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 polymerases. The duplex product DNA orientation differs by 40 degrees between the polymerizing mode and editing mode structures. The role of the thumb in this DNA motion provides a model for editing in the pol alpha family.     

Proofreading dynamics of a processive DNA polymerase

Replicative DNA polymerases present an intrinsic proofreading activity during which the DNA primer chain is transferred between the polymerization and exonuclease sites of the protein. The dynamics of this primer transfer reaction during active polymerization remain poorly understood. Here we describe a single‐molecule mechanical method to investigate the conformational dynamics of the intramolecular DNA primer transfer during the processive replicative activity of the Φ29 DNA polymerase and two of its mutants. We find that mechanical tension applied to a single polymerase–DNA complex promotes the intramolecular transfer of the primer in a similar way to the incorporation of a mismatched nucleotide. The primer transfer is achieved through two novel intermediates, one a tension‐sensitive and functional polymerization conformation and a second non‐active state that may work as a fidelity check point for the proofreading reaction.     

Magnesium-induced assembly of a complete DNA polymerase catalytic complex

The molecular details of the nucleotidyl transferase reaction have remained speculative, as strategies to trap catalytic intermediates for structure determination utilize substrates lacking the primer terminus 3'-OH and catalytic Mg2+, resulting in an incomplete and distorted active site geometry. Since the geometric arrangement of these essential atoms will impact chemistry, structural insight into fidelity strategies has been hampered. Here, we present a crystal structure of a precatalytic complex of a DNA polymerase with bound substrates that include the primer 3'-OH and catalytic Mg2+. This catalytic intermediate was trapped with a nonhydrolyzable deoxynucleotide analog. Comparison with two new structures of DNA polymerase beta lacking the 3'-OH or catalytic Mg2+ is described. These structures provide direct evidence that both atoms are required to achieve a proper geometry necessary for an in-line nucleophilic attack of O3' on the alphaP of the incoming nucleotide.     

Dissociation and reconstitution of a DNA polymerase alpha-primase complex

The conditions for dissociation of the DNA polymerase alpha-primase complex (DNA polymerase alpha 1) have been examined. It was revealed that 50% ethylene glycol effectively dissociated the complex. The dissociated DNA polymerase and primase were purified to eliminate cross-contaminating activities by column chromatography using buffers containing 50% ethylene glycol. The sedimentation coefficients of the purified DNA polymerase and primase were 7.1S and 5.7S, respectively. These two enzymes were mixed in the presence of 20% ethylene glycol and the mixture was sedimented through a glycerol gradient containing no ethylene glycol. The DNA polymerase and primase activities co-sedimented at 9.1S which corresponds to the S value of intact alpha 1, indicating the reconstitution of the DNA polymerase alpha-primase complex.     

DNA polymerase alpha is associated with replicating SV40 nucleoprotein complexes.

Simian virus 40 (SV40) nucleoprotein complexes were extracted from nuclei of infected monkey cells and fractionated on neutral sucrose density gradients. Complexes which contained replicating SV40 DNA (95S) separated well from those containing closed circular supercoiled viral DNA (75S). DNA polymerase activity was associated with the replicating nucleoprotein complexes but not with the slower sedimenting complexes. This DNA polymerase activity coprecipitated with the nucleoprotein complexes in the presence of MgCl2 and remained associated with the 95S complexes. This DNA polymerase activity has been identified as primarily DNA polymerase alpha on the basis of its sedimentation behavior, optimum salt concentration, and sensitivity to N-ethylmaleimide. DNA polymerase gamma activity was also detected in the complexes, but DNA polymerase beta was not associated with the complexes.     

Conformational dynamics of DNA polymerase probed with a novel fluorescent DNA base analogue

DNA polymerases discriminate between correct and incorrect nucleotide substrates during a "nonchemical" step that precedes phosphodiester bond formation in the enzymatic cycle of nucleotide incorporation. Despite the importance of this process in polymerase fidelity, the precise nature of the molecular events involved remains unknown. Here we report a fluorescence resonance energy transfer (FRET) system that monitors conformational changes of a polymerase-DNA complex during selection and binding of nucleotide substrates. This system utilizes the fluorescent base analogue 1,3-diaza-2-oxophenothiazine (tC) as the FRET donor and Alexa-555 (A555) as the acceptor. The tC donor was incorporated within a model DNA primer/template in place of a normal base, adjacent to the primer 3' terminus, while the A555 acceptor was attached to an engineered cysteine residue (C751) located in the fingers subdomain of the Klenow fragment (KF) polymerase. The FRET efficiency increased significantly following binding of a correct nucleotide substrate to the KF-DNA complex, showing that the fingers had closed over the active site. Fluorescence anisotropy titrations utilizing tC as a reporter indicated that the DNA was more tightly bound by the polymerase under these conditions, consistent with the formation of a closed ternary complex. The rate of the nucleotide-induced conformational transition, measured in stopped-flow FRET experiments, closely matched the rate of correct nucleotide incorporation, measured in rapid quench-flow experiments, indicating that the conformational change was the rate-limiting step in the overall cycle of nucleotide incorporation for the labeled KF-DNA system. Taken together, these results indicate that the FRET system can be used to probe enzyme conformational changes that are linked to the biochemical function of DNA polymerase.     

Structure of a preternary complex involving a prokaryotic NHEJ DNA polymerase

In many prokaryotes, a specific DNA primase/polymerase (PolDom) is required for nonhomologous end joining (NHEJ) repair of DNA double-strand breaks (DSBs). Here, we report the crystal structure of a catalytically active conformation of Mycobacterium tuberculosis PolDom, consisting of a polymerase bound to a DNA end with a 3' overhang, two metal ions, and an incoming nucleotide but, significantly, lacking a primer strand. This structure represents a polymerase:DNA complex in a preternary intermediate state. This polymerase complex occurs in solution, stabilizing the enzyme on DNA ends and promoting nucleotide extension of short incoming termini. We also demonstrate that the invariant Arg(220), contained in a conserved loop (loop 2), plays an essential role in catalysis by regulating binding of a second metal ion in the active site. We propose that this NHEJ intermediate facilitates extension reactions involving critically short or noncomplementary DNA ends, thus promoting break repair and minimizing sequence loss during DSB repair.     

DNA polymerase I and DNA primase complex in yeast

Chromatographic analysis of poly(dT) replication activity in fresh yeast extracts showed that the activities required co-fractionate with the yeast DNA polymerase I. Since poly(dT) replication requires both a primase and a DNA polymerase, the results of the fractionation studies suggest that these two enzymes might exist as a complex in the yeast extract. Sucrose gradient analysis of concentrated purified yeast DNA polymerase I preparations demonstrates that the yeast DNA polymerase I does sediment as a complex with DNA primase activity. Two DNA polymerase I peptides estimated at 78,000 and 140,000 Da were found in the complex that were absent from the primase-free DNA polymerase fraction. Rabbit anti-yeast DNA polymerase I antibody inhibits DNA polymerase I but not DNA primase although rabbit antibodies are shown to remove DNA primase activity from solution by binding to the complex. Mouse monoclonal antibody to yeast DNA polymerase I binds to free yeast DNA polymerase I as well as the complex, but not to the free DNA primase activity. These results suggest that these two activities exist as a complex and reside on the different polypeptides. Replication of poly(dT) and single-stranded circular phage DNA by yeast DNA polymerase I and primase requires ATP and dNTPs. The size of the primer produced is 8 to 9 nucleotides in the presence of dNTPs and somewhat larger in the absence of dNTPs. Aphidicolin, an inhibitor of yeast DNA polymerase I, is not inhibitory to the yeast DNA primase activity. The primase activity is inhibited by adenosine 5'-(3-thio)tri-phosphate but not by alpha-amanitin. The association of yeast DNA polymerase I and yeast DNA primase can be demonstrated directly by isolation of the complex on a column containing yeast DNA polymerase I mouse monoclonal antibody covalently linked to Protein A-Sepharose. Both DNA polymerase I and DNA primase activities are retained by the column and can be eluted with 3.5 M MgCl2. Part of the primase activity can be dissociated from DNA polymerase on the column with 1 M MgCl2 and this free primase activity can be detected as poly(dT) replication activity in the presence of Escherichia coli polymerase I.     

Purification and characterization of two forms of DNA polymerase alpha from mouse FM3A cells: a DNA polymerase alpha-primase complex and a free DNA polymerase alpha

Two forms of DNA polymerase alpha, alpha 1 and alpha 2, have been partially purified from mouse FM3A cells by discriminating one form from the other on the basis of the association of primase activity. The primase activity in the most purified alpha 1 fraction co-sedimented with the DNA polymerase activity in a glycerol gradient, and almost no primase activity was detected in the most purified alpha 2 fraction. The primase activity associated with DNA polymerase alpha was assayed indirectly by measuring ATP-dependent DNA synthesis with poly (dT) as template. Characterization of the assay system was performed with the purified alpha 1. The system was absolutely dependent on the presence of ATP and a divalent cation. Mn2+ was much more effective than Mg2+, and 5-fold higher activity was observed with Mn2+ than with Mg2+ at their optimal concentrations. The primase activity assayed by the above system showed sensitivity to (NH4)2SO4 very similar to that of free primase reported by Tseng and Ahlem (J. Biol. Chem. 258, 9845-9849, 1983). The activity was inhibited by more than 50% by 20 mM (NH4)2SO4. alpha 1 and alpha 2 were very similar as DNA polymerases in their sensitivity to several inhibitors and their preference for template-primers, except that alpha 1 had a slightly greater preference for poly (dT) X (rA)10 than alpha 2 did. The major difference between the two forms was observed in their S values, 8.2 and 6.4 S for alpha 1 and alpha 2, respectively.     

In vitro DNA synthesis by an alpha-like DNA polymerase bound to replicating simian virus 40 chromosomes.

Simian virus 40 chromosomes carry out replicative DNA synthesis in vitro which is sensitive to aphidicolin and to N-ethylmaleimide, resistant to 2',3'-dideoxythymidine-5'-triphosphate, and proportional to the amount of chromosome-associated alpha-like polymerase. Thus, an alpha-like DNA polymerase (alpha polymerase or delta polymerase) is responsible for in vitro DNA synthesis.     

DNA polymerase alpha--primase complex of Physarum polycephalum.

DNA polymerase alpha and DNA polymerase alpha--primase complex of Physarum polycephalum were purified by rapid methods, and antibodies were raised against the complex. In crude extracts, immune-reactive polypeptides of 220 kDa, 180 kDa, 150 kDa, 140 kDa, 110 kDa, 86 kDa, 57 kDa and 52 kDa were identified. The structural relationships between the 220 kDa, 110 kDa and 140 kDa (the most abundant form) was investigated by peptide mapping. The 140 kDa form was active DNA polymerase alpha. The 57 kDa and the 52 kDa polypeptides were identified as primase subunits by auto-catalytic labelling. In amoebae, the immune-reactive 140 kDa polypeptide was replaced by a 135 kDa active DNA polymerase alpha.     

Movement of replicating DNA through a stationary replisome

We found that DNA is replicated at a central stationary polymerase, and each replicated region moves away from the replisome. In Bacillus subtilis, DNA polymerase is predominantly located at or near midcell. When replication was blocked in a specific chromosomal region, that region was centrally located with DNA polymerase. Upon release of the block, each copy of the duplicated region was located toward opposite cell poles, away from the central replisome. In a roughly synchronous population of cells, a region of chromosome between origin and terminus moved to the replisome prior to duplication. Thus, the polymerase at the replication forks is stationary, and the template is pulled in and released outward during duplication. We propose that B. subtilis, and probably many bacteria, harness energy released during nucleotide condensation by a stationary replisome to facilitate chromosome partitioning.     

The complex of DNA gyrase and quinolone drugs on DNA forms a barrier to the T7 DNA polymerase replication complex

Quinolone drugs can inhibit bacterial DNA replication, via interaction with the type II topoisomerase DNA gyrase. Using a DNA template containing a preferred site for quinolone-induced gyrase cleavage, we have demonstrated that the passage of the bacteriophage T7 replication complex is blocked in vitro by the formation of a gyrase-drug-DNA complex. The majority of the polymerase is arrested approximately 10 bp upstream of this preferred site, although other minor sites of blocking have been observed. The ability of mutant gyrase proteins to arrest DNA replication in vitro has been investigated. Gyrase containing mutations in the A subunit at either the active-site tyrosine (Tyr122) or Ser83 (a residue known to be involved in quinolone interaction) failed to halt the progress of the polymerase. A low-level, quinolone-resistant mutation in the B subunit of gyrase showed reduced blocking compared to wild-type. We have demonstrated that DNA cleavage and replication blocking occur on similar time-scales and we conclude that formation of the cleavable complex is a prerequisite for polymerase blocking. Additionally, we have shown that collision of the replication proteins with the gyrase-drug-DNA complex is not sufficient to render this complex irreversible and that further factors must be involved in processing this stalled complex.     

Affinity labeling the DNA polymerase alpha complex. I. Pyridoxal 5'-phosphate inhibition of DNA polymerase and DNA primase activities of the DNA polymerase alpha complex from Drosophila melanogaster embryos

DNA polymerase alpha from Drosophila melanogaster embryos is a multisubunit enzyme complex which can exhibit DNA polymerase, 3'----5' exonuclease, and DNA primase activities. Pyridoxal 5'-phosphate (PLP) inhibition of DNA polymerase activity in this complex is time dependent and exhibits saturation kinetics. Inhibition can be reversed by incubation with an excess of a primary amine unless the PLP-enzyme conjugate is first reduced with NaBH4. These results indicate that PLP inhibition occurs via imine formation at a specific site(s) on the enzyme. Results from substrate protection experiments are most consistent with inhibition of DNA polymerase activity by PLP binding to either one of two sites. One site (PLP site 1) can be protected from PLP inhibition by any nucleoside triphosphate in the absence or presence of template-primer, suggesting that PLP site 1 defines a nucleotide-binding site which is important for DNA polymerase activity but which is distinct from the DNA polymerase active site. PLP also inhibits DNA primase activity of the DNA polymerase alpha complex, and primase activity can be protected from PLP inhibition by nucleotide alone, arguing that PLP site 1 lies within the DNA primase active site. The second inhibitory PLP-binding site (PLP site 2) is only protected from PLP inhibition when the enzyme is bound to both template-primer and correct dNTP in a stable ternary complex. Since binding of PLP at site 2 is mutually exclusive with template-directed dNTP binding at the DNA polymerase active site, PLP site 2 appears to define the dNTP binding domain of the active site. Results from initial velocity analysis of PLP inhibition argue that there is a rate-limiting step in the polymerization cycle during product release and/or translocation.     

Fidelity of DNA polymerase I and the DNA polymerase I-DNA primase complex from Saccharomyces cerevisiae.

We have determined the fidelity of DNA synthesis by DNA polymerase I (yPol I) from Saccharomyces cerevisiae. To determine whether subunits other than the polymerase catalytic subunit influence fidelity, we measured the accuracy of yPol I purified by conventional procedures, which yields DNA polymerase with a partially proteolyzed catalytic subunit and no associated primase activity, and that of yPol I purified by immunoaffinity chromatography, which yields polymerase having a single high-molecular-weight species of the catalytic subunit, as well as three additional polypeptides and DNA primase activity. In assays that score polymerase errors within the lacZ alpha-complementation gene in M13mp2 DNA, yPol I and the yPol I-primase complex produced single-base substitutions, single-base frameshifts, and larger deletions. For specific errors and template positions, the two forms of polymerase exhibited differences in fidelity that could be as large as 10-fold. Nevertheless, results for the overall error frequency and the spectrum of errors suggest that the yPol I-DNA primase complex is not highly accurate and that, just as for the polymerase alone, its fidelity is not sufficient to account for a low spontaneous mutation rate in vivo. The specificity data also suggest models to explain -1 base frameshifts in nonrepeated sequences and certain complex deletions by a direct repeat mechanism involving aberrant loop-back synthesis.     

Accessory proteins assist exonuclease-deficient bacteriophage T4 DNA polymerase in replicating past an abasic site

Replicative DNA polymerases, such as T4 polymerase, possess both elongation and 3'-5' exonuclease proofreading catalytic activities. They arrest at the base preceding DNA damage on the coding DNA strand and specialized DNA polymerases have evolved to replicate across the lesion by a process known as TLS (translesion DNA synthesis). TLS is considered to take place in two steps that often require different enzymes, insertion of a nucleotide opposite the damaged template base followed by extension from the inserted nucleotide. We and others have observed that inactivation of the 3'-5' exonuclease function of T4 polymerase enables TLS across a single site-specific abasic [AP (apurinic/apyrimidinic)] lesion. In the present study we report a role for auxiliary replicative factors in this reaction. When replication is performed with a large excess of DNA template over DNA polymerase in the absence of auxiliary factors, the exo- polymerase (T4 DNA polymerase deficient in the 3'-5' exonuclease activity) inserts one nucleotide opposite the AP site but does not extend past the lesion. Addition of the clamp processivity factor and the clamp loader complex restores primer extension across an AP lesion on a circular AP-containing DNA substrate by the exo- polymerase, but has no effect on the wild-type enzyme. Hence T4 DNA polymerase exhibits a variety of responses to DNA damage. It can behave as a replicative polymerase or (in the absence of proofreading activity) as a specialized DNA polymerase and carry out TLS. As a specialized polymerase it can function either as an inserter or (with the help of accessory proteins) as an extender. The capacity to separate these distinct functions in a single DNA polymerase provides insight into the biochemical requirements for translesion DNA synthesis.