Primer-template DNA Research Articles

Using microsecond single-molecule FRET to determine the assembly pathways of T4 ssDNA binding protein onto model DNA replication forks

DNA replication is a core biological process that occurs in prokaryotic cells at high speeds (∼1 nucleotide residue added per millisecond) and with high fidelity (fewer than one misincorporation event per 107 nucleotide additions). The ssDNA binding protein [gene product 32 (gp32)] of the T4 bacteriophage is a central integrating component of the replication complex that must continuously bind to and unbind from transiently exposed template strands during DNA synthesis. We here report microsecond single-molecule FRET (smFRET) measurements on Cy3/Cy5-labeled primer-template (p/t) DNA constructs in the presence of gp32. These measurements probe the distance between Cy3/Cy5 fluorophores that label the ends of a short (15-nt) segment of ssDNA attached to a model p/t DNA construct and permit us to track the stochastic interconversion between various protein bound and unbound states. The length of the 15-nt ssDNA lattice is sufficient to accommodate up to two cooperatively bound gp32 proteins in either of two positions. We apply a unique multipoint time correlation function analysis to the microsecond-resolved smFRET data obtained to determine and compare the kinetics of various possible reaction pathways for the assembly of cooperatively bound gp32 protein onto ssDNA sequences located at the replication fork. The results of our analysis reveal the presence and translocation mechanisms of short-lived intermediate bound states that are likely to play a critical role in the assembly mechanisms of ssDNA binding proteins at replication forks and other ss duplex junctions.

Bypass of DNA-Protein Cross-links Conjugated to the 7-Deazaguanine Position of DNA by Translesion Synthesis Polymerases

DNA-protein cross-links (DPCs) are bulky DNA lesions that form both endogenously and following exposure to bis-electrophiles such as common antitumor agents. The structural and biological consequences of DPCs have not been fully elucidated due to the complexity of these adducts. The most common site of DPC formation in DNA following treatment with bis-electrophiles such as nitrogen mustards and cisplatin is the N7 position of guanine, but the resulting conjugates are hydrolytically labile and thus are not suitable for structural and biological studies. In this report, hydrolytically stable structural mimics of N7-guanine-conjugated DPCs were generated by reductive amination reactions between the Lys and Arg side chains of proteins/peptides and aldehyde groups linked to 7-deazaguanine residues in DNA. These model DPCs were subjected to in vitro replication in the presence of human translesion synthesis DNA polymerases. DPCs containing full-length proteins (11-28 kDa) or a 23-mer peptide blocked human polymerases η and κ. DPC conjugates to a 10-mer peptide were bypassed with nucleotide insertion efficiency 50-100-fold lower than for native G. Both human polymerase (hPol) κ and hPol η inserted the correct base (C) opposite the 10-mer peptide cross-link, although small amounts of T were added by hPol η. Molecular dynamics simulation of an hPol κ ternary complex containing a template-primer DNA with dCTP opposite the 10-mer peptide DPC revealed that this bulky lesion can be accommodated in the polymerase active site by aligning with the major groove of the adducted DNA within the ternary complex of polymerase and dCTP.

Conformational States of HIV-1 Reverse Transcriptase for Nucleotide Incorporation vs Pyrophosphorolysis-Binding of Foscarnet.

HIV-1 reverse transcriptase (RT) catalytically incorporates individual nucleotides into a viral DNA strand complementing an RNA or DNA template strand; the polymerase active site of RT adopts multiple conformational and structural states while performing this task. The states associated are dNTP binding at the N site, catalytic incorporation of a nucleotide, release of a pyrophosphate, and translocation of the primer 3'-end to the P site. Structural characterization of each of these states may help in understanding the molecular mechanisms of drug activity and resistance and in developing new RT inhibitors. Using a 38-mer DNA template-primer aptamer as the substrate mimic, we crystallized an RT/dsDNA complex that is catalytically active, yet translocation-incompetent in crystals. The ability of RT to perform dNTP binding and incorporation in crystals permitted obtaining a series of structures: (I) RT/DNA (P-site), (II) RT/DNA/AZTTP ternary, (III) RT/AZT-terminated DNA (N-site), and (IV) RT/AZT-terminated DNA (N-site)/foscarnet complexes. The stable N-site complex permitted the binding of foscarnet as a pyrophosphate mimic. The Mg(2+) ions dissociated after catalytic addition of AZTMP in the pretranslocated structure III, whereas ions A and B had re-entered the active site to bind foscarnet in structure IV. The binding of foscarnet involves chelation with the Mg(2+) (B) ion and interactions with K65 and R72. The analysis of interactions of foscarnet and the recently discovered nucleotide-competing RT inhibitor (NcRTI) α-T-CNP in two different conformational states of the enzyme provides insights for developing new classes of polymerase active site RT inhibitors.

Secondary Interaction Interfaces with PCNA Control Conformational Switching of DNA Polymerase PolB from Polymerization to Editing.

Replicative DNA polymerases (Pols) frequently possess two distinct DNA processing activities: DNA synthesis (polymerization) and proofreading (3'-5' exonuclease activity). The polymerase and exonuclease reactions are performed alternately and are spatially separated in different protein domains. Thus, the growing DNA primer terminus has to undergo dynamic conformational switching between two distinct functional sites on the polymerase. Furthermore, the transition from polymerization (pol) mode to exonuclease (exo) mode must occur in the context of a DNA Pol holoenzyme, wherein the polymerase is physically associated with processivity factor proliferating cell nuclear antigen (PCNA) and primer-template DNA. The mechanism of this conformational switching and the role that PCNA plays in it have remained obscure, largely due to the dynamic nature of ternary Pol/PCNA/DNA assemblies. Here, we present computational models of ternary assemblies for archaeal polymerase PolB. We have combined all available structural information for the binary complexes with electron microscopy data and have refined atomistic models for ternary PolB/PCNA/DNA assemblies in pol and exo modes using molecular dynamics simulations. In addition to the canonical PIP-box/interdomain connector loop (IDCL) interface of PolB with PCNA, contact analysis of the simulation trajectories revealed new secondary binding interfaces, distinct between the pol and exo states. Using targeted molecular dynamics, we explored the conformational transition from pol to exo mode. We identified a hinge region between the thumb and palm domains of PolB that is critical for conformational switching. With the thumb domain anchored onto the PCNA surface, the neighboring palm domain executed rotational motion around the hinge, bringing the core of PolB down toward PCNA to form a new interface with the clamp. A helix from PolB containing a patch of arginine residues was involved in the binding, locking the complex in the exo mode conformation. Together, these results provide a structural view of how the transition between the pol and exo states of PolB is coordinated through PCNA to achieve efficient proofreading.

Interaction between the Rev1 C-Terminal Domain and the PolD3 Subunit of Polζ Suggests a Mechanism of Polymerase Exchange upon Rev1/Polζ-Dependent Translesion Synthesis.

Translesion synthesis (TLS) is a mutagenic branch of cellular DNA damage tolerance that enables bypass replication over DNA lesions carried out by specialized low-fidelity DNA polymerases. The replicative bypass of most types of DNA damage is performed in a two-step process of Rev1/Polζ-dependent TLS. In the first step, a Y-family TLS enzyme, typically Polη, Polι, or Polκ, inserts a nucleotide across a DNA lesion. In the second step, a four-subunit B-family DNA polymerase Polζ (Rev3/Rev7/PolD2/PolD3 complex) extends the distorted DNA primer-template. The coordinated action of error-prone TLS enzymes is regulated through their interactions with the two scaffold proteins, the sliding clamp PCNA and the TLS polymerase Rev1. Rev1 interactions with all other TLS enzymes are mediated by its C-terminal domain (Rev1-CT), which can simultaneously bind the Rev7 subunit of Polζ and Rev1-interacting regions (RIRs) from Polη, Polι, or Polκ. In this work, we identified a previously unknown RIR motif in the C-terminal part of PolD3 subunit of Polζ whose interaction with the Rev1-CT is among the tightest mediated by RIR motifs. Three-dimensional structure of the Rev1-CT/PolD3-RIR complex determined by NMR spectroscopy revealed a structural basis for the relatively high affinity of this interaction. The unexpected discovery of PolD3-RIR motif suggests a mechanism of "inserter" to "extender" DNA polymerase switch upon Rev1/Polζ-dependent TLS, in which the PolD3-RIR binding to the Rev1-CT (i) helps displace the "inserter" Polη, Polι, or Polκ from its complex with Rev1, and (ii) facilitates assembly of the four-subunit "extender" Polζ through simultaneous interaction of Rev1-CT with Rev7 and PolD3 subunits.

Structural Insights into the Processing of Nucleobase-Modified Nucleotides by DNA Polymerases.

The DNA polymerase-catalyzed incorporation of modified nucleotides is employed in many biological technologies of prime importance, such as next-generation sequencing, nucleic acid-based diagnostics, transcription analysis, and aptamer selection by systematic enrichment of ligands by exponential amplification (SELEX). Recent studies have shown that 2'-deoxynucleoside triphosphates (dNTPs) that are functionalized with modifications at the nucleobase such as dyes, affinity tags, spin and redox labels, or even oligonucleotides are substrates for DNA polymerases, even if modifications of high steric demand are used. The position at which the modification is introduced in the nucleotide has been identified as crucial for retaining substrate activity for DNA polymerases. Modifications are usually attached at the C5 position of pyrimidines and the C7 position of 7-deazapurines. Furthermore, it has been shown that the nature of the modification may impact the efficiency of incorporation of a modified nucleotide into the nascent DNA strand by a DNA polymerase. This Account places functional data obtained in studies of the incorporation of modified nucleotides by DNA polymerases in the context of recently obtained structural data. Crystal structure analysis of a Thermus aquaticus (Taq) DNA polymerase variant (namely, KlenTaq DNA polymerase) in ternary complex with primer-template DNA and several modified nucleotides provided the first structural insights into how nucleobase-modified triphosphates are tolerated. We found that bulky modifications are processed by KlenTaq DNA polymerase as a result of cavities in the protein that enable the modification to extend outside the active site. In addition, we found that the enzyme is able to adapt to different modifications in a flexible manner and adopts different amino acid side-chain conformations at the active site depending on the nature of the nucleotide modification. Different "strategies" (i.e., hydrogen bonding, cation-π interactions) enable the protein to stabilize the respective protein-substrate complex without significantly changing the overall structure of the complex. Interestingly, it was also discovered that a modified nucleotide may be more efficiently processed by KlenTaq DNA polymerase when the 3'-primer terminus is also a modified nucleotide instead of a nonmodified natural one. Indeed, the modifications of two modified nucleotides at adjacent positions can interact with each other (i.e., by π-π interactions) and thereby stabilize the enzyme-substrate complex, resulting in more efficient transformation. Several studies have indicated that archeal DNA polymerases belonging to sequence family B are better suited for the incorporation of nucleobase-modified nucleotides than enzymes from family A. However, significantly less structural data are available for family B DNA polymerases. In order to gain insights into the preference for modified substrates by members of family B, we succeeded in obtaining binary structures of 9°N and KOD DNA polymerases bound to primer-template DNA. We found that the major groove of the archeal family B DNA polymerases is better accessible than in family A DNA polymerases. This might explain the observed superiority of family B DNA polymerases in polymerizing nucleotides that bear bulky modifications located in the major groove, such as modification at C5 of pyrimidines and C7 of 7-deazapurines. Overall, this Account summarizes our recent findings providing structural insight into the mechanism by which modified nucleotides are processed by DNA polymerases. It provides guidelines for the design of modified nucleotides, thus supporting future efforts based on the acceptance of modified nucleotides by DNA polymerases.

Measurement of DNA Polymerase Incorporation Kinetics of Dye-Labeled Nucleotides Using Total Internal Reflection Fluorescence Microscopy.

We report a method for the rapid and automated measurements of the incorporation kinetics of fluorescent dye-labeled nucleotides by DNA polymerases without using stopped-flow and quench-flow methods. Total internal reflection fluorescence microscopy is used to monitor the incorporation of fluorescently labeled nucleotides by DNA polymerase into surface-bound primed DNA templates, and a microfluidic system is used to perform the reactions. We successfully demonstrated the method using Bst DNA polymerase and a set of coumarin-labeled nucleotides. Our method allows the rapid acquisition of polymerase kinetics for implementing and improving DNA sequencing technologies that rely on labeled nucleotides and DNA polymerases.

11 Checkpoints controlled by PCNA, DNA and ATP direct the timing and order of events in the clamp loading mechanism

PCNA (Proliferating Cell Nuclear Antigen) clamp is an essential ring shaped protein that is loaded around the primer-template (p/t) DNA junction to serve as a mobile tether for DNA polymerase and e...

Creating cellular diversity through transcription factor competition.

The development of blood cells has long served as a model system to study the generation of diverse mature cells from multipotent progenitors. The article by Org et al (2015) reveals how transcription factor competition on primed DNA templates may contribute to embryonic blood cell specification during the early stages of mesoderm development. The study not only provides new insights into the functionality of the key haematopoietic transcription factor Scl/Tal1, but also provides a potentially widely applicable framework for transcription factor‐mediated cell fate specification.

Dynamic Interactions between DNA and the T4 Single-Stranded Binding Protein gp32: Multi-Dimensional Correlation Analysis of Microsecond Single-Molecule FRET and Linear Dichroism Fluctuations

Protein-nucleic acid interactions are of central importance in genome-regulatory processes. The DNA replication system of the T4 bacteriophage is an excellent model system to study DNA replication in higher organisms, since the T4-coded replication machinery utilizes many of the same components. Here we present studies of the kinetics of binding of gp32, the single-stranded (ss) DNA binding protein of the T4 system, whose roles include the functional integration of the other components of the T4 replication complex as well as protecting the transiently-exposed single-stranded DNA template sequences from DNA nucleases and melting out unfavorable secondary structures potentially formed by the lagging strand during DNA replication. We have performed single-molecule measurements of internally-labelled Cy3/Cy5 labeled primer-template DNA constructs in the presence of gp32 by simultaneously monitoring single-molecule Forster resonance energy transfer (smFRET) and single-molecule fluorescence-detected linear-dichroism (smFLD) on the microsecond time scale. smFRET measurements probe the distance between the fluorophores, while smFLD measurements are sensitive to local orientations of the Cy3-labeled sugar-phosphate backbones of the DNA construct. Multiple transient FRET states are observed when adding gp32 protein to tethered model replication fork DNA constructs, permitting us to track the dynamics of millisecond interconversions between various configurations of the gp32 / ssDNA system. The distribution of FRET states changes as a function of gp32 concentration, suggesting that we may be monitoring fluctuations that reflect changes associated with the progression from isolated to cooperative gp32 binding. We apply a multi-dimensional correlation function analysis to our microsecond-resolved smFRET and smLD data to reveal statistically relevant mechanistic information about the reaction pathways of the ssDNA-gp32 system at the replication fork.

The interplay of primer-template DNA phosphorylation status and single-stranded DNA binding proteins in directing clamp loaders to the appropriate polarity of DNA.

Sliding clamps are loaded onto DNA by clamp loaders to serve the critical role of coordinating various enzymes on DNA. Clamp loaders must quickly and efficiently load clamps at primer/template (p/t) junctions containing a duplex region with a free 3′OH (3′DNA), but it is unclear how clamp loaders target these sites. To measure the Escherichia coli and Saccharomyces cerevisiae clamp loader specificity toward 3′DNA, fluorescent β and PCNA clamps were used to measure clamp closing triggered by DNA substrates of differing polarity, testing the role of both the 5′phosphate (5′P) and the presence of single-stranded binding proteins (SSBs). SSBs inhibit clamp loading by both clamp loaders on the incorrect polarity of DNA (5′DNA). The 5′P groups contribute selectivity to differing degrees for the two clamp loaders, suggesting variations in the mechanism by which clamp loaders target 3′DNA. Interestingly, the χ subunit of the E. coli clamp loader is not required for SSB to inhibit clamp loading on phosphorylated 5′DNA, showing that χ·SSB interactions are dispensable. These studies highlight a common role for SSBs in directing clamp loaders to 3′DNA, as well as uncover nuances in the mechanisms by which SSBs perform this vital role.

Internally labeled Cy3/Cy5 DNA constructs show greatly enhanced photo-stability in single-molecule FRET experiments

DNA constructs labeled with cyanine fluorescent dyes are important substrates for single-molecule (sm) studies of the functional activity of protein–DNA complexes. We previously studied the local DNA backbone fluctuations of replication fork and primer–template DNA constructs labeled with Cy3/Cy5 donor–acceptor Förster resonance energy transfer (FRET) chromophore pairs and showed that, contrary to dyes linked ‘externally’ to the bases with flexible tethers, direct ‘internal’ (and rigid) insertion of the chromophores into the sugar-phosphate backbones resulted in DNA constructs that could be used to study intrinsic and protein-induced DNA backbone fluctuations by both smFRET and sm Fluorescent Linear Dichroism (smFLD). Here we show that these rigidly inserted Cy3/Cy5 chromophores also exhibit two additional useful properties, showing both high photo-stability and minimal effects on the local thermodynamic stability of the DNA constructs. The increased photo-stability of the internal labels significantly reduces the proportion of false positive smFRET conversion ‘background’ signals, thereby simplifying interpretations of both smFRET and smFLD experiments, while the decreased effects of the internal probes on local thermodynamic stability also make fluctuations sensed by these probes more representative of the unperturbed DNA structure. We suggest that internal probe labeling may be useful in studies of many DNA–protein interaction systems.

Role of the LEXE Motif of Protein-primed DNA Polymerases in the Interaction with the Incoming Nucleotide

The LEXE motif, conserved in eukaryotic type DNA polymerases, is placed close to the polymerization active site. Previous studies suggested that the second Glu was involved in binding a third noncatalytic ion in bacteriophage RB69 DNA polymerase. In the protein-primed DNA polymerase subgroup, the LEXE motif lacks the first Glu in most cases, but it has a conserved Phe/Trp and a Gly preceding that position. To ascertain the role of those residues, we have analyzed the behavior of mutants at the corresponding ϕ29 DNA polymerase residues Gly-481, Trp-483, Ala-484, and Glu-486. We show that mutations at Gly-481 and Trp-483 hamper insertion of the incoming dNTP in the presence of Mg(2+) ions, a reaction highly improved when Mn(2+) was used as metal activator. These results, together with previous crystallographic resolution of ϕ29 DNA polymerase ternary complex, allow us to infer that Gly-481 and Trp-483 could form a pocket that orients Val-250 to interact with the dNTP. Mutants at Glu-486 are also defective in polymerization and, as mutants at Gly-481 and Trp-483, in the pyrophosphorolytic activity with Mg(2+). Recovery of both reactions with Mn(2+) supports a role for Glu-486 in the interaction with the pyrophosphate moiety of the dNTP.

Formosusin A, a novel specific inhibitor of mammalian DNA polymerase β from the fungus Paecilomyces formosus

Variotin (1) and three novel compounds, formosusin A (2), B (3), and C (4), were isolated from the cultures of the fungus Paecilomyces formosus, and their structures were determined by spectroscopic analyses. Compound 2 is (6Z,8E,10E)-variotin, a new cis-olefin analog of compound 1. Compound 2 selectively inhibited the activity of mammalian DNA polymerase β (pol β) in vitro, with an IC50 of 35.6μM. By contrast, compounds 1, 3, and 4 did not influence the activity of pol β. These four compounds showed no effect on the activities of other 10 mammalian pols (i.e., pols α, γ, δ, ε, η, ι, κ, λ, and μ, and terminal deoxynucleotidyl transferase). These compounds also did not inhibit the activities of fish, insect, plant, and prokaryotic pols and other DNA metabolic enzymes tested. These results suggested that compound 2 could be a selective inhibitor of mammalian pol β. The compound 2-induced inhibition of rat pol β activity was competitive and non-competitive with respect to the DNA template–primer substrate and the dNTP substrate, respectively. On the basis of these results, the relationship between the three-dimensional structure and pol β inhibitory mechanism of compound 2 is discussed.

Measuring and Modeling the Kinetics of Individual DNA–DNA Polymerase Complexes on a Nanopore

The assembly of a DNA-DNA polymerase binary complex is the precursory step in genome replication, in which the enzyme binds to the 3' junction created when a primer binds to its complementary substrate. In this study, we use an active control method for observing the binding interaction between Klenow fragment (exo-) (KF) in the bulk-phase chamber above an α-hemolysin (α-HL) nanopore and a single DNA molecule tethered noncovalently in the nanopore. Specifically, the control method regulates the temporal availability of the primer-template DNA to KF binding and unbinding above the nanopore, on millisecond-to-second time scales. Our nanopore measurements support a model that incorporates two mutually exclusive binding states of KF to DNA at the primer-template junction site, termed "weakly bound" and "strongly bound" states. The composite binding affinity constant, the equilibrium constant between the weak and strong states, and the unbound-to-strong association rate are quantified from the data using derived modeling analysis. The results support that the strong state is in the nucleotide incorporation pathway, consistent with other nanopore assays. Surprisingly, the measured unbound-to-strong association process does not fit a model that admits binding of only free (unbound) KF to the tethered DNA but does fit an association rate that is proportional to the total (unbound and DNA-bound) KF concentration in the chamber above the nanopore. Our method provides a tool for measuring pre-equilibrium kinetics one molecule at a time, serially and for tens of thousands of single-molecule events, and can be used for other polynucleotide-binding enzymes.

Proofreading exonuclease on a tether: the complex between the E. coli DNA polymerase III subunits α, ε, θ and β reveals a highly flexible arrangement of the proofreading domain

A complex of the three (αεθ) core subunits and the β2 sliding clamp is responsible for DNA synthesis by Pol III, the Escherichia coli chromosomal DNA replicase. The 1.7 Å crystal structure of a complex between the PHP domain of α (polymerase) and the C-terminal segment of ε (proofreading exonuclease) subunits shows that ε is attached to α at a site far from the polymerase active site. Both α and ε contain clamp-binding motifs (CBMs) that interact simultaneously with β2 in the polymerization mode of DNA replication by Pol III. Strengthening of both CBMs enables isolation of stable αεθ:β2 complexes. Nuclear magnetic resonance experiments with reconstituted αεθ:β2 demonstrate retention of high mobility of a segment of 22 residues in the linker that connects the exonuclease domain of ε with its α-binding segment. In spite of this, small-angle X-ray scattering data show that the isolated complex with strengthened CBMs has a compact, but still flexible, structure. Photo-crosslinking with p-benzoyl-L-phenylalanine incorporated at different sites in the α-PHP domain confirm the conformational variability of the tether. Structural models of the αεθ:β2 replicase complex with primer-template DNA combine all available structural data.

Single-molecule Investigation of Substrate Binding Kinetics and Protein Conformational Dynamics of a B-family Replicative DNA Polymerase

Replicative DNA polymerases use a complex, multistep mechanism for efficient and accurate DNA replication as uncovered by intense kinetic and structural studies. Recently, single-molecule fluorescence spectroscopy has provided new insights into real time conformational dynamics utilized by DNA polymerases during substrate binding and nucleotide incorporation. We have used single-molecule Förster resonance energy transfer techniques to investigate the kinetics and conformational dynamics of Sulfolobus solfataricus DNA polymerase B1 (PolB1) during DNA and nucleotide binding. Our experiments demonstrate that this replicative polymerase can bind to DNA in at least three conformations, corresponding to an open and closed conformation of the finger domain as well as a conformation with the DNA substrate bound to the exonuclease active site of PolB1. Additionally, our results show that PolB1 can transition between these conformations without dissociating from a primer-template DNA substrate. Furthermore, we show that the closed conformation is promoted by a matched incoming dNTP but not by a mismatched dNTP and that mismatches at the primer-template terminus lead to an increase in the binding of the DNA to the exonuclease site. Our analysis has also revealed new details of the biphasic dissociation kinetics of the polymerase-DNA binary complex. Notably, comparison of the results obtained in this study with PolB1 with those from similar single-molecule studies with an A-family DNA polymerase suggests mechanistic differences between these polymerases. In summary, our findings provide novel mechanistic insights into protein conformational dynamics and substrate binding kinetics of a high fidelity B-family DNA polymerase.

Impact of Individual PCNA-DNA Contacts on Clamp Loading and Function on DNA

Ring-shaped clamp proteins encircle DNA and affect the work of many proteins, notably processive replication by DNA polymerases. Crystal structures of clamps show several cationic residues inside the ring, and in a co-crystal of E. coli β clamp-DNA they directly contact the tilted duplex passing through. To investigate the role of these contacts in reactions involving circular clamps, we examined single arginine/lysine mutants of S. cerevisiae PCNA in RFC-catalyzed loading of the clamp onto primer-template DNA (ptDNA). Previous kinetic analysis has shown that ptDNA entry inside an ATP-activated RFC-PCNA complex accelerates clamp opening and ATP hydrolysis, which is followed by slow PCNA closure around DNA and product dissociation. Here we directly measured multiple steps in the reaction--PCNA opening, ptDNA binding, PCNA closure, phosphate release and complex dissociation to determine if mutation of PCNA residues R14, K20, R80, K146, R149 or K217 to alanine affects the reaction mechanism. Contrary to earlier steady state analysis of these mutants, pre-steady state data show that loss of single cationic residues can alter the rates of all DNA-linked steps in the reaction as well as movement of PCNA on DNA. These results explain an earlier finding that individual arginines and lysines inside human PCNA are essential for pol δ processivity. Mutations in the N-terminal domain have greater impact than in the C-terminal domain, indicating a positional bias in PCNA-DNA contacts that can influence its functions on DNA.

Time Dependence of Partitoning between Polymerization and Editing Sites by Klenow Polymerase

Klenow polymerase is the large fragment of DNA polymerase I from E. coli. Klenow possesses 5'-3' polymerase and intrinsic 3'-5' exonuclease activities with two distinct active sites that are located ∼30 Ǻ apart. During DNA replication, proofreading activity enhances replication fidelity by excising misincorporated nucleotides from the 3' end of primer strand. The first step of the proofreading process is sending the 3'-primer terminus to the proofreading site, which requires separation of the primer terminus from the template strand. Here we examined the binding of Klenow to matched and mismatched primed-template DNA (pt-DNA) by monitoring the steady state fluorescence intensity change of a single 2-aminopurine base site-specifically substituted in the template strand within the duplex part of pt-DNA. The changes in fluorescence intensity allow us to follow shuttling of the primer terminus between the polymerization and proofreading sites.We have found that the rate of partitioning of the primer between the two active sites depends on: 1) the number of mismatched bases at the primer-template junction, 2) the presence or absence of divalent ions, and 3) the type of divalent ions. Magnesium and calcium ions have opposite effects on the direction of the shift between the pol and exo sites. Substitution of the normal phosphodiester linkage between the last two bases of the primer strand with a non-hydrolysable phosphorothioate linkage also has significant effects on the partitioning between sites.

Impact of Individual Proliferating Cell Nuclear Antigen-DNA Contacts on Clamp Loading and Function on DNA

Ring-shaped clamp proteins encircle DNA and affect the work of many proteins, notably processive replication by DNA polymerases. Crystal structures of clamps show several cationic residues inside the ring, and in a co-crystal of Escherichia coli β clamp-DNA, they directly contact the tilted duplex passing through (Georgescu, R. E., Kim, S. S., Yurieva, O., Kuriyan, J., Kong, X. P., and O'Donnell, M. (2008) Structure of a sliding clamp on DNA. Cell 132, 43-54). To investigate the role of these contacts in reactions involving circular clamps, we examined single arginine/lysine mutants of Saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA) in replication factor C (RFC)-catalyzed loading of the clamp onto primer template DNA (ptDNA). Previous kinetic analysis has shown that ptDNA entry inside an ATP-activated RFC-PCNA complex accelerates clamp opening and ATP hydrolysis, which is followed by slow PCNA closure around DNA and product dissociation. Here we directly measured multiple steps in the reaction (PCNA opening, ptDNA binding, PCNA closure, phosphate release, and complex dissociation) to determine whether mutation of PCNA residues Arg-14, Lys-20, Arg-80, Lys-146, Arg-149, or Lys-217 to alanine affects the reaction mechanism. Contrary to earlier steady state analysis of these mutants (McNally, R., Bowman, G. D., Goedken, E. R., O'Donnell, M., and Kuriyan, J. (2010) Analysis of the role of PCNA-DNA contacts during clamp loading. BMC Struct. Biol. 10, 3), our pre-steady state data show that loss of single cationic residues can alter the rates of all DNA-linked steps in the reaction, as well as movement of PCNA on DNA. These results explain an earlier finding that individual arginines and lysines inside human PCNA are essential for polymerase δ processivity (Fukuda, K., Morioka, H., Imajou, S., Ikeda, S., Ohtsuka, E., and Tsurimoto, T. (1995) Structure-function relationship of the eukaryotic DNA replication factor, proliferating cell nuclear antigen. J. Biol. Chem. 270, 22527-22534). Mutations in the N-terminal domain have greater impact than in the C-terminal domain, indicating a positional bias in PCNA-DNA contacts that can influence its functions on DNA.