Role of nucleobase-specific interactions in the binding and bending of DNA by human male sex determination factor SRY.

Y-chromosome-encoded master transcription factor SRY functions in the embryogenesis of therian mammals to initiate male development. Through interactions of its conserved high-mobility group box within a widened DNA minor groove, SRY and related Sox factors induce sharp bends at specific DNA target sites. Here, we present the crystal structure of the SRY high-mobility group domain bound to a DNA site containing consensus element 5'-ATTGTT. The structure contains three complexes in the asymmetric unit; in each complex, SRY forms 10 hydrogen bonds with minor-groove base atoms in 5'-CATTGT/ACAATG-3', shifting the recognition sequence by one base pair (italics). These nucleobase interactions involve conserved residues Arg7, Asn10, and Tyr74 on one side of intercalated Ile13 (the cantilever) and Arg20, Asn32, and Ser36 on the other. Unlike the less-bent NMR structure, DNA bend angles (69-84°) of the distinct box-DNA complexes are similar to those observed in homologous Sox domain-DNA structures. Electrophoretic studies indicate that respective substitutions of Asn32, Ser36, or Tyr74 by Ala exhibit slightly attenuated specific DNA-binding affinity and bend angles (70-73°) relative to WT (79°). By contrast, respective substitutions of Arg7, Asn10, or Arg20 by Ala markedly impaired DNA-binding affinity in association with much smaller DNA bend angles (53-65°). In a rodent cell-based model of the embryonic gonadal ridge, full-length SRY variants bearing these respective Ala substitutions exhibited significantly decreased transcriptional activation of SRY's principal target gene (Sox9). Together, our findings suggest that nucleobase-specific hydrogen bonds by SRY are critical for specific DNA binding, bending, and transcriptional activation.

Activation of Gcn2 by small molecules designed to be inhibitors.

The integrated stress response (ISR) is an important mechanism by which cells confer protection against environmental stresses. Central to the ISR is a collection of related protein kinases that monitor stress conditions, such as Gcn2 (EIF2AK4) that recognizes nutrient limitations, inducing phosphorylation of eukaryotic translation initiation factor 2 (eIF2). Gcn2 phosphorylation of eIF2 lowers bulk protein synthesis, conserving energy and nutrients, coincident with preferential translation of stress-adaptive gene transcripts, such as that encoding the Atf4 transcriptional regulator. While Gcn2 is central for cell protection to nutrient stress and its depletion in humans leads to pulmonary disorders, Gcn2 can also contribute to the progression of cancers and facilitate neurological disorders during chronic stress. Consequently, specific ATP-competitive inhibitors of Gcn2 protein kinase have been developed. In this study, we report that one such Gcn2 inhibitor, Gcn2iB, can activate Gcn2, and we probe the mechanism by which this activation occurs. Low concentrations of Gcn2iB increase Gcn2 phosphorylation of eIF2 and enhance Atf4 expression and activity. Of importance, Gcn2iB can activate Gcn2 mutants devoid of functional regulatory domains or with certain kinase domain substitutions derived from Gcn2-deficient human patients. Other ATP-competitive inhibitors can also activate Gcn2, although there are differences in their mechanisms of activation. These results provide a cautionary note about the pharmacodynamics of eIF2 kinase inhibitors in therapeutic applications. Compounds designed to be kinase inhibitors that instead directly activate Gcn2, even loss of function variants, may provide tools to alleviate deficiencies in Gcn2 and other regulators of the ISR.

Mapping immunological and host receptor binding determinants of SARS-CoV spike protein utilizing the Qubevirus platform.

The motifs involved in tropism and immunological interactions of SARS-CoV spike (S) protein were investigated utilizing the Qubevirus platform. We showed that separately, 14 overlapping peptide fragments representing the S protein (F1-14 of 100 residues each) could be inserted into the C terminus of A1 on recombinant Qubevirus without affecting its viability. Additionally, recombinant phage expression resulted in the surface exposure of different engineered fragments in an accessible manner. The F6 from S425-525 was found to contain the binding determinant of the recombinant human angiotensin-converting enzyme 2, with the shortest active binding motif situated between residues S437-492. Upstream, another fragment, F7, containing an overlapping portion of F6 would not bind to recombinant human angiotensin-converting enzyme 2, confirming that a contiguous stretch of residues could adopt the appropriate structural orientation of F6 as an insertion within the Qubevirus. The F6 (S441-460) and other inserts, including F7/F8 (S601-620) and F10 (S781-800), were demonstrated to contain important immunological determinants through recognition and binding of S protein specific (anti-S) antibodies. An engineered chimeric insert bearing the fusion of all three anti-S reactive epitopes improved substantially the recognition and binding to their cognate antibodies. These results provide insights into humoral immune relevant epitopes and tropism characteristics of the S protein with implications for the development of subunit vaccines or other biologics against SARS-CoV.

New Ref-1/APE1 targeted inhibitors demonstrating improved potency for clinical applications in multiple cancer types.

AP endonuclease-1/Redox factor-1 (APE1/Ref-1 or Ref-1) is a multifunctional protein that is overexpressed in most aggressive cancers and impacts various cancer cell signaling pathways. Ref-1's redox activity plays a significant role in activating transcription factors (TFs) such as NFκB, HIF1α, STAT3 and AP-1, which are crucial contributors to the development of tumors and metastatic growth. Therefore, development of potent, selective inhibitors to target Ref-1 redox function is an appealing approach for therapeutic intervention. A first-generation compound, APX3330 successfully completed phase I clinical trial in adults with progressing solid tumors with favorable response rate, pharmacokinetics (PK), and minimal toxicity. These positive results prompted us to develop more potent analogs of APX3330 to effectively target Ref-1 in solid tumors. In this study, we present structure-activity relationship (SAR) identification and validation of lead compounds that exhibit a greater potency and a similar or better safety profile to APX3330. In order to triage and characterize the most potent and on-target second-generation Ref-1 redox inhibitors, we assayed for PK, mouse and human S9 fraction metabolic stability, in silico ADMET properties, ligand-based WaterLOGSY NMR measurements, pharmacodynamic markers, cell viability in multiple cancer cell types, and two distinct 3-dimensional (3D) cell killing assays (Tumor-Microenvironment on a Chip and 3D spheroid). To characterize the effects of Ref-1 inhibition in vivo, global proteomics was used following treatment with the top four analogs. This study identified and characterized more potent inhibitors of Ref-1 redox function (that outperformed APX3330 by 5-10-fold) with PK studies demonstrating efficacious doses for translation to clinic.

Structural and genome-wide analyses suggest that transposon-derived protein SETMAR alters transcription and splicing.

Extensive portions of the human genome have unknown function, including those derived from transposable elements. One such element, the DNA transposon Hsmar1, entered the primate lineage approximately 50 million years ago leaving behind terminal inverted repeat (TIR) sequences and a single intact copy of the Hsmar1 transposase, which retains its ancestral TIR-DNA-binding activity, and is fused with a lysine methyltransferase SET domain to constitute the chimeric SETMAR gene. Here, we provide a structural basis for recognition of TIRs by SETMAR and investigate the function of SETMAR through genome-wide approaches. As elucidated in our 2.37 Å crystal structure, SETMAR forms a dimeric complex with each DNA-binding domain bound specifically to TIR-DNA through the formation of 32 hydrogen bonds. We found that SETMAR recognizes primarily TIR sequences (∼5000 sites) within the human genome as assessed by chromatin immunoprecipitation sequencing analysis. In two SETMAR KO cell lines, we identified 163 shared differentially expressed genes and 233 shared alternative splicing events. Among these genes are several pre-mRNA-splicing factors, transcription factors, and genes associated with neuronal function, and one alternatively spliced primate-specific gene, TMEM14B, which has been identified as a marker for neocortex expansion associated with brain evolution. Taken together, our results suggest a model in which SETMAR impacts differential expression and alternative splicing of genes associated with transcription and neuronal function, potentially through both its TIR-specific DNA-binding and lysine methyltransferase activities, consistent with a role for SETMAR in simian primate development.

Two distinct rotations of bithiazole DNA intercalation revealed by direct comparison of crystal structures of Co(III)•bleomycin A2 and B2 bound to duplex 5'-TAGTT sites.

Bleomycins constitute a family of anticancer natural products that bind DNA through intercalation of a C-terminal tail/bithiazole moiety and hydrogen-bonding interactions between the remainder of the drug and the minor groove. The clinical utility of the bleomycins is believed to result from single- and double-strand DNA cleavage mediated by the HOO-Fe(III) form of the drug. The bleomycins also serve as a model system to understand the nature of complex drug-DNA interactions that may guide future DNA-targeted drug discovery. In this study, the impact of the C-terminal tail on bleomycin-DNA interactions was investigated. Toward this goal, we determined two crystal structures of HOO-Co(III)•BLMA2 "green" (a stable structural analogue of the active HOO-Fe(III) drug) bound to duplex DNA containing 5'-TAGTT, one in which the entire drug is bound (fully bound) and a second with only the C-terminal tail/bithiazole bound (partially bound). The structures reported here were captured by soaking HOO-Co(III)•BLMA2 into preformed host-guest crystals including a preferred DNA-binding site. While the overall structure of DNA-bound BLMA2 was found to be similar to those reported earlier at the same DNA site for BLMB2, the intercalated bithiazole of BLMB2 is "flipped" 180˚ relative to DNA-bound BLMA2. This finding highlights an unidentified role for the C-terminal tail in directing the intercalation of the bithiazole. In addition, these analyses identified specific bond rotations within the C-terminal domain of the drug that may be relevant for its reorganization and ability to carry out a double-strand DNA cleavage event.

A New Drug Discovery Platform: Application to DNA Polymerase Eta and Apurinic/Apyrimidinic Endonuclease 1.

The ability to quickly discover reliable hits from screening and rapidly convert them into lead compounds, which can be verified in functional assays, is central to drug discovery. The expedited validation of novel targets and the identification of modulators to advance to preclinical studies can significantly increase drug development success. Our SaXPyTM ("SAR by X-ray Poses Quickly") platform, which is applicable to any X-ray crystallography-enabled drug target, couples the established methods of protein X-ray crystallography and fragment-based drug discovery (FBDD) with advanced computational and medicinal chemistry to deliver small molecule modulators or targeted protein degradation ligands in a short timeframe. Our approach, especially for elusive or "undruggable" targets, allows for (i) hit generation; (ii) the mapping of protein-ligand interactions; (iii) the assessment of target ligandability; (iv) the discovery of novel and potential allosteric binding sites; and (v) hit-to-lead execution. These advances inform chemical tractability and downstream biology and generate novel intellectual property. We describe here the application of SaXPy in the discovery and development of DNA damage response inhibitors against DNA polymerase eta (Pol η or POLH) and apurinic/apyrimidinic endonuclease 1 (APE1 or APEX1). Notably, our SaXPy platform allowed us to solve the first crystal structures of these proteins bound to small molecules and to discover novel binding sites for each target.

Visualizing "Alternative Isoinformational Engineered" DNA in A- and B-Forms at High Resolution.

A fundamental property of DNA built from four informational nucleotide units (GCAT) is its ability to adopt different helical forms within the context of the Watson-Crick pair. Well-characterized examples include A-, B-, and Z-DNA. For this study, we created an isoinformational biomimetic polymer, built (like standard DNA) from four informational "letters", but with the building blocks being artificial. This ALternative Isoinformational ENgineered (ALIEN) DNA was hypothesized to support two nucleobase pairs, the P:Z pair matching 2-amino-imidazo-[1,2a]-1,3,5-triazin-[8H]-4-one with 6-amino-3-5-nitro-1H-pyridin-2-one and the B:S pair matching 6-amino-4-hydroxy-5-1H-purin-2-one with 3-methyl-6-amino-pyrimidin-2-one. We report two structures of ALIEN DNA duplexes at 1.2 Å resolution and a third at 1.65 Å. All of these are built from a single self-complementary sequence (5'-CTSZZPBSBSZPPBAG) that includes 12 consecutive ALIEN nucleotides. We characterized the helical, nucleobase pair, and dinucleotide step parameters of ALIEN DNA in these structures. In addition to showing that ALIEN pairs retain basic Watson-Crick pairing geometry, two of the ALIEN DNA structures are characterized as A-form DNA and one as B-form DNA. We identified parameters that map differences effecting the transition between the two helical forms; these same parameters distinguish helical forms of isoinformational natural DNA. Collectively, our analyses suggest that ALIEN DNA retains essential structural features of natural DNA, not only its information density and Watson-Crick pairing but also its ability to adopt two canonical forms.

Bis-aryl-α,ß-unsaturated ketone (ABK) chaperonin inhibitors exhibit selective cytotoxicity to colorectal cancer cells that correlates with levels of aberrant HSP60 in the cytosol.

While many studies have established the importance of protein homeostasis in tumor progression, little effort has been made to examine the therapeutic potential of targeting the HSP60 chaperonin system. In healthy cells, HSP60 is localized to the mitochondrial matrix; however, emerging evidence indicates HSP60 can be over-expressed and mis-localized to the cytosol of cancer cells, which is hypothesized to promote tumor cell survival and proliferation. This opens a potential avenue to selectively target the aberrant HSP60 in the cytosol as a chemotherapeutic strategy. In the present work, we examined a series of bis-aryl-α,ß-unsaturated ketone (ABK) HSP60 inhibitors for their ability to selectively target cancerous vs non-cancerous colon and intestine cells. We found that lead analogs inhibited migration and clonogenicity of cancer cells, with cytotoxicity correlating with the level of aberrant HSP60 in the cytosol.

Building better polymerases: Engineering the replication of expanded genetic alphabets.

DNA polymerases are today used throughout scientific research, biotechnology, and medicine, in part for their ability to interact with unnatural forms of DNA created by synthetic biologists. Here especially, natural DNA polymerases often do not have the "performance specifications" needed for transformative technologies. This creates a need for science-guided rational (or semi-rational) engineering to identify variants that replicate unnatural base pairs (UBPs), unnatural backbones, tags, or other evolutionarily novel features of unnatural DNA. In this review, we provide a brief overview of the chemistry and properties of replicative DNA polymerases and their evolved variants, focusing on the Klenow fragment of Taq DNA polymerase (Klentaq). We describe comparative structural, enzymatic, and molecular dynamics studies of WT and Klentaq variants, complexed with natural or noncanonical substrates. Combining these methods provides insight into how specific amino acid substitutions distant from the active site in a Klentaq DNA polymerase variant (ZP Klentaq) contribute to its ability to replicate UBPs with improved efficiency compared with Klentaq. This approach can therefore serve to guide any future rational engineering of replicative DNA polymerases.

Snapshots of an evolved DNA polymerase pre- and post-incorporation of an unnatural nucleotide.

The next challenge in synthetic biology is to be able to replicate synthetic nucleic acid sequences efficiently. The synthetic pair, 2-amino-8-(1-beta-d-2'- deoxyribofuranosyl) imidazo [1,2-a]-1,3,5-triazin-[8H]-4-one (trivially designated P) with 6-amino-3-(2'-deoxyribofuranosyl)-5-nitro-1H-pyridin-2-one (trivially designated Z), is replicated by certain Family A polymerases, albeit with lower efficiency. Through directed evolution, we identified a variant KlenTaq polymerase (M444V, P527A, D551E, E832V) that incorporates dZTP opposite P more efficiently than the wild-type enzyme. Here, we report two crystal structures of this variant KlenTaq, a post-incorporation complex that includes a template-primer with P:Z trapped in the active site (binary complex) and a pre-incorporation complex with dZTP paired to template P in the active site (ternary complex). In forming the ternary complex, the fingers domain exhibits a larger closure angle than in natural complexes but engages the template-primer and incoming dNTP through similar interactions. In the binary complex, although many of the interactions found in the natural complexes are retained, there is increased relative motion of the thumb domain. Collectively, our analyses suggest that it is the post-incorporation complex for unnatural substrates that presents a challenge to the natural enzyme and that more efficient replication of P:Z pairs requires a more flexible polymerase.

Consecutive non-natural PZ nucleobase pairs in DNA impact helical structure as seen in 50 µs molecular dynamics simulations.

Z: Little is known about the influence of multiple consecutive 'non-standard' ( , 6-amino-5-nitro-2(1H)-pyridone, and , 2-amino-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one) nucleobase pairs on the structural parameters of duplex DNA. nucleobase pairs follow standard rules for Watson-Crick base pairing but have rearranged hydrogen bonding donor and acceptor groups. Using the X-ray crystal structure as a starting point, we have modeled the motions of a DNA duplex built from a self-complementary oligonucleotide (5΄-CTTATPPPZZZATAAG-3΄) in water over a period of 50 µs and calculated DNA local parameters, step parameters, helix parameters, and major/minor groove widths to examine how the presence of multiple, consecutive nucleobase pairs might impact helical structure. In these simulations, the -containing DNA duplex exhibits a significantly wider major groove and greater average values of stagger, slide, rise, twist and h-rise than observed for a 'control' oligonucleotide in which nucleobase pairs are replaced by . The molecular origins of these structural changes are likely associated with at least two differences between and . First, the electrostatic properties of differ from in terms of density distribution and dipole moment. Second, differences are seen in the base stacking of pairs in dinucleotide steps, arising from energetically favorable stacking of the nitro group in with π-electrons of the adjacent base.

"Skinny" and "Fat" DNA: Two New Double Helices.

According to the iconic model, the Watson-Crick double helix exploits nucleobase pairs that are both size complementary (big purines pair with small pyrimidines) and hydrogen bond complementary (hydrogen bond donors pair with hydrogen bond acceptors). Using a synthetic biology strategy, we report here the discovery of two new DNA-like systems that appear to support molecular recognition with the same proficiency as standard Watson-Crick DNA. However, these both violate size complementarity (big pairs with small), retaining hydrogen bond complementarity (donors pair with acceptors) as their only specificity principle. They exclude mismatches as well as standard Watson-Crick DNA excludes mismatches. In crystal structures, these "skinny" and "fat" systems form the expected hydrogen bonds, while conferring novel minor groove properties to the resultant duplex regions of the DNA oligonucleotides. Further, computational tools, previously tested primarily on natural DNA, appear to work well for these two new molecular recognition systems, offering a validation of the power of modern computational biology. These new molecular recognition systems may have application in materials science and synthetic biology, and in developing our understanding of alternative ways that genetic information might be stored and transmitted.

Toward an Expanded Genome: Structural and Computational Characterization of an Artificially Expanded Genetic Information System.

Although the fundamental properties of DNA as first proposed by Watson and Crick in 1953 provided a basic understanding of how duplex DNA was organized and might be replicated, it was not until the first crystal structures of DNA (Z-DNA in 1979, B-DNA in 1980, and A-DNA in 1982) that the true complexity of the molecule began to be appreciated. Many crystal structures of oligonucleotides have since shed light on the helical forms that "Watson-Crick" DNA can adopt, their associated groove widths, and the properties of the nucleobase pairs and their interactions in all three helical forms. Additional understanding of the properties of Watson-Crick DNA has been provided by computational studies employing a variety of theoretical methods. Together with these studies devoted to understanding Watson-Crick DNA, recent efforts to expand the genetic alphabet have founded a new field in synthetic biology. One of these efforts, the artificially expanded genetic information system (AEGIS) developed by Steven Benner and co-workers, takes advantage of orthogonal hydrogen bonding to produce DNA comprised of six nucleobase pairs, of which the most extensively studied is referred to as P:Z with P being 2-amino-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one) and Z being 6-amino-5-nitro-2(1H)-pyridone. P:Z forms three edge-on hydrogen bonds that differ from standard Watson-Crick pairs in the arrangement of acceptors and donor groups; P presents acceptor, acceptor, donor, and Z presents donor, donor, acceptor. Z is unique among the AEGIS nucleobases in having a nitro group present in the major groove. PZ-containing DNA has been exploited in a number of clinical applications and is being used to develop receptors and catalysts. Ultimately, the grand challenge will be to create a semisynthetic organism with an expanded genome. Furthermore, just as our understanding of the properties of natural DNA have benefited from structural and computational characterization, so too will our understanding of artificial DNA. This Account focuses on the structural and biophysical properties of AEGIS DNA containing P:Z pairs. We begin with the fundamental properties of P:Z nucleobase pairs, including their electrostatic potential and hydrogen-bonding energies, as elucidated by quantum mechanical calculations. We then examine the impact of including multiple consecutive P:Z pairs into duplex DNA providing an opportunity to investigate stacking interactions between P:Z pairs. The self-complementary 5'-CTTATPPTAZZATAAG was crystallized in B-form using the host-guest system along with analogous natural sequences including Gs or As. Use of the host-guest system to characterize B-DNA obviates a number of limitations on the structural characterization of sequences of interest; these include the ability to crystallize the desired sequences and to distinguish structural effects imparted by the lattice constraints from those inherent in the sequence itself. On the other hand, 3/6ZP, 5'-CTTATPPPZZZATAAG, was crystallized in A-form in a DNA-only lattice allowing a comparative analysis of P:Z pairs in two of the biologically relevant helical forms: A- and B-DNA. Computational studies on the 3/6ZP sequence starting in A-form provide additional evidence for a more energetically favorable stacking interaction, which we term the "slide" conformer, observed in the A-form crystal structure; this unusual stacking interaction plays a major role in altering the conformational dynamics observed for the PZ-containing duplex as compared to a GC-containing "control" duplex in long time scale molecular dynamics simulations. This combined use of structural and computational strategies paves the way for obtaining a detailed description of artificial DNA, both in how it differs from Watson-Crick DNA and in the rational discovery of proteins, such as endonucleases, transcription factors, and polymerases, which can specifically manipulate DNA containing AEGIS nucleobase pairs.

Crystal structures of GCN2 protein kinase C-terminal domains suggest regulatory differences in yeast and mammals.

In response to amino acid starvation, GCN2 phosphorylation of eIF2 leads to repression of general translation and initiation of gene reprogramming that facilitates adaptation to nutrient stress. GCN2 is a multidomain protein with key regulatory domains that directly monitor uncharged tRNAs which accumulate during nutrient limitation, leading to activation of this eIF2 kinase and translational control. A critical feature of regulation of this stress response kinase is its C-terminal domain (CTD). Here, we present high resolution crystal structures of murine and yeast CTDs, which guide a functional analysis of the mammalian GCN2. Despite low sequence identity, both yeast and mammalian CTDs share a core subunit structure and an unusual interdigitated dimeric form, albeit with significant differences. Disruption of the dimeric form of murine CTD led to loss of translational control by GCN2, suggesting that dimerization is critical for function as is true for yeast GCN2. However, although both CTDs bind single- and double-stranded RNA, murine GCN2 does not appear to stably associate with the ribosome, whereas yeast GCN2 does. This finding suggests that there are key regulatory differences between yeast and mammalian CTDs, which is consistent with structural differences.

The DDN catalytic motif is required for Metnase functions in non-homologous end joining (NHEJ) repair and replication restart.

Metnase (or SETMAR) arose from a chimeric fusion of the Hsmar1 transposase downstream of a protein methylase in anthropoid primates. Although the Metnase transposase domain has been largely conserved, its catalytic motif (DDN) differs from the DDD motif of related transposases, which may be important for its role as a DNA repair factor and its enzymatic activities. Here, we show that substitution of DDN(610) with either DDD(610) or DDE(610) significantly reduced in vivo functions of Metnase in NHEJ repair and accelerated restart of replication forks. We next tested whether the DDD or DDE mutants cleave single-strand extensions and flaps in partial duplex DNA and pseudo-Tyr structures that mimic stalled replication forks. Neither substrate is cleaved by the DDD or DDE mutant, under the conditions where wild-type Metnase effectively cleaves ssDNA overhangs. We then characterized the ssDNA-binding activity of the Metnase transposase domain and found that the catalytic domain binds ssDNA but not dsDNA, whereas dsDNA binding activity resides in the helix-turn-helix DNA binding domain. Substitution of Asn-610 with either Asp or Glu within the transposase domain significantly reduces ssDNA binding activity. Collectively, our results suggest that a single mutation DDN(610) → DDD(610), which restores the ancestral catalytic site, results in loss of function in Metnase.

Structural basis for a six nucleotide genetic alphabet.

Expanded genetic systems are most likely to work with natural enzymes if the added nucleotides pair with geometries that are similar to those displayed by standard duplex DNA. Here, we present crystal structures of 16-mer duplexes showing this to be the case with two nonstandard nucleobases (Z, 6-amino-5-nitro-2(1H)-pyridone and P, 2-amino-imidazo[1,2-a]-1,3,5-triazin-4(8H)one) that were designed to form a Z:P pair with a standard "edge on" Watson-Crick geometry, but joined by rearranged hydrogen bond donor and acceptor groups. One duplex, with four Z:P pairs, was crystallized with a reverse transcriptase host and adopts primarily a B-form. Another contained six consecutive Z:P pairs; it crystallized without a host in an A-form. In both structures, Z:P pairs fit canonical nucleobase hydrogen-bonding parameters and known DNA helical forms. Unique features include stacking of the nitro group on Z with the adjacent nucleobase ring in the A-form duplex. In both B- and A-duplexes, major groove widths for the Z:P pairs are approximately 1 Å wider than those of comparable G:C pairs, perhaps to accommodate the large nitro group on Z. Otherwise, ZP-rich DNA had many of the same properties as CG-rich DNA, a conclusion supported by circular dichroism studies in solution. The ability of standard duplexes to accommodate multiple and consecutive Z:P pairs is consistent with the ability of natural polymerases to biosynthesize those pairs. This, in turn, implies that the GACTZP synthetic genetic system can explore the entire expanded sequence space that additional nucleotides create, a major step forward in this area of synthetic biology.

High-resolution crystal structures reveal plasticity in the metal binding site of apurinic/apyrimidinic endonuclease I.

Apurinic/apyrimidinic endonuclease I (APE1) is an essential base excision repair enzyme that catalyzes a Mg²âº-dependent reaction in which the phosphodiester backbone is cleaved 5' of an abasic site in duplex DNA. This reaction has been proposed to involve either one or two metal ions bound to the active site. In the present study, we report crystal structures of Mg²âº, Mn²âº, and apo-APE1 determined at 1.4, 2.2, and 1.65 Å, respectively, representing two of the highest resolution structures yet reported for APE1. In our structures, a single well-ordered Mn²âº ion was observed coordinated by D70 and E96; the Mg²âº site exhibited disorder modeled as two closely positioned sites coordinated by D70 and E96 or E96 alone. Direct metal binding analysis of wild-type, D70A, and E96A APE1, as assessed by differential scanning fluorimetry, indicated a role for D70 and E96 in binding of Mg²âº or Mn²âº to APE1. Consistent with the disorder exhibited by Mg²âº bound to the active site, two different conformations of E96 were observed coordinated to Mg²âº. A third conformation for E96 in the apo structure is similar to that observed in the APE1-DNA-Mg²âº complex structure. Thus, binding of Mg²âº in three different positions within the active site of APE1 in these crystal structures corresponds directly with three different conformations of E96. Taken together, our results are consistent with the initial capture of metal by D70 and E96 and repositioning of Mg²âº facilitated by the structural plasticity of E96 in the active site.

The structure of an authentic spore photoproduct lesion in DNA suggests a basis for recognition.

The spore photoproduct lesion (SP; 5-thymine-5,6-dihydrothymine) is the dominant photoproduct found in UV-irradiated spores of some bacteria such as Bacillus subtilis. Upon spore germination, this lesion is repaired in a light-independent manner by a specific repair enzyme: the spore photoproduct lyase (SP lyase). In this work, a host-guest approach in which the N-terminal fragment of Moloney murine leukemia virus reverse transcriptase (MMLV RT) serves as the host and DNA as the guest was used to determine the crystal structures of complexes including 16 bp oligonucleotides with and without the SP lesion at 2.14 and 1.72 Šresolution, respectively. In contrast to other types of thymine-thymine lesions, the SP lesion retains normal Watson-Crick hydrogen bonding to the adenine bases of the complementary strand, with shorter hydrogen bonds than found in the structure of the undamaged DNA. However, the lesion induces structural changes in the local conformation of what is otherwise B-form DNA. The region surrounding the lesion differs significantly in helical form from B-DNA, and the minor groove is widened by almost 3 Šcompared with that of the undamaged DNA. Thus, these unusual structural features associated with SP lesions may provide a basis for recognition by the SP lyase.

Inhibition of apurinic/apyrimidinic endonuclease I's redox activity revisited.

The essential base excision repair protein, apurinic/apyrimidinic endonuclease 1 (APE1), plays an important role in redox regulation in cells and is currently targeted for the development of cancer therapeutics. One compound that binds APE1 directly is (E)-3-[2-(5,6-dimethoxy-3-methyl-1,4-benzoquinonyl)]-2-nonylpropenoic acid (E3330). Here, we revisit the mechanism by which this negatively charged compound interacts with APE1 and inhibits its redox activity. At high concentrations (millimolar), E3330 interacts with two regions in the endonuclease active site of APE1, as mapped by hydrogen-deuterium exchange mass spectrometry. However, this interaction lowers the melting temperature of APE1, which is consistent with a loss of structure in APE1, as measured by both differential scanning fluorimetry and circular dichroism. These results are consistent with other findings that E3330 concentrations of >100 µM are required to inhibit APE1's endonuclease activity. To determine the role of E3330's negatively charged carboxylate in redox inhibition, we converted the carboxylate to an amide by synthesizing (E)-2-[(4,5-dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)methylene]-N-methoxy-undecanamide (E3330-amide), a novel uncharged derivative. E3330-amide has no effect on the melting temperature of APE1, suggesting that it does not interact with the fully folded protein. However, E3330-amide inhibits APE1's redox activity in in vitro electrophoretic mobility shift redox and cell-based transactivation assays, producing IC(50) values (8.5 and 7 µM) lower than those produced with E3330 (20 and 55 µM, respectively). Thus, E3330's negatively charged carboxylate is not required for redox inhibition. Collectively, our results provide additional support for a mechanism of redox inhibition involving interaction of E3330 or E3330-amide with partially unfolded APE1.