Bret D. Freudenthal

Observing a DNA polymerase choose right from wrong.

DNA polymerase (pol) β is a model polymerase involved in gap-filling DNA synthesis utilizing two metals to facilitate nucleotidyl transfer. Previous structural studies have trapped catalytic intermediates by utilizing substrate analogs (dideoxy-terminated primer or nonhydrolysable incoming nucleotide). To identify additional intermediates during catalysis, we now employ natural substrates (correct and incorrect nucleotides) and follow product formation in real time with 15 different crystal structures. We are able to observe molecular adjustments at the active site that hasten correct nucleotide insertion and deter incorrect insertion not appreciated previously. A third metal binding site is transiently formed during correct, but not incorrect, nucleotide insertion. Additionally, long incubations indicate that pyrophosphate more easily dissociates after incorrect, compared to correct, nucleotide insertion. This appears to be coupled to subdomain repositioning that is required for catalytic activation/deactivation. The structures provide insights into a fundamental chemical reaction that impacts polymerase fidelity and genome stability.

Uncovering the polymerase-induced cytotoxicity of an oxidized nucleotide

Oxidative stress promotes genomic instability and human diseases. A common oxidized nucleoside is 8-oxo-7,8-dihydro-2′-deoxyguanosine, which is found both in DNA (8-oxo-G) and as a free nucleotide (8-oxo-dGTP). Nucleotide pools are especially vulnerable to oxidative damage. Therefore cells encode an enzyme (MutT/MTH1) that removes free oxidized nucleotides. This cleansing function is required for cancer cell survival and to modulate Escherichia coli antibiotic sensitivity in a DNA polymerase (pol)-dependent manner. How polymerases discriminate between damaged and non-damaged nucleotides is not well understood. This analysis is essential given the role of oxidized nucleotides in mutagenesis, cancer therapeutics, and bacterial antibiotics. Even with cellular sanitizing activities, nucleotide pools contain enough 8-oxo-dGTP to promote mutagenesis. This arises from the dual coding potential where 8-oxo-dGTP(anti) base pairs with cytosine and 8-oxo-dGTP(syn) uses its Hoogsteen edge to base pair with adenine. Here we use time-lapse crystallography to follow 8-oxo-dGTP insertion opposite adenine or cytosine with human pol β, to reveal that insertion is accommodated in either the syn- or anti-conformation, respectively. For 8-oxo-dGTP(anti) insertion, a novel divalent metal relieves repulsive interactions between the adducted guanine base and the triphosphate of the oxidized nucleotide. With either templating base, hydrogen-bonding interactions between the bases are lost as the enzyme reopens after catalysis, leading to a cytotoxic nicked DNA repair intermediate. Combining structural snapshots with kinetic and computational analysis reveals how 8-oxo-dGTP uses charge modulation during insertion that can lead to a blocked DNA repair intermediate.

Suicidal cross-linking of PARP-1 to AP site intermediates in cells undergoing base excision repair

Poly(ADP-ribose) polymerase-1 (PARP-1) is an abundant nuclear enzyme in mammalian cells. The enzyme synthesizes polymers of ADP-ribose from the coenzyme NAD+ and plays multifaceted roles in cellular responses to genotoxic stress, including DNA repair. It had been shown that mouse fibroblasts treated with a DNA methylating agent in combination with a PARP inhibitor exhibit higher cytotoxicity than cells treated with methylating agent alone. This lethality of the PARP inhibitor is dependent on apurinic/apyrimidinic (AP) sites in the DNA and the presence of PARP-1. Here, we show that purified PARP-1 is capable of forming a DNA-protein cross-link (DPC) by covalently attaching to the AP site. This DPC formation is specific to the presence of the natural AP site in DNA and is accompanied by a single-strand DNA incision. Cellular studies confirm the formation of PARP-1 DPCs during alkylating agent-induced base excision repair (BER) and formation of DPCs is enhanced by a PARP inhibitor. Using an N-terminal and C-terminal truncated PARP-1 we show that a polypeptide fragment comprising the zinc 3 and BRCT sub-domains is sufficient for DPC formation. The covalent attachment of PARP-1 to AP site-containing DNA appears to be a suicidal event when BER is overwhelmed or disrupted.

Intrinsic mutagenic properties of 5-chlorocytosine: A mechanistic connection between chronic inflammation and cancer.

Significance Chronic inflammation is a significant risk factor for cancer and other human diseases. During chronic inflammation, cells exposed to neutrophil-derived hypochlorous acid accumulate in their genomes the DNA lesion 5-chlorocytosine (5ClC). Using a battery of chemical, structural, and genetic tools, the present study demonstrates that 5ClC is a mutagenic lesion, suggesting that genomic 5ClC accumulation could have very serious biological consequences. 5ClC induces C→T transitions, a type of mutation commonly observed in tissues under inflammatory stress as well as in the genomes of inflammation-driven cancers. Thus, the mutagenic properties of 5ClC represent an appealing molecular mechanism by which chronic inflammation induces the genetic changes that potentially enable and stimulate carcinogenesis. During chronic inflammation, neutrophil-secreted hypochlorous acid can damage nearby cells inducing the genomic accumulation of 5-chlorocytosine (5ClC), a known inflammation biomarker. Although 5ClC has been shown to promote epigenetic changes, it has been unknown heretofore if 5ClC directly perpetrates a mutagenic outcome within the cell. The present work shows that 5ClC is intrinsically mutagenic, both in vitro and, at a level of a single molecule per cell, in vivo. Using biochemical and genetic approaches, we have quantified the mutagenic and toxic properties of 5ClC, showing that this lesion caused C→T transitions at frequencies ranging from 3–9% depending on the polymerase traversing the lesion. X-ray crystallographic studies provided a molecular basis for the mutagenicity of 5ClC; a snapshot of human polymerase β replicating across a primed 5ClC-containing template uncovered 5ClC engaged in a nascent base pair with an incoming dATP analog. Accommodation of the chlorine substituent in the template major groove enabled a unique interaction between 5ClC and the incoming dATP, which would facilitate mutagenic lesion bypass. The type of mutation induced by 5ClC, the C→T transition, has been previously shown to occur in substantial amounts both in tissues under inflammatory stress and in the genomes of many inflammation-associated cancers. In fact, many sequence-specific mutational signatures uncovered in sequenced cancer genomes feature C→T mutations. Therefore, the mutagenic ability of 5ClC documented in the present study may constitute a direct functional link between chronic inflammation and the genetic changes that enable and promote malignant transformation.

DNA polymerase minor groove interactions modulate mutagenic bypass of a templating 8-oxoguanine lesion

A major base lesion resulting from oxidative stress is 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxoG) that has ambiguous coding potential. Error-free DNA synthesis involves 8-oxoG adopting an anti-conformation to base pair with cytosine whereas mutagenic bypass involves 8-oxoG adopting a syn-conformation to base pair with adenine. Left unrepaired the syn-8-oxoG/dAMP base pair results in a G–C to T–A transversion. During base excision repair of this mispair, DNA polymerase (pol) β is confronted with gap filling opposite 8-oxoG. To determine how pol β discriminates between anti- and syn-8-oxoG, we introduced a point mutation (R283K) to alter insertion specificity. Kinetic studies demonstrate that this substitution results in an increased fidelity opposite 8-oxoG. Structural studies with R283K pol β show that the binary DNA complex has 8-oxoG in equilibrium between anti- and syn-forms. Ternary complexes with incoming dCTP resemble the wild-type enzyme, with templating anti-8-oxoG base pairing with incoming cytosine. In contrast to wild-type pol β, the ternary complex of the R283K mutant with an incoming dATP-analogue and templating 8-oxoG resembles a G–A mismatched structure with 8-oxoG adopting an anti-conformation. These results demonstrate that the incoming nucleotide is unable to induce a syn-8-oxoG conformation without minor groove DNA polymerase interactions that influence templating (anti-/syn-equilibrium) of 8-oxoG while modulating fidelity.

Requirement for transient metal ions revealed through computational analysis for DNA polymerase going in reverse.

Significance DNA polymerases use a general two-metal ion mechanism for DNA synthesis. Recent time-lapse crystallographic studies identified additional adjunct metal ions in the polymerase active site. One of these ions correlates with appearance of pyrophosphate and was proposed to be involved in pyrophosphorolysis (reverse reaction of DNA synthesis). Because DNA polymerases can use pyrophosphorolysis to remove chain-terminating nucleotides during chemotherapies, a better understanding of this reaction is warranted. Through site-directed mutagenesis, pyrophosphorolysis measurements, and computational analysis, we examine the role of metal ions in the reverse reaction. The results indicate that the product-associated metal ion facilitates pyrophosphorolysis during the early stages of the reaction but deters the reaction at later stages, suggesting dynamic metal behavior that can modulate the chemical equilibrium. DNA polymerases facilitate faithful insertion of nucleotides, a central reaction occurring during DNA replication and repair. DNA synthesis (forward reaction) is “balanced,” as dictated by the chemical equilibrium by the reverse reaction of pyrophosphorolysis. Two closely spaced divalent metal ions (catalytic and nucleotide-binding metals) provide the scaffold for these reactions. The catalytic metal lowers the pKa of O3′ of the growing primer terminus, and the nucleotide-binding metal facilitates substrate binding. Recent time-lapse crystallographic studies of DNA polymerases have identified an additional metal ion (product metal) associated with pyrophosphate formation, leading to the suggestion of its possible involvement in the reverse reaction. Here, we establish a rationale for a role of the product metal using quantum mechanical/molecular mechanical calculations of the reverse reaction in the confines of the DNA polymerase β active site. Additionally, site-directed mutagenesis identifies essential residues and metal-binding sites necessary for pyrophosphorolysis. The results indicate that the catalytic metal site must be occupied by a magnesium ion for pyrophosphorolysis to occur. Critically, the product metal site is occupied by a magnesium ion early in the pyrophosphorolysis reaction path but must be removed later. The proposed dynamic nature of the active site metal ions is consistent with crystallographic structures. The transition barrier for pyrophosphorolysis was estimated to be significantly higher than that for the forward reaction, consistent with kinetic activity measurements of the respective reactions. These observations provide a framework to understand how ions and active site changes could modulate the internal chemical equilibrium of a reaction that is central to genome stability.

PARP1 changes from three-dimensional DNA damage searching to one-dimensional diffusion after auto-PARylation or in the presence of APE1

Abstract PARP1-dependent poly-ADP-ribosylation (PARylation) participates in the repair of many forms of DNA damage. Here, we used atomic force microscopy (AFM) and single molecule fluorescence microscopy to examine the interactions of PARP1 with common DNA repair intermediates. AFM volume analysis indicates that PARP1 binds to DNA at nicks, abasic (AP) sites, and ends as a monomer. Single molecule DNA tightrope assays were used to follow the real-time dynamic behavior of PARP1 in the absence and presence of AP endonuclease (APE1) on AP DNA damage arrays. These experiments revealed that PARP1 conducted damage search mostly through 3D diffusion. Co-localization of APE1 with PARP1 on DNA was found capable of inducing 1D diffusion of otherwise nonmotile PARP1, while excess APE1 also facilitated the dissociation of DNA-bound PARP1. Moreover, auto-PARylation of PARP1 allowed the protein to switch its damage search strategy by causing a 3-fold increase in linear diffusion. Finally, we demonstrated that PARP inhibitor olaparib did not significantly alter the rate of PARP1 dissociation from DNA, but instead resulted in more motility of DNA-bound PARP1 molecules.

New structural snapshots provide molecular insights into the mechanism of high fidelity DNA synthesis

Time-lapse X-ray crystallography allows visualization of intermediate structures during the DNA polymerase catalytic cycle. Employing time-lapse crystallography with human DNA polymerase β has recently allowed us to capture and solve novel intermediate structures that are not stable enough to be analyzed by traditional crystallography. The structures of these intermediates reveals exciting surprises about active site metal ions and enzyme conformational changes as the reaction proceeds from the ground state to product release. In this perspective, we provide an overview of recent advances in understanding the DNA polymerase nucleotidyl transferase reaction and highlight both the significance and mysteries of enzyme efficiency and specificity that remain to be solved.

Insertion of oxidized nucleotide triggers rapid DNA polymerase opening.

A novel mechanism is unveiled to explain why a pro-mutagenic nucleotide lesion (oxidized guanine, 8-oxoG) causes the mammalian DNA repair polymerase-β (pol-β) to rapidly transition to an inactive open conformation. The mechanism involves unexpected features revealed recently in time-lapse crystallography. Specifically, a delicate water network associated with a lesion-stabilizing auxilliary product ion Mg(p) triggers a cascade of events that leads to poor active site geometry and the rupture of crucial molecular interactions between key residues in both the anti(8-oxoG:C) and syn(8-oxoG:A) systems. Once the base pairs in these lesioned systems are broken, dislocation of both Asp192 (a metal coordinating ligand) and the oxoG phosphate group (PO4) interfere with the hydrogen bonding between Asp192 and Arg258, whose rotation toward Asp192 is crucial to the closed-to-open enzyme transition. Energetically, the lesioned open states are similar in energy to those of the corresponding closed complexes after chemistry, in marked contrast to the unlesioned pol-β anti(G:C) system, whose open state is energetically higher than the closed state. The delicate surveillance system offers a fundamental protective mechanism in the cell that triggers DNA repair events which help deter insertion of oxidized lesions.

Optimal and Variant Metal-Ion Routes in DNA Polymerase β’s Conformational Pathways

To interpret recent structures of the R283K mutant of human DNA repair enzyme DNA polymerase β (pol β) differing in the number of Mg(2+) ions, we apply transition path sampling (TPS) to assess the effect of differing ion placement on the transition from the open one-metal to the closed two-metal state. We find that the closing pathway depends on the initial ion position, both in terms of the individual transition states and associated energies. The energy barrier of the conformational pathway varies from 25 to 58 kJ/mol, compared to the conformational energy barrier of 42 kJ/mol for the wild-type pol β reported previously. Moreover, we find a preferred ion route located in the center of the enzyme, parallel to the DNA. Within this route, the conformational pathway is similar to that of the overall open to closed transition of pol β, but outside it, especially when the ion starts near active site residues Arg258 and Asp190, the conformational pathway diverges significantly. Our findings should apply generally to pol β, since R283K is relatively far from the active site; further experimental and computational work are required to confirm this. Our studies also underscore the common feature that less active mutants have less stable closed states than their open states, in marked contrast to the wild-type enzyme, where the closed state is significantly more stable than the open form.