Promoter dependent RNA polymerase II bypass of the epimerizable DNA lesion, Fapy•dG and 8-Oxo-2'-deoxyguanosine.

Formamidopyrimidine (Fapy•dG) is a major lesion arising from oxidation of dG that is produced from a common chemical precursor of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OxodGuo). In human cells, replication of single-stranded shuttle vectors containing Fapy•dG is more mutagenic than 8-OxodGuo. Here, we present the first data regarding promoter dependent RNA polymerase II bypass of Fapy•dG. 8-OxodGuo bypass was examined side-by-side. Experiments were carried out using double-stranded shuttle vectors in HeLa cell nuclear lysates and in HEK 293T cells. The lesions do not significantly block transcriptional bypass efficiency. Less than 2% adenosine incorporation occurred in cells when the lesions were base paired with dC. Inhibiting base excision repair in HEK 293T cells significantly increased adenosine incorporation, particularly from Fapy•dG:dC bypass which yielded ∼25% adenosine incorporation. No effect was detected upon transcriptional bypass of either lesion in nucleotide excision repair deficient cells. Transcriptional mutagenesis was significantly higher when shuttle vectors containing dA opposite one of the lesions were employed. For Fapy•dG:dA bypass, adenosine incorporation was greater than 85%; whereas 8-OxodGuo:dA yielded >20% point mutations. The combination of more frequent replication mistakes and greater error-prone Pol II bypass suggest that Fapy•dG is more mutagenic than 8-OxodGuo.

Biochemical and structural characterization of Fapy•dG replication by Human DNA polymerase ß.

N6-(2-deoxy-α,ß-d-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamido-pyrimidine (Fapy•dG) is formed from a common intermediate and in comparable amounts to the well-studied mutagenic DNA lesion 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OxodGuo). Fapy•dG preferentially gives rise to G → T transversions and G → A transitions. However, the molecular basis by which Fapy•dG is processed by DNA polymerases during this mutagenic process remains poorly understood. To address this we investigated how DNA polymerase ß (Pol ß), a model mammalian polymerase, bypasses a templating Fapy•dG, inserts Fapy•dGTP, and extends from Fapy•dG at the primer terminus. When Fapy•dG is present in the template, Pol ß incorporates TMP less efficiently than either dCMP or dAMP. Kinetic analysis revealed that Fapy•dGTP is a poor substrate but is incorporated ∼3-times more efficiently opposite dA than dC. Extension from Fapy•dG at the 3'-terminus of a nascent primer is inefficient due to the primer terminus being poorly positioned for catalysis. Together these data indicate that mutagenic bypass of Fapy•dG is likely to be the source of the mutagenic effects of the lesion and not Fapy•dGTP. These experiments increase our understanding of the promutagenic effects of Fapy•dG.

Molecular Mechanism of RNA Polymerase II Transcriptional Mutagenesis by the Epimerizable DNA Lesion, Fapy·dG.

Oxidative DNA lesions cause significant detrimental effects on a living species. Two major DNA lesions resulting from dG oxidation, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OxodGuo) and formamidopyrimidine (Fapy·dG), are produced from a common chemical intermediate. Fapy·dG is formed in comparable yields under oxygen-deficient conditions. Replicative bypass of Fapy·dG in human cells is more mutagenic than that of 8-OxodGuo. Despite the biological importance of transcriptional mutagenesis, there are no reports of the effects of Fapy·dG on RNA polymerase II (Pol II) activity. Here we perform comprehensive kinetic studies to investigate the impact of Fapy·dG on three key transcriptional fidelity checkpoint steps by Pol II: insertion, extension, and proofreading steps. The ratios of error-free versus error-prone incorporation opposite Fapy·dG are significantly reduced in comparison with undamaged dG. Similarly, Fapy·dG:A mispair is extended with comparable efficiency as that of the error-free, Fapy·dG:C base pair. The α- and ß-configurational isomers of Fapy·dG have distinct effects on Pol II insertion and extension. Pol II can preferentially cleave error-prone products by proofreading. To further understand the structural basis of transcription processing of Fapy·dG, five different structures were solved, including Fapy·dG template-loading state (apo), error-free cytidine triphosphate (CTP) binding state (prechemistry), error-prone ATP binding state (prechemistry), error-free Fapy·dG:C product state (postchemistry), and error-prone Fapy·dG:A product state (postchemistry), revealing distinctive nucleotide binding and product states. Taken together, our study provides a comprehensive mechanistic framework for better understanding how Fapy·dG lesions impact transcription and subsequent pathological consequences.

Quantification of Intracellular DNA-Protein Cross-Links with N7-Methyl-2'-Deoxyguanosine and Their Contribution to Cytotoxicity.

The major product of DNA-methylating agents, N7-methyl-2'-deoxyguanosine (MdG), is a persistent lesion in vivo, but it is not believed to have a large direct physiological impact. However, MdG reacts with histone proteins to form reversible DNA-protein cross-links (DPCMdG), a family of DNA lesions that can significantly threaten cell survival. In this paper, we developed a tandem mass spectrometry method for quantifying the amounts of MdG and DPCMdG in nuclear DNA by taking advantage of their chemical lability and the concurrent release of N7-methylguanine. Using this method, we determined that DPCMdG is formed in less than 1% yield based upon the levels of MdG in methyl methanesulfonate (MMS)-treated HeLa cells. Despite its low chemical yield, DPCMdG contributes to MMS cytotoxicity. Consequently, cells that lack efficient DPC repair by the DPC protease SPRTN are hypersensitive to MMS. This investigation shows that the downstream chemical and biochemical effects of initially formed DNA damage can have significant biological consequences. With respect to MdG formation, the initial DNA lesion is only the beginning.

Histone Deacetylase 1 Inhibition by Peptides Containing a DNA Damage-Induced, Nonenzymatic, Histone Covalent Modification.

Treatment of HeLa cells with the DNA damaging agent, bleomycin (BLM), results in the formation of a nonenzymatic 5-methylene-2-pyrrolone histone covalent modification on lysine residues (KMP). KMP is much more electrophilic than other N-acyllysine covalent modifications and post-translational modifications, including N-acetyllysine (KAc). Using histone peptides containing KMP, we show that this modification inhibits the class I histone deacetylase, HDAC1, by reacting with a conserved cysteine (C261) located near the active site. HDAC1 is inhibited by histone peptides whose corresponding N-acetylated sequences are known deacetylation substrates, but not one containing a scrambled sequence. The HDAC1 inhibitor, trichostatin A, competes with covalent modification by the KMP-containing peptides. HDAC1 is also covalently modified by a KMP-containing peptide in a complex milieu. These data indicate that peptides containing KMP are recognized by HDAC1 and are bound in the active site. The effects on HDAC1 indicate that KMP formation in cells may contribute to the biological effects of DNA damaging agents, such as BLM, that form this nonenzymatic covalent modification.

DNA-Histone Cross-Link Formation via Hole Trapping in Nucleosome Core Particles.

Radical cations (holes) produced in DNA by ionizing radiation and other oxidants yield DNA-protein cross-links (DPCs). Detailed studies of DPC formation in chromatin via this process are lacking. We describe here a comprehensive examination of DPC formation within nucleosome core particles (NCPs), which are the monomeric component of chromatin. DNA holes are introduced at defined sites within NCPs that are constructed from the bottom-up. DPCs form at DNA holes in yields comparable to those of alkali-labile DNA lesions that result from water trapping. DPC-forming efficiency and site preference within the NCP are dependent on translational and rotational positioning. Mass spectrometry and the use of mutant histones reveal that lysine residues in histone N-terminal tails and amino termini are responsible for the DPC formation. These studies are corroborated by computational simulation at the microsecond time scale, showing a wide range of interactions that can precede DPC formation. Three consecutive dGs, which are pervasive in the human genome, including G-quadruplex-forming sequences, are sufficient to produce DPCs that could impact gene expression.

Participation of Histones in DNA Damage and Repair within Nucleosome Core Particles: Mechanism and Applications.

DNA is damaged by various endogenous and exogenous sources, leading to a diverse group of reactive intermediates that yield a complex mixture of products. The initially formed products are often metastable and can react to yield lesions that are more biologically deleterious. Mechanistic studies are frequently carried out on free DNA as the substrate. The observations do not necessarily reflect the reaction environment inside human cells where genomic DNA is condensed as chromatin in the nucleus. Chromatin is made up of monomeric structural units called nucleosomes, which are comprised of DNA wrapped around an octameric core of histone proteins (two copies each of histones H2A, H2B, H3, and H4).This account presents a summary of our work in the past decade on the mechanistic studies of DNA damage and repair in reconstituted nucleosome core particles (NCPs). A series of metastable lesions and reactive intermediates, such as abasic sites (AP), N7-methyl-2'-deoxyguanosine (MdG), and 2'-deoxyadenosin-N6-yl radical (dA•), have been independently generated in a site-specific manner in bottom-up-synthesized NCPs. Detailed mechanistic studies on these NCPs revealed that histones actively participate in DNA damage and repair processes in diverse ways. For instance, nucleophilic residues in the flexible histone N-terminal tails, such as Lys and N-terminal α-amine, react with electrophilic DNA damage and reactive intermediates. In some cases, transient intermediates are produced, leading to the promotion or suppression of damage and repair processes. In other examples, reactions with histones yield reversible or stable DNA-protein cross-links (DPCs). Histones also utilize acidic and basic residues, such as histidine and aspartic acid, to catalyze DNA strand cleavage through general acid/base catalysis. Alternatively, a Tyr in histone plays a vital role in nucleosomal DNA damage and repair via radical transfer. Finally, the reactivity discovered during the mechanistic studies has facilitated the development of new reagents and methods with applications in biotechnology.This research has enriched our knowledge of the roles of histone proteins in DNA damage and repair and their contributions to epigenetics and may have significant biological implications. The residues in histone N-terminal tails that react with DNA lesions also play pivotal roles in regulating the structure and function of chromatin, indicating that there may be cross-talk between DNA damage and repair in eukaryotic cells and epigenetic regulation. Also, in view of the biased amino acid composition of histones, these results provide hints about how the proteins have evolved to minimize their deleterious effects but maximize beneficial ones for maintaining genome integrity. Finally, previously unreported DPCs and histone post-translational modifications have been discovered through this research. The effects of these newly identified lesions on the structure and function of chromatin and their fates inside cells remain to be elucidated.

8-Oxo-2'-deoxyguanosine Replication in Mutational Hot Spot Sequences of the p53 Gene in Human Cells Is Less Mutagenic than That of the Corresponding Formamidopyrimidine.

7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-OxodGuo) is a ubiquitous DNA damage formed by oxidation of 2'-deoxyguanosine. In this study, plasmid DNA containing 8-OxodGuo located in three mutational hot spots of human cancers, codons 248, 249, and 273 of the Tp53 tumor suppressor gene, was replicated in HEK 293T cells. 8-OxodGuo was only a weak block of replication, and the bypass was largely error-free. The mutations (1-5%) were primarily G → T transversions, and the mutation frequency was generally lower than that of the chemically related Fapy·dG. A unique 8-OxodGuo mutation spectrum was observed at each site, as reflected by replication in translesion synthesis (TLS) polymerase- or hPol λ-deficient cells. In codon 248 (CG*G) and 249 (AG*G), where G* denotes 8-OxodGuo, hPol η and hPol ζ carried out largely error-free bypass of the lesion, whereas hPol κ and hPol ι were involved mostly in error-prone TLS, resulting in G → T mutations. 8-OxodGuo bypass in codon 273 (CG*T) was unlike the other two sites, as hPol κ participated in the mostly error-free bypass of the lesion. Yet, in all three sites, including codon 273, simultaneous deficiency of hpol κ and hPol ι resulted in reduction of G → T transversions. This indicates a convincing role of these two TLS polymerases in error-prone bypass of 8-OxodGuo. Although the dominant mutation was G → T in each site, in codon 249, and to a lesser extent in codon 248, significant semi-targeted single-base deletions also occurred, which suggests that 8-OxodGuo can initiate slippage of a base near the lesion site. This study underscores the importance of sequence context in 8-OxodGuo mutagenesis in human cells. It also provides a more comprehensive comparison between 8-OxodGuo and the sister lesion, Fapy·dG. The greater mutagenicity of the latter in the same sequence contexts indicates that Fapy·dG is a biologically significant lesion and biomarker on par with 8-OxodGuo.

Photochemical and Single Electron Transfer Generation of 2'-Deoxycytidin-N4-yl Radical from Oxime Esters.

A 2'-deoxycytidin-N4-yl radical (dC·), a strong oxidant that also abstracts hydrogen atoms from carbon-hydrogen bonds, is produced in a variety of DNA damaging processes. We describe here the independent generation of dC· from oxime esters under UV-irradiation or single electron transfer conditions. Support for this σ-type iminyl radical generation is provided by product studies carried out under aerobic and anaerobic conditions, as well as electron spin resonance (ESR) characterization of dC· in a homogeneous glassy solution at low temperature. Density functional theory (DFT) calculations also support fragmentation of the corresponding radical anions of oxime esters 2d and 2e to dC· and subsequent hydrogen atom abstraction from organic solvents. The corresponding 2'-deoxynucleotide triphosphate (dNTP) of isopropyl oxime ester 2c (5) is incorporated opposite 2'-deoxyadenosine and 2'-deoxyguanosine by a DNA polymerase with approximately equal efficiency. Photolysis experiments of DNA containing 2c support dC· generation and indicate that the radical produces tandem lesions when flanked on the 5'-side by 5'-d(GGT). These experiments suggest that oxime esters are reliable sources of nitrogen radicals in nucleic acids that will be useful mechanistic tools and possibly radiosensitizing agents when incorporated in DNA.

Light-controlled twister ribozyme with single-molecule detection resolves RNA function in time and space.

Small ribozymes such as Oryza sativa twister spontaneously cleave their own RNA when the ribozyme folds into its active conformation. The coupling between twister folding and self-cleavage has been difficult to study, however, because the active ribozyme rapidly converts to product. Here, we describe the synthesis of a photocaged nucleotide that releases guanosine within microseconds upon photosolvolysis with blue light. Application of this tool to O. sativa twister achieved the spatial (75 µm) and temporal (≤30 ms) control required to resolve folding and self-cleavage events when combined with single-molecule fluorescence detection of the ribozyme folding pathway. Real-time observation of single ribozymes after photo-deprotection showed that the precleaved folded state is unstable and quickly unfolds if the RNA does not react. Kinetic analysis showed that Mg2+ and Mn2+ ions increase ribozyme efficiency by making transitions to the high energy active conformation more probable, rather than by stabilizing the folded ground state or the cleaved product. This tool for light-controlled single RNA folding should offer precise and rapid control of other nucleic acid systems.

Local Alteration of Ionic Strength in a Nucleosome Core Particle and Its Effect on N7-Methyl-2'-deoxyguanosine Depurination.

Positively charged N-terminal histone tails play important roles in maintaining the nucleosome (and chromatin) structure and function. Charge alteration, including those imposed by post-translational modifications, impacts chromatin dynamics, protein binding, and the fate of DNA damage. There is evidence that N-terminal histone tails affect the local ionic environment within a nucleosome core particle (NCP), but this phenomenon is not well understood. Determining the modulation of the local ionic environment within an NCP by histone tails could help uncover the underlying mechanisms of their functions and effects. Utilizing bottom-up syntheses of NCPs containing wild-type or mutated histones and a fluorescent probe that is sensitive to the local ionic environment, we show that interaction with positively charged N-terminal tails increases the local ionic strength near nucleosomal DNA. The effect is diminished by replacing positively charged residues with neutral ones or deleting a tail in its entirety. Replacing the fluorescent probe with the major DNA methylation product, N7-methyl-2'-deoxyguanosine (MdG), revealed changes in the depurination rate constant varying inversely with local ionic strength. These data indicate that the MdG hydrolysis rates depend on and also inform on local ionic strength in an NCP. Overall, histone tail charge contributes to the complexity of the NCP structure and function by modulating the local ionic strength.

Intracellular Formation of a DNA Damage-Induced, Histone Post-Translational Modification Following Bleomycin Treatment.

Evaluating the significance of various forms of DNA damage is complicated by discoveries that some lesions inactivate repair enzymes or produce more deleterious forms of damage. Histone lysines within nucleosomes react with the commonly produced C4'-oxidized abasic site (C4-AP) to concomitantly yield an electrophilic modification (KMP) on lysine and DNA strand scission. We developed a chemoproteomic approach to identify KMP in HeLa cells. More than 60 000 KMP-modified histones are produced per cell. Using LC-MS/MS, we detected KMP at 17 of the 57 lysine residues distributed throughout the four core histone proteins. Therefore, KMP constitutes a DNA damage-induced, nonenzymatic histone post-translational modification. KMP formation suggests that downstream processes resulting from DNA damage could have ramifications on cells.

Structural Dynamics of a Common Mutagenic Oxidative DNA Lesion in Duplex DNA and during DNA Replication.

N6-(2-Deoxy-α,ß-d-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamido pyrimidine (Fapy•dG) is a prevalent form of genomic DNA damage. Fapy•dG is formed in greater amounts under anoxic conditions than the well-studied, chemically related 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodGuo). Fapy•dG is more mutagenic in mammalian cells than 8-oxodGuo. A distinctive property of Fapy•dG is facile epimerization, but prior works with Fapy•dG analogues have precluded determining its effect on chemistry. We present crystallographic characterization of natural Fapy•dG in duplex DNA and as the template base for DNA polymerase ß (Pol ß). Fapy•dG adopts the ß-anomer when base paired with cytosine but exists as a mixture of α- and ß-anomers when promutagenically base paired with adenine. Rotation about the bond between the glycosidic nitrogen atom and the pyrimidine ring is also affected by the opposing nucleotide. Sodium cyanoborohydride soaking experiments trap the ring-opened Fapy•dG, demonstrating that ring opening and epimerization occur in the crystalline state. Ring opening and epimerization are facilitated by propitious water molecules that are observed in the structures. Determination of Fapy•dG mutagenicity in wild type and Pol ß knockdown HEK 293T cells indicates that Pol ß contributes to G → T transversions but also suppresses G → A transitions. Complementary kinetic studies have determined that Fapy•dG promotes mutagenesis by decreasing the catalytic efficiency of dCMP insertion opposite Fapy•dG, thus reducing polymerase fidelity. Kinetic studies have determined that dCMP incorporation opposite the ß-anomer is ∼90 times faster than the α-anomer. This research identifies the importance of anomer dynamics, a feature unique to formamidopyrimidines, when considering the incorporation of nucleotides opposite Fapy•dG and potentially the repair of this structurally unusual lesion.

Covalent Modification of Bromodomain Proteins by Peptides Containing a DNA Damage-Induced, Histone Post-Translational Modification.

An electrophilic 5-methylene-2-pyrrolone modification (KMP ) is produced at lysine residues of histone proteins in nucleosome core particles upon reaction with a commonly formed DNA lesion (C4-AP). The nonenzymatic KMP modification is also generated in the histones of HeLa cells treated with the antitumor agent, bleomycin that oxidizes DNA and forms C4-AP. This nonenzymatic covalent histone modification has the same charge as the N-acetyllysine (KAc ) modification but is more electrophilic. In this study we show that KMP -containing histone peptides are recognized by, and covalently modify bromodomain proteins that are KAc readers. Distinct selectivity preferences for covalent bromodomain modification are observed following incubation with KMP -containing peptides of different sequence. MS/MS analysis of 3 covalently modified bromodomain proteins confirmed that Cys adduction was selective. The modified Cys was not always proximal to the KAc binding site, indicating that KMP -containing peptide interaction with bromodomain protein is distinct from the former. Analysis of protein adduction yields as a function of bromodomain pH at which the protein charge is zero (pI) or cysteine solvent accessible surface area are also consistent with non-promiscuous interaction between the proteins and electrophilic peptides. These data suggest that intracellular formation of KMP could affect cellular function and viability by modifying proteins that regulate genetic expression.

Selective Inhibition of DNA Polymerase ß by a Covalent Inhibitor.

DNA polymerase ß (Pol ß) plays a vital role in DNA repair and has been closely linked to cancer. Selective inhibitors of this enzyme are lacking. Inspired by DNA lesions produced by antitumor agents that inactivate Pol ß, we have undertaken the development of covalent small-molecule inhibitors of this enzyme. Using a two-stage process involving chemically synthesized libraries, we identified a potent irreversible inhibitor (14) of Pol ß (KI = 1.8 ± 0.45 µM, kinact = (7.0 ± 1.0) × 10-3 s-1). Inhibitor 14 selectively inactivates Pol ß over other DNA polymerases. LC-MS/MS analysis of trypsin digests of Pol ß treated with 14 identified two lysines within the polymerase binding site that are covalently modified, one of which was previously determined to play a role in DNA binding. Fluorescence anisotropy experiments show that pretreatment of Pol ß with 14 prevents DNA binding. Experiments using a pro-inhibitor (pro-14) in wild type mouse embryonic fibroblasts (MEFs) indicate that the inhibitor (5 µM) is itself not cytotoxic but works synergistically with the DNA alkylating agent, methylmethanesulfonate (MMS), to kill cells. Moreover, experiments in Pol ß null MEFs indicate that pro-14 is selective for the target enzyme. Finally, pro-14 also works synergistically with MMS and bleomycin to kill HeLa cells. The results suggest that pro-14 is a potentially useful tool in studies of the role of Pol ß in disease.

Identifying Poly(ADP-ribose)-Binding Proteins with Photoaffinity-Based Proteomics.

Post-translational modification of proteins with poly(ADP-ribose) (PAR) is an important component of the DNA damage response. Four PAR synthesis inhibitors have recently been approved for the treatment of breast, ovarian, and prostate cancers. Despite the clinical significance of PAR, a molecular understanding of its function, including its binding partners, remains incomplete. In this work, we synthesized a PAR photoaffinity probe that captures and isolates endogenous PAR binders. Our method identified dozens of known PAR-binding proteins and hundreds of novel candidates involved in DNA repair, RNA processing, and metabolism. PAR binding by eight candidates was confirmed using pull-down and/or electrophoretic mobility shift assays. Using PAR probes of defined lengths, we detected proteins that preferentially bind to 40-mer versus 8-mer PAR, indicating that polymer length may regulate the outcome and timing of PAR signaling pathways. This investigation produces the first census of PAR-binding proteins, provides a proteomics analysis of length-selective PAR binding, and associates PAR binding with RNA metabolism and the formation of biomolecular condensates.

Reactivity and DNA Damage by Independently Generated 2'-Deoxycytidin-N4-yl Radical.

Oxidative stress produces a variety of radicals in DNA, including pyrimidine nucleobase radicals. The nitrogen-centered DNA radical 2'-deoxycytidin-N4-yl radical (dC·) plays a role in DNA damage mediated by one electron oxidants, such as HOCl and ionizing radiation. However, the reactivity of dC· is not well understood. To reduce this knowledge gap, we photochemically generated dC· from a nitrophenyl oxime nucleoside and within chemically synthesized oligonucleotides from the same precursor. dC· formation is confirmed by transient UV-absorption spectroscopy in laser flash photolysis (LFP) experiments. LFP and duplex DNA cleavage experiments indicate that dC· oxidizes dG. Transient formation of the dG radical cation (dG+•) is observed in LFP experiments. Oxidation of the opposing dG in DNA results in hole transfer when the opposing dG is part of a dGGG sequence. The sequence dependence is attributed to a competition between rapid proton transfer from dG+• to the opposing dC anion formed and hole transfer. Enhanced hole transfer when less acidic O6-methyl-2'-deoxyguanosine is opposite dC· supports this proposal. dC· produces tandem lesions in sequences containing thymidine at the 5'-position by abstracting a hydrogen atom from the thymine methyl group. The corresponding thymidine peroxyl radical completes tandem lesion formation by reacting with the 5'-adjacent nucleotide. As dC· is reduced to dC, its role in the process is traceless and is only detectable because of the ability to independently generate it from a stable precursor. These experiments reveal that dC· oxidizes neighboring nucleotides, resulting in deleterious tandem lesions and hole transfer in appropriate sequences.

Protein Domain Specific Covalent Inhibition of Human DNA Polymerase ß.

DNA polymerase ß (Pol ß) is a frequently overexpressed and/or mutated bifunctional repair enzyme. Pol ß possesses polymerase and lyase active sites, that are employed in two steps of base excision repair. Pol ß is an attractive therapeutic target for which there is a need for inhibitors. Two mechanistically inspired covalent inhibitors (1, IC50 =21.0 µM; 9, IC50 =18.7 µM) that modify lysine residues in different Pol ß active sites are characterized. Despite modifying lysine residues in different active sites, 1 and 9 inactivate the polymerase and lyase activities of Pol ß. Fluorescence anisotropy experiments indicate that they do so by preventing DNA binding. Inhibitors 1 and 9 provide the basis for a general approach to preparing domain selective inhibitors of bifunctional polymerases. Such molecules could prove to be useful tools for studying the role of wild type and mutant forms of Pol ß and other polymerases in DNA repair.

A guardian residue hinders insertion of a Fapy•dGTP analog by modulating the open-closed DNA polymerase transition.

4,6-Diamino-5-formamidopyrimidine (Fapy•dG) is an abundant form of oxidative DNA damage that is mutagenic and contributes to the pathogenesis of human disease. When Fapy•dG is in its nucleotide triphosphate form, Fapy•dGTP, it is inefficiently cleansed from the nucleotide pool by the responsible enzyme in Escherichia coli MutT and its mammalian homolog MTH1. Therefore, under oxidative stress conditions, Fapy•dGTP could become a pro-mutagenic substrate for insertion into the genome by DNA polymerases. Here, we evaluated insertion kinetics and high-resolution ternary complex crystal structures of a configurationally stable Fapy•dGTP analog, ß-C-Fapy•dGTP, with DNA polymerase ß. The crystallographic snapshots and kinetic data indicate that binding of ß-C-Fapy•dGTP impedes enzyme closure, thus hindering insertion. The structures reveal that an active site residue, Asp276, positions ß-C-Fapy•dGTP so that it distorts the geometry of critical catalytic atoms. Removal of this guardian side chain permits enzyme closure and increases the efficiency of ß-C-Fapy•dG insertion opposite dC. These results highlight the stringent requirements necessary to achieve a closed DNA polymerase active site poised for efficient nucleotide incorporation and illustrate how DNA polymerase ß has evolved to hinder Fapy•dGTP insertion.

Histone tails decrease N7-methyl-2'-deoxyguanosine depurination and yield DNA-protein cross-links in nucleosome core particles and cells.

Monofunctional alkylating agents preferentially react at the N7 position of 2'-deoxyguanosine in duplex DNA. Methylated DNA, such as that produced by methyl methanesulfonate (MMS) and temozolomide, exists for days in organisms. The predominant consequence of N7-methyl-2'-deoxyguanosine (MdG) is widely believed to be abasic site (AP) formation via hydrolysis, a process that is slow in free DNA. Examination of MdG reactivity within nucleosome core particles (NCPs) provided two general observations. MdG depurination rate constants are reduced in NCPs compared with when the identical DNA sequence is free in solution. The magnitude of the decrease correlates with proximity to the positively charged histone tails, and experiments in NCPs containing histone variants reveal that positively charged amino acids are responsible for the decreased rate of abasic site formation from MdG. In addition, the lysine-rich histone tails form DNA-protein cross-links (DPCs) with MdG. Cross-link formation is reversible and is ascribed to nucleophilic attack at the C8 position of MdG. DPC and retarded abasic site formation are observed in NCPs randomly damaged by MMS, indicating that these are general processes. Histone-MdG cross-links were also detected by mass spectrometry in chromatin isolated from V79 Chinese hamster lung cells treated with MMS. The formation of DPCs following damage by a monofunctional alkylating agent has not been reported previously. These observations reveal the possibility that such DPCs may contribute to the cytotoxicity of monofunctional alkylating agents, such as MMS, N-methyl-N-nitrosourea, and temozolomide.