields uaa triplets.the selex method of in vitro selection was used to isolate rnas that bind the rb69 rega translational repressor protein immobilized on ni-nta agarose. after five rounds of selex, the pool of selected rna displayed striking sequence uniformity: uaauaauaauaaua was clearly enriched in the 14 nucleotides that underwent selection. individual, cloned molecules displayed a repeating (uaa) sequence, with only two rnas having a 3' aug. removing the 3' aug slightly reduced binding in gel shift assays, mov ...200515866068
molecular dynamics of a food carcinogen-dna adduct in a replicative dna polymerase suggest hindered nucleotide incorporation and extension.2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (phip) is the most abundant of the carcinogenic heterocyclic aromatic amines in the human diet, and the major mutagenic effect of dietary phip is g-->t transversions. the major phip-derived dna adduct is to c8 of guanine. we have investigated this adduct in a phip-induced mutational hotspot 5'-ggga-3' of the apc tumor suppressor gene, frequently mutated in mammalian colon tumors. we have carried out a molecular dynamics study to elucidate on a stru ...200516167826
transcription and rna processing during expression of genes preceding dna ligase gene 30 in t4-related bacteriophages.early gene expression in bacteriophage t4 is controlled primarily by the unique early promoters, while t4-encoded regb endoribonuclease promotes degradation of many early messages contributing to the rapid shift of gene expression from the early to middle stages. the regulatory region for the genes clustered upstream of dna ligase gene 30 of t4 was known to carry two strong early promoters and two putative regb sites. here, we present the comparative analysis of the regulatory events in this reg ...200616225899
effect of a and b metal ion site occupancy on conformational changes in an rb69 dna polymerase ternary complex.rapid chemical quench assays, as well as equilibrium and stopped-flow fluorescence experiments, were performed with an rb69 dna polymerase (rb69 pol)-primer-template (p/t) complex containing 2-aminopurine (dap) and a metal exchange-inert rh(iii) derivative of a deoxynucleoside triphosphate (rh.dttp). the objective was to determine the effect of catalytic metal ion (a site) occupancy on the affinity of an incoming rh.dttp for the rb69 pol-p/t binary complex and on the rate of the conformational c ...200919228037
rb69 dna polymerase mutants with expanded nascent base-pair-binding pockets are highly efficient but have reduced base selectivity.we have investigated the effect of systematically enlarging the nascent base-pair-binding pocket (nbp) of a replicative dna polymerase from bacteriophage rb69 (rb69 pol) on the incorporation efficiency (k(pol)/k(d,app)) for both correct and incorrect dnmps. accordingly, we replaced residues l561, y567, and s565 in the nbp with ala, ala, and gly, respectively. we combined l561a and y567a to give a double mutant and then introduced the s565g mutation to give a triple mutant. the efficiency of inco ...200919522539
structure of the small outer capsid protein, soc: a clamp for stabilizing capsids of t4-like phages.many viruses need to stabilize their capsid structure against dna pressure and for survival in hostile environments. the 9-kda outer capsid protein (soc) of bacteriophage t4, which stabilizes the virus, attaches to the capsid during the final stage of maturation. there are 870 soc molecules that act as a "glue" between neighboring hexameric capsomers, forming a "cage" that stabilizes the t4 capsid against extremes of ph and temperature. here we report a 1.9 a resolution crystal structure of soc ...201019835886
kinetics of mismatch formation opposite lesions by the replicative dna polymerase from bacteriophage rb69.the fidelity of dna replication is under constant threat from the formation of lesions within the genome. oxidation of dna bases leads to the formation of altered dna bases such as 8-oxo-7,8-dihydroguanine, commonly called 8-oxog, and 2-hydroxyadenine, or 2-oha. in this work we have examined the incorporation kinetics opposite these two oxidatively derived lesions as well as an abasic site analogue by the replicative dna polymerase from bacteriophage rb69. we compared the kinetic parameters for ...201020166748
crystal structure of a replicative dna polymerase bound to the oxidized guanine lesion guanidinohydantoin.the oxidation of guanine generates one of the most common dna lesions, 8-oxo-7,8-dihydroguanine (8-oxog). the further oxidation of 8-oxog can produce either guanidinohydantoin (gh) in duplex dna or spiroiminodihydantoin (sp) in nucleosides and ssdna. although gh can be a strong block for replicative dna polymerases such as rb69 dna polymerase, this lesion is also mutagenic: dna polymerases bypass gh by preferentially incorporating a purine with a slight preference for adenine, which results in g ...201020166752
the roles of tyr391 and tyr619 in rb69 dna polymerase replication fidelity.in the family-b dna polymerase of bacteriophage rb69, the conserved aromatic palm-subdomain residues tyr391 and tyr619 interact with the last primer-template base-pair. tyr619 interacts via a water-mediated hydrogen bond with the phosphate of the terminal primer nucleotide. the main-chain amide of tyr391 interacts with the corresponding template nucleotide. a hydrogen bond has been postulated between tyr391 and the hydroxyl group of tyr567, a residue that plays a key role in base discrimination. ...200717321543
substitution of ala for tyr567 in rb69 dna polymerase allows damp and dgmp to be inserted opposite guanidinohydantoin .continuous oxidative damage inflicted on dna produces 7,8-dihydro-8-oxoguanine (8-oxog), a commonly occurring lesion that can potentially cause cancer by producing g → t transversions during dna replication. mild oxidation of 8-oxog leads to the formation of hydantoins, specifically guanidinohydantoin (gh) and spiroiminodihydantoin (sp), which are 100% mutagenic because they encode almost exclusively the insertion of damp and dgmp (encoding g → t and g → c transversions, respectively). the wild- ...201020795733
genome analysis of phage js98 defines a fourth major subgroup of t4-like phages in escherichia coli.numerous t4-like escherichia coli phages were isolated from human stool and environmental wastewater samples in bangladesh and switzerland. the sequences of the major head gene (g23) revealed that these coliphages could be placed into four subgroups, represented by the phages t4, rb69, rb49, and js98. thus, js98 defines a new major subgroup of e. coli t4-like phages. we conducted an analysis of the 169-kb js98 genome sequence. overall, 198 of the 266 js98 open reading frames (orfs) shared amino ...200717693496
caught bending the a-rule: crystal structures of translesion dna synthesis with a non-natural nucleotide.damage to dna involving excision of the nucleobase at the n-glycosidic bond forms abasic sites. if a nucleotide becomes incorporated opposite an unrepaired abasic site during dna synthesis, most b family polymerases obey the a-rule and preferentially incorporate damp without instruction from the template. in addition to being potentially mutagenic, abasic sites provide strong blocks to dna synthesis. a previous crystal structure of an exonuclease deficient variant of the replicative b family dna ...200717718515
insights into base selectivity from the 1.8 å resolution structure of an rb69 dna polymerase ternary complex.bacteriophage rb69 dna polymerase (rb69 pol) has served as a model for investigating how b family polymerases achieve a high level of fidelity during dna replication. we report here the structure of an rb69 pol ternary complex at 1.8 å resolution, extending the resolution from our previously reported structure at 2.6 å [franklin, m. c., et al. (2001) cell 105, 657-667]. in the structure presented here, a network of five highly ordered, buried water molecules can be seen to interact with the n3 a ...201021158418
use of 2-aminopurine fluorescence to study the role of the beta hairpin in the proofreading pathway catalyzed by the phage t4 and rb69 dna polymerases.for dna polymerases to proofread a misincorporated nucleotide, the terminal 3-4 nucleotides of the primer strand must be separated from the template strand before being bound in the exonuclease active center. genetic and biochemical studies of the bacteriophage t4 dna polymerase revealed that a prominent beta-hairpin structure in the exonuclease domain is needed to efficiently form the strand-separated exonuclease complexes. we present here further mutational analysis of the loop region of the t ...200818481871
characterization of a replicative dna polymerase mutant with reduced fidelity and increased translesion synthesis capacity.changing a highly conserved amino acid in motif a of any of the four yeast family b dna polymerases, dna polymerase alpha, delta, epsilon or zeta, results in yeast strains with elevated mutation rates. in order to better understand this phenotype, we have performed structure-function studies of homologous mutants of rb69 dna polymerase (rb69 pol), a structural model for family b members. when leu415 in rb69 pol is replaced with phenylalanine or glycine, the mutant polymerases retain high-catalyt ...200818503083
reaction mechanism of the epsilon subunit of e. coli dna polymerase iii: insights into active site metal coordination and catalytically significant residues.the 28 kda epsilon subunit of escherichia coli dna polymerase iii is the exonucleotidic proofreader responsible for editing polymerase insertion errors. here, we study the mechanism by which epsilon carries out the exonuclease activity. we performed quantum mechanics/molecular mechanics calculations on the n-terminal domain containing the exonuclease activity. both the free-epsilon and a complex epsilon bound to a theta homologue (hot) were studied. for the epsilon-hot complex mg(2+) or mn(2+) w ...200919119875
the reopening rate of the fingers domain is a determinant of base selectivity for rb69 dna polymerase.two divalent metal ions are required for nucleotide incorporation by dna polymerases. here we use the bacteriophage rb69 dna polymerase (rb69 pol) and the metal ion exchange-inert nucleotide analogue rhodium(iii) deoxythymidine triphosphate (rh.dttp) to investigate the requirements of metal binding to the "a" site and to the "b" site, independently. we show that while binding of a metal ion to the a site is required for the nucleotidyl transfer reaction to occur, this metal binding is insufficie ...200919228036
structure of the 2-aminopurine-cytosine base pair formed in the polymerase active site of the rb69 y567a-dna polymerase.the adenine base analogue 2-aminopurine (2ap) is a potent base substitution mutagen in prokaryotes because of its enhanceed ability to form a mutagenic base pair with an incoming dctp. despite more than 50 years of research, the structure of the 2ap-c base pair remains unclear. we report the structure of the 2ap-dctp base pair formed within the polymerase active site of the rb69 y567a-dna polymerase. a modified wobble 2ap-c base pair was detected with one h-bond between n1 of 2ap and a proton fr ...201122023103
engineering of a chimeric rb69 dna polymerase sensitive to drugs targeting the cytomegalovirus enzyme.detailed structural and biochemical studies with the human cytomegalovirus (hcmv ul54) dna polymerase are hampered by difficulties to obtain this enzyme in large quantities. the crystal structure of the related rb69 dna polymerase (gp43) is often used as a model system to explain mechanisms of inhibition of dna synthesis and drug resistance. however, here we demonstrate that gp43 is approximately 400-fold less sensitive to the pyrophosphate analog foscarnet, when compared with ul54. the rb69 enz ...200919622750
substitution of ala for tyr567 in rb69 dna polymerase allows damp to be inserted opposite 7,8-dihydro-8-oxoguanine .accurate copying of the genome by dna polymerases is challenging due in part to the continuous damage inflicted on dna, which results from its contact with reactive oxygen species (ros), producing lesions such as 7,8-dihydro-8-oxoguanine (8-oxog). the deleterious effects of 8-oxog can be attributed to its dual coding potential that leads to g --> t transversions. the wild-type (wt) pol alpha family dna polymerase from bacteriophage rb69 (rb69pol) prefers to insert dcmp as opposed to damp when si ...201020411947
mutational clusters generated by non-processive polymerases: a case study using dna polymerase betain vitro.available dna mutational spectra reveal that the number of mutants with multiple mutations ("multiples") is usually greater than expected from a random distribution of mutations among mutants. these overloads imply the occurrence of non-random clusters of mutations, probably generated during episodes of low-fidelity dna synthesis. excess multiples have been reported not only for viruses, bacteria, and eukaryotic cells but also for the dna polymerases of phages t4 and rb69 in vitro. in the simple ...201020627824
reversal of a mutator activity by a nearby fidelity-neutral substitution in the rb69 dna polymerase binding pocket.phage rb69 b-family dna polymerase is responsible for the overall high fidelity of rb69 dna synthesis. fidelity is compromised when conserved tyr567, one of the residues that form the nascent polymerase base-pair binding pocket, is replaced by alanine. the y567a mutator mutant has an enlarged binding pocket and can incorporate and extend mispairs efficiently. ser565 is a nearby conserved residue that also contributes to the binding pocket, but a s565g replacement has only a small impact on dna r ...201020950625
structural analysis of bacteriophage t4 dna replication: a review in the virology journal series on bacteriophage t4 and its relatives.the bacteriophage t4 encodes 10 proteins, known collectively as the replisome, that are responsible for the replication of the phage genome. the replisomal proteins can be subdivided into three activities; the replicase, responsible for duplicating dna, the primosomal proteins, responsible for unwinding and okazaki fragment initiation, and the okazaki repair proteins. the replicase includes the gp43 dna polymerase, the gp45 processivity clamp, the gp44/62 clamp loader complex, and the gp32 singl ...201021129204
variation in mutation rates caused by rb69pol fidelity mutants can be rationalized on the basis of their kinetic behavior and crystal structures.we have previously observed that stepwise replacement of amino acid residues in the nascent base-pair binding pocket of rb69 dna polymerase (rb69pol) with ala or gly expanded the space in this pocket, resulting in a progressive increase in misincorporation. however, in vivo results with similar rb69pol nascent base-pair binding pocket mutants showed that mutation rates, as determined by the t4 phage ri forward assay and rii reversion assay, were significantly lower for the rb69pol s565g/y567a do ...201121216248
t4-like genome organization of the escherichia coli o157:h7 lytic phage ar1.we report the genome organization and analysis of the first completely sequenced t4-like phage ar1 of e. coli o157:h7. unlike most of the other sequenced phages of o157:h7 strain that belong to the temperate podoviridae and siphoviridae families, ar1 is a t4-like phage known to efficiently infect this pathogenic bacterial strain. the 167,435-bp ar1 genome is currently the largest among all the sequenced e. coli o157:h7 phages. it encodes a total of 281 potential orfs and ten putative trna genes. ...201121507986
phosphonoformic acid inhibits viral replication by trapping the closed form of the dna polymerase.phosphonoformic acid (pfa, foscarnet) belongs to a class of antiviral drugs which inhibit the human cytomegalovirus (hcmv) dna polymerase (ul54) by mimicking the pyrophosphate leaving group of the nucleotide transfer reaction. difficulties expressing ul54 have hampered investigation of the precise structural requirements rendering inhibition by this drug. however, a previously engineered chimeric dna polymerase, constructed by mutating the homologous polymerase from bacteriophage rb69 (gp43) to ...201121566148
hydrogen-bonding capability of a templating difluorotoluene nucleotide residue in an rb69 dna polymerase ternary complex.results obtained using 2,4-difluorotoluene nucleobase (df) as a nonpolar thymine isostere by kool and colleagues challenged the watson-crick dogma that hydrogen bonds between complementary bases are an absolute requirement for accurate dna replication. here, we report crystal structure of an rb69 dna polymerase l561a/s565g/y567a triple mutant ternary complex with a templating df opposite dttp at 1.8 Å-resolution. in this structure, direct hydrogen bonds were observed between: (i) df and the inc ...201121667997
a crystallographic study of the role of sequence context in thymine glycol bypass by a replicative dna polymerase serendipitously sheds light on the exonuclease complex.thymine glycol (tg) is the most common oxidation product of thymine and is known to be a strong block to replicative dna polymerases. a previously solved structure of the bacteriophage rb69 dna polymerase (rb69 gp43) in complex with tg in the sequence context 5'-g-tg-g sheds light on how tg blocks primer elongation: the protruding methyl group of the oxidized thymine displaces the adjacent 5'-g, which can no longer serve as a template for primer elongation [aller, p., rould, m. a., hogg, m, wall ...201121781974
structural insights into complete metal ion coordination from ternary complexes of b family rb69 dna polymerase.we have captured a preinsertion ternary complex of rb69 dna polymerase (rb69pol) containing the 3' hydroxyl group at the terminus of an extendable primer (pto3') and a nonhydrolyzable 2'-deoxyuridine 5'-α,β-substituted triphosphate, dupxpp, where x is either nh or ch(2), opposite a complementary templating da nucleotide residue. here we report four structures of these complexes formed by three different rb69pol variants with catalytically inert ca(2+) and four other structures with catalytically ...201121923197
the miscoding potential of 5-hydroxycytosine arises due to template instability in the replicative polymerase active site.5-hydroxycytosine (5-ohc) is a stable oxidation product of cytosine associated with an increased frequency of c → t transition mutations. when this lesion escapes recognition by the base excision repair pathway and persists to serve as a templating base during dna synthesis, replicative dna polymerases often misincorporate damp at the primer terminus, which can lead to fixation of mutations and subsequent disease. to characterize the dynamics of dna synthesis opposite 5-ohc, we initiated a compa ...201122026756
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