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The eukaryotic DNA replication machinery is conserved from yeast to humans and requires the actions of multiple DNA polymerases. In addition to replicative DNA polymerases for duplication of the leading and lagging DNA strands, another group of specialized polymerases is required for DNA repair and/or translesion DNA synthesis (TLS). We emphasize here recent findings that accelerate our understanding of the structure and mechanisms of these remarkable enzymes. We also highlight growing evidence on the role of DNA polymerases in the origin of certain cancers, and paradoxically as emerging targets for cancer therapy., (Copyright © 2018 Elsevier Ltd. All rights reserved.) more...

The idea that signal-dependent transcription might involve the generation of transient DNA nicks or even breaks in the regulatory regions of genes, accompanied by activation of DNA damage repair pathways, would seem to be counterintuitive, as DNA damage is usually considered harmful to cellular integrity. However, recent studies have generated a substantial body of evidence that now argues that programmed DNA single- or double-strand breaks can, at least in specific cases, have a role in transcription regulation. Here, we discuss the emerging functions of DNA breaks in the relief of DNA torsional stress and in promoter and enhancer activation. more...

N1-methyl-deoxyadenosine (1-MeA) is formed by methylation of deoxyadenosine at the N1 atom. 1-MeA presents a block to replicative DNA polymerases due to its inability to participate in Watson-Crick (W-C) base pairing. Here we determine how human DNA polymerase-ι (Polι) promotes error-free replication across 1-MeA. Steady state kinetic analyses indicate that Polι is ~100 fold more efficient in incorporating the correct nucleotide T versus the incorrect nucleotide C opposite 1-MeA. To understand the basis of this selectivity, we determined ternary structures of Polι bound to template 1-MeA and incoming dTTP or dCTP. In both structures, template 1-MeA rotates to the syn conformation but pairs differently with dTTP versus dCTP. Thus, whereas dTTP partakes in stable Hoogsteen base pairing with 1-MeA, dCTP fails to gain a "foothold" and is largely disordered. Together, our kinetic and structural studies show how Polι maintains discrimination between correct and incorrect incoming nucleotide opposite 1-MeA in preserving genome integrity. more...

The creation of restriction enzymes with programmable DNA-binding and -cleavage specificities has long been a goal of modern biology. The recently discovered Type IIL MmeI family of restriction-and-modification (RM) enzymes that possess a shared target recognition domain provides a framework for engineering such new specificities. However, a lack of structural information on Type IIL enzymes has limited the repertoire that can be rationally engineered. We report here a crystal structure of MmeI in complex with its DNA substrate and an S-adenosylmethionine analog (Sinefungin). The structure uncovers for the first time the interactions that underlie MmeI-DNA recognition and methylation (5'-TCCRAC-3'; R = purine) and provides a molecular basis for changing specificity at four of the six base pairs of the recognition sequence (5'-TCCRAC-3'). Surprisingly, the enzyme is resilient to specificity changes at the first position of the recognition sequence (5'-TCCRAC-3'). Collectively, the structure provides a basis for engineering further derivatives of MmeI and delineates which base pairs of the recognition sequence are more amenable to alterations than others. more...

The Polα/primase complex assembles the short RNA-DNA fragments for priming of lagging and leading strand DNA replication in eukaryotes. As such, the Polα polymerase subunit encounters two types of substrates during primer synthesis: an RNA:DNA helix and a DNA:DNA helix. The engagement of the polymerase subunit with the DNA:DNA helix has been suggested as the of basis for primer termination in eukaryotes. However, there is no structural information on how the Polα polymerase subunit actually engages with a DNA:DNA helix during primer synthesis. We present here the first crystal structure of human Polα polymerase subunit in complex with a DNA:DNA helix. Unexpectedly, we find that portion of the DNA:DNA helix in contact with the polymerase is not in a B-form but in a hybrid A-B form. Almost all of the contacts observed previously with an RNA primer are preserved with a DNA primer--with the same set of polymerase residues tracking the sugar-phosphate backbone of the DNA or RNA primer. Thus, rather than loss of specific contacts, the free energy cost of distorting DNA from B- to hybrid A-B form may augur the termination of primer synthesis in eukaryotes. more...

Type III R-M enzymes were identified >40 years ago and yet there is no structural information on these multisubunit enzymes. Here we report the structure of a Type III R-M system, consisting of the entire EcoP15I complex (Mod2Res1) bound to DNA. The structure suggests how ATP hydrolysis is coupled to long-range diffusion of a helicase on DNA, and how a dimeric methyltransferase functions to methylate only one of the two DNA strands. We show that the EcoP15I motor domains are specifically adapted to bind double-stranded DNA and to facilitate DNA sliding via a novel 'Pin' domain. We also uncover unexpected 'division of labour', where one Mod subunit recognizes DNA, while the other Mod subunit methylates the target adenine--a mechanism that may extend to adenine N6 RNA methylation in mammalian cells. Together the structure sheds new light on the mechanisms of both helicases and methyltransferases in DNA and RNA metabolism. more...

EcoP15I is the prototype of the Type III restriction enzyme family, composed of two modification (Mod) subunits to which two (or one) restriction (Res) subunits are then added. The Mod subunits are responsible for DNA recognition and methylation, while the Res subunits are responsible for ATP hydrolysis and cleavage. Despite extensive biochemical and genetic studies, there is still no structural information on Type III restriction enzymes. We present here small-angle X-ray scattering (SAXS) and analytical ultracentrifugation analysis of the EcoP15I holoenzyme and the Mod(2) subcomplex. We show that the Mod(2) subcomplex has a relatively compact shape with a radius of gyration (R(G)) of ∼37.4 Å and a maximal dimension of ∼110 Å. The holoenzyme adopts an elongated crescent shape with an R(G) of ∼65.3 Å and a maximal dimension of ∼218 Å. From reconstructed SAXS envelopes, we postulate that Mod(2) is likely docked in the middle of the holoenzyme with a Res subunit at each end. We discuss the implications of our model for EcoP15I action, whereby the Res subunits may come together and form a "sliding clamp" around the DNA., (Copyright © 2012 Elsevier Ltd. All rights reserved.) more...

Type IIL restriction enzymes have rejuvenated the search for user-specified DNA binding and cutting. By aligning and contrasting the highly comparable amino-acid sequences yet diverse recognition specificities across the family of enzymes, amino acids involved in DNA binding have been identified and mutated to produce alternative binding specificities. To date, the specificity of MmeI (a type IIL restriction enzyme) has successfully been altered at positions 3, 4 and 6 of the asymmetric TCCRAC (where R is a purine) DNA-recognition sequence. To further understand the structural basis of MmeI DNA-binding specificity, the enzyme has been crystallized in complex with its DNA substrate. The crystal belonged to space group P1, with unit-cell parameters a = 61.73, b = 94.96, c = 161.24 Å, α = 72.79, β = 89.12, γ = 71.68°, and diffracted to 2.6 Å resolution when exposed to synchrotron radiation. The structure promises to reveal the basis of MmeI DNA-binding specificity and will complement efforts to create enzymes with novel specificities., (© 2011 International Union of Crystallography. All rights reserved.) more...

Interferon regulatory factors IRF-3 and IRF-7 are transcription factors essential in the activation of interferon-β (IFN-β) gene in response to viral infections. Although, both proteins recognize the same consensus IRF binding site AANNGAAA, they have distinct DNA binding preferences for sites in vivo. The X-ray structures of IRF-3 and IRF-7 DNA binding domains (DBDs) bound to IFN-β promoter elements revealed flexibility in the loops (L1-L3) and the residues that make contacts with the target sequence. To characterize the conformational changes that occur on DNA binding and how they differ between IRF family members, we have solved the X-ray structures of IRF-3 and IRF-7 DBDs in the absence of DNA. We found that loop L1, carrying the conserved histidine that interacts with the DNA minor groove, is disordered in apo IRF-3 but is ordered in apo IRF-7. This is reflected in differences in DNA binding affinities when the conserved histidine in loop L1 is mutated to alanine in the two proteins. The stability of loop L1 in IRF-7 derives from a unique combination of hydrophobic residues that pack against the protein core. Together, our data show that differences in flexibility of loop L1 are an important determinant of differential IRF-DNA binding. more...

Restriction enzymes share little or no sequence homology with the exception of isoschizomers, or enzymes that recognize and cleave the same DNA sequence. We present here the structure of a BamHI isoschizomer, OkrAI, bound to the same DNA sequence (TATGGATCCATA) as that cocrystallized with BamHI. We show that OkrAI is a more minimal version of BamHI, lacking not only the N- and C-terminal helices but also an internal 3(10) helix and containing β-strands that are shorter than those in BamHI. Despite these structural differences, OkrAI recognizes the DNA in a remarkably similar manner to BamHI, including asymmetric contacts via C-terminal 'arms' that appear to 'compete' for the minor groove. However, the arms are shorter than in BamHI. We observe similar DNA-binding affinities between OkrAI and BamHI but OkrAI has higher star activity (at 37°C) compared to BamHI. Together, the OkrAI and BamHI structures offer a rare opportunity to compare two restriction enzymes that work on exactly the same DNA substrate. more...

DNA polymerase delta (Pol delta) is a high-fidelity polymerase that has a central role in replication from yeast to humans. We present the crystal structure of the catalytic subunit of yeast Pol delta in ternary complex with a template primer and an incoming nucleotide. The structure, determined at 2.0-A resolution, catches the enzyme in the act of replication, revealing how the polymerase and exonuclease domains are juxtaposed relative to each other and how a correct nucleotide is selected and incorporated. The structure also reveals the 'sensing' interactions near the primer terminus, which signal a switch from the polymerizing to the editing mode. Taken together, the structure provides a chemical basis for the bulk of DNA synthesis in eukaryotic cells and a framework for understanding the effects of cancer-causing mutations in Pol delta. more...

Y-family DNA polymerases have proven to be remarkably diverse in their functions and in strategies for replicating through DNA lesions. The structure of yeast Rev1 ternary complex has revealed the most radical replication strategy, where the polymerase itself dictates the identity of the incoming nucleotide, as well as the identity of the templating base. We show here that many of the key elements of this highly unusual strategy are conserved between yeast and human Rev1, including the eviction of template G from the DNA helix and the pairing of incoming deoxycytidine 5'-triphosphate with a surrogate arginine residue. We also show that the catalytic core of human Rev1 is uniquely augmented by two large inserts, I1 and I2, wherein I1 extends >20 A away from the active site and may serve as a platform for protein-protein interactions specific for Rev1's role in translesion DNA synthesis in human cells, and I2 acts as a "flap" on the hydrophobic pocket accommodating template G. We suggest that these novel structural features are important for providing human Rev1 greater latitude in promoting efficient and error-free translesion DNA synthesis through the diverse array of bulky and potentially carcinogenic N(2)-deoxyguanosine DNA adducts in human cells. more...

The recognition of specific DNA-binding sites by transcription factors is a critical yet poorly understood step in the control of gene expression. Members of the Hox family of transcription factors bind DNA by making nearly identical major groove contacts via the recognition helices of their homeodomains. In vivo specificity, however, often depends on extended and unstructured regions that link Hox homeodomains to a DNA-bound cofactor, Extradenticle (Exd). Using a combination of structure determination, computational analysis, and in vitro and in vivo assays, we show that Hox proteins recognize specific Hox-Exd binding sites via residues located in these extended regions that insert into the minor groove but only when presented with the correct DNA sequence. Our results suggest that these residues, which are conserved in a paralog-specific manner, confer specificity by recognizing a sequence-dependent DNA structure instead of directly reading a specific DNA sequence. more...

DNA recognition by proteins is essential for specific expression of genes in a living organism. En route to a target DNA site, a protein will often sample noncognate DNA sites through nonspecific protein-DNA interactions, resulting in a variety of conformationally different binding states. We present here the crystal structure of endonuclease BstYI bound to a noncognate DNA. Surprisingly, the structure reveals the enzyme in a "hemispecific" binding state on the pathway between nonspecific and specific recognition. A single base pair change in the DNA abolishes binding of only one monomer, with the second monomer bound specifically. We show that the enzyme binds essentially as a rigid body, and that one end of the DNA is accommodated loosely in the binding cleft while the other end is held tightly. Another intriguing feature of the structure is Ser172, which has a dual role in establishing nonspecific and specific contacts. Taken together, the structure provides a snapshot of an enzyme in a "paused" intermediate state that may be part of a more general mechanism of scanning DNA. more...

Human DNA polymerase kappa (Pol kappa) is a proficient extender of mispaired primer termini on undamaged DNAs and is implicated in the extension step of lesion bypass. We present here the structure of Pol kappa catalytic core in ternary complex with DNA and an incoming nucleotide. The structure reveals encirclement of the DNA by a unique "N-clasp" at the N terminus of Pol kappa, which augments the conventional right-handed grip on the DNA by the palm, fingers, and thumb domains and the PAD and provides additional thermodynamic stability. The structure also reveals an active-site cleft that is constrained by the close apposition of the N-clasp and the fingers domain, and therefore can accommodate only a single Watson-Crick base pair. Together, DNA encirclement and other structural features help explain Pol kappa's ability to extend mismatches and to promote replication through various minor groove DNA lesions, by extending from the nucleotide incorporated opposite the lesion by another polymerase. more...

Many reactions in cells proceed via the sequestration of two DNA molecules in a synaptic complex. SfiI is a member of a growing family of restriction enzymes that can bind and cleave two DNA sites simultaneously. We present here the structures of tetrameric SfiI in complex with cognate DNA. The structures reveal two different binding states of SfiI: one with both DNA-binding sites fully occupied and the other with fully and partially occupied sites. These two states provide details on how SfiI recognizes and cleaves its target DNA sites, and gives insight into sequential binding events. The SfiI recognition sequence (GGCCNNNN[downward arrow]NGGCC) is a subset of the recognition sequence of BglI (GCCNNNN[downward arrow]NGGC), and both enzymes cleave their target DNAs to leave 3-base 3' overhangs. We show that even though SfiI is a tetramer and BglI is a dimer, and there is little sequence similarity between the two enzymes, their modes of DNA recognition are unusually similar. more...

The type II restriction endonuclease BstYI recognizes the degenerate sequence 5'-RGATCY-3' (where R = A/G and Y = C/T), which overlaps with both BamHI (GGATCC) and BglII (AGATCT), and thus raises the question of whether BstYI DNA recognition will be more BamHI-like or BglII-like. We present here the structure of BstYI bound to a cognate DNA sequence (AGATCT). We find the complex to be more BglII-like with similarities mapping to DNA conformation, domain organization, and residues involved in catalysis. However, BstYI is unique in containing an extended arm subdomain, and the mechanism of DNA capture has both BglII-like and BamHI-like elements. Further, DNA recognition is more minimal than BglII and BamHI, where only two residues mediate recognition of the entire core sequence. Taken together, the structure reveals a mechanism of degenerate DNA recognition and offers insights into the possibilities and limitations in altering specificities of closely related restriction enzymes. more...

We have recently published the crystal structure of the adeno-associated virus type 2 superfamily 3 (SF3) helicase Rep40. Although based on its biochemical properties it is unlikely that Rep40 plays a central role as a replicative helicase the involvement of this motor protein in DNA packaging has recently been demonstrated. Here we focused our attention on residues that fall within and adjacent to the B' motif of SF3 helicases that directly interact with single-stranded DNA during translocation of the motor protein. In vitro, alanine substitution at positions Lys-404 or Lys-406 abrogated the ability of the protein to interact with single-stranded DNA as demonstrated by electrophoretic mobility shift assay and fluorescence anisotropy, and accordingly these mutants could not unwind a partially duplex DNA substrate. Despite this loss of helicase activity, basal ATPase activity in these mutants remained intact. However, unlike the wild-type protein, K404A and K406A ATPase activity was not stimulated by DNA. As predicted, disruption of motor activity through interference with DNA binding resulted in an inability of Rep40 to package adeno-associated virus DNA in a tissue culture-based assay. Taken together, we characterized, for the first time in an SF3 helicase family member, residues that are directly involved in single-stranded DNA binding and that are critical for the Rep motor activity. Based on our findings we propose B' as the signature motif of SF3 helicases that is responsible for the complex interactions required for the coupling of DNA binding and ATP hydrolysis. more...

Almost all DNA polymerases show a strong preference for incorporating the nucleotide that forms the correct Watson-Crick base pair with the template base. In addition, the catalytic efficiencies with which any given polymerase forms the four possible correct base pairs are roughly the same. Human DNA polymerase-iota (hPoliota), a member of the Y family of DNA polymerases, is an exception to these rules. hPoliota incorporates the correct nucleotide opposite a template adenine with a several hundred to several thousand fold greater efficiency than it incorporates the correct nucleotide opposite a template thymine, whereas its efficiency for correct nucleotide incorporation opposite a template guanine or cytosine is intermediate between these two extremes. Here we present the crystal structure of hPoliota bound to a template primer and an incoming nucleotide. The structure reveals a polymerase that is 'specialized' for Hoogsteen base-pairing, whereby the templating base is driven to the syn conformation. Hoogsteen base-pairing offers a basis for the varied efficiencies and fidelities of hPoliota opposite different template bases, and it provides an elegant mechanism for promoting replication through minor-groove purine adducts that interfere with replication. more...

The molecular mechanism by which DNA-binding proteins find their specific binding sites is still unclear. To gain insights into structural and energetic elements of this mechanism, we used the crystal structure of the nonspecific BamHI-DNA complex as a template to study the dominant electrostatic interaction in the nonspecific association of protein with DNA, and the possible sliding pathways that could be sustained by such an interaction. Based on calculations using the nonlinear Poisson-Boltzmann method and Brownian dynamics, a model is proposed for the initial nonspecific binding of BamHI to B-form DNA that differs from that seen in the crystal structure of the nonspecific complex. The model is electrostatically favorable, and the salt dependence as well as other thermodynamic parameters calculated for this model are in good agreement with experimental results. Several residues in BamHI are identified for their important contribution to the energy in the nonspecific binding model, and specific mutagenesis experiments are proposed to test the model on this basis. We show that a favorable sliding pathway of the protein along DNA is helical. more...

The Ets and IRF transcription factor families contain structurally divergent members, PU.1, Spi-B and IRF-4 (Pip), IRF-8 (ICSBP), respectively, which have evolved to cooperatively assemble on composite DNA elements and regulate gene expression in the immune system. Whereas PU.1 recruits IRF-4 or IRF-8 to DNA, it exhibits an anticooperative interaction with IRF-1 and IRF-2. We report here the structure of the ternary complex formed with the DNA binding domains of PU.1 and IRF-4 on a composite DNA element. The DNA in the complex contorts into an unusual S shape that juxtaposes PU.1 and IRF-4 for selective electrostatic and hydrophobic interactions across the central minor groove. Together, the protein-protein and protein-DNA interactions provide insights into the stereochemical basis of cooperativity and anti-cooperativity between Ets and IRF factors. more...

PU.1 and IRF-4, members of the Ets and interferon regulatory families of transcription factors, respectively, form a cooperative ternary complex that is implicated in the regulation of B-cell-specific gene expression. A portion of the cooperativity involves interaction between the PU.1 and IRF-4 DNA-binding domains. We report here the crystallization and preliminary characterization of PU.1 and IRF-4 DNA-binding domains bound to a 21-mer DNA site from the lambdaB element of immunoglobulin light chain lambda(2-4) enhancer. The crystals belong to space group P2(1) with unit cell dimensions of a=40.7A, b=62.3A, c=67.9A, beta=95.6 degrees with one complex molecule per asymmetric unit. Crystals diffract to a resolution of 3A using X-rays from a rotating anode generator but improve to 2.3A with synchrotron radiation. The crystals are highly mosaic, but the mosaicity can be improved following a series of steps involving the addition of additives, use of peritone oil as a cryoprotectant, slow flash-cooling in the cryostream, and several cycles of crystal annealing. more...

Upon viral infection, NF-kappaB translocates to the nucleus and activates the IFN-beta gene by binding to the PRDII element. Strikingly, NF-kappaB loses its ability to activate the IFN-beta gene when the PRDII element is substituted by closely related sites. We report here the crystal structure of NF-kappaB p50/p65 heterodimer bound to the PRDII element from the IFN-beta promoter. The structure reveals an unexpected alteration in configuration, in which the p50 specificity domain moves by as much as approximately 9 A when compared to NF-kappaB heterodimer bound to the immunoglobulin kappaB site (Ig-kappaB) while maintaining the same base-specific contacts with the DNA. Taken together, the structure offers new insights into the allosteric effects of closely related DNA sites on the configuration of NF-kappaB and its transcriptional selectivity. more...

Enhancer activation serves as the main mechanism regulating signal-dependent transcriptional programs, ensuring cellular plasticity, yet central questions persist regarding their mechanism of activation. Here, by successfully mapping topoisomerase I-DNA covalent complexes genome-wide, we find that most, if not all, acutely activated enhancers, including those induced by 17β-estradiol, dihydrotestosterone, tumor necrosis factor alpha and neuronal depolarization, are hotspots for topoisomerase I-DNA covalent complexes, functioning as epigenomic signatures read by the classic DNA damage sensor protein, Ku70. Ku70 in turn nucleates a heterochromatin protein 1 gamma (HP1γ)-mediator subunit Med26 complex to facilitate acute, but not chronic, transcriptional activation programs. Together, our data uncover a broad, unappreciated transcriptional code, required for most, if not all, acute signal-dependent enhancer activation events in both mitotic and postmitotic cells. more...

A short prokaryotic Argonaute (pAgo) TIR-APAZ (SPARTA) defense system, activated by invading DNA to unleash its TIR domain for NAD(P)+ hydrolysis, was recently identified in bacteria. We report the crystal structure of SPARTA heterodimer in the absence of guide-RNA/target-ssDNA (2.66 Å) and a cryo-EM structure of the SPARTA oligomer (tetramer of heterodimers) bound to guide-RNA/target-ssDNA at nominal 3.15–3.35 Å resolution. The crystal structure provides a high-resolution view of SPARTA, revealing the APAZ domain as equivalent to the N, L1, and L2 regions of long pAgos and the MID domain containing a unique insertion (insert57). Cryo-EM structure reveals regions of the PIWI (loop10-9) and APAZ (helix αN) domains that reconfigure for nucleic-acid binding and decrypts regions/residues that reorganize to expose a positively charged pocket for higher-order assembly. The TIR domains amass in a parallel-strands arrangement for catalysis. We visualize SPARTA before and after RNA/ssDNA binding and uncover the basis of its active assembly leading to abortive infection. The SPARTA defense system, activated by invading DNA for NAD(P)+ hydrolysis, was recently identified in bacteria. Here, authors visualize SPARTA before and after nucleic acid binding and uncover the basis of its active assembly leading to abortive infection. [ABSTRACT FROM AUTHOR] more...

PrimPol is a human DNA polymerase-primase that localizes to mitochondria and nucleus and bypasses the major oxidative lesion 7,8-dihydro-8-oxoguanine (oxoG) via translesion synthesis, in mostly error-free manner. We present structures of PrimPol insertion complexes with a DNA template-primer and correct dCTP or erroneous dATP opposite the lesion, as well as extension complexes with C or A as a 3′−terminal primer base. We show that during the insertion of C and extension from it, the active site is unperturbed, reflecting the readiness of PrimPol to accommodate oxoG(anti). The misinsertion of A opposite oxoG(syn) also does not alter the active site, and is likely less favorable due to lower thermodynamic stability of the oxoG(syn)•A base-pair. During the extension step, oxoG(syn) induces an opening of its base-pair with A or misalignment of the 3′-A primer terminus. Together, the structures show how PrimPol accurately synthesizes DNA opposite oxidatively damaged DNA in human cells. The human DNA primase and DNA polymerase PrimPol replicates through the major oxidative DNA damage lesion 7,8-dihydro-8-oxoguanine (oxoG) via translesion synthesis in a mostly error-free manner thus suppressing oxoG-induced mutagenesis in mitochondria and the nucleus. Here, the authors present crystal structures of PrimPol in complex with an oxoG lesion in different contexts that provide mechanistic insights into how PrimPol performs predominantly accurate synthesis on oxidative-damaged DNAs in human cells. [ABSTRACT FROM AUTHOR] more...

DNA polymerase ε (Polε) is a multi-subunit polymerase that contributes to genomic stability via its roles in leading strand replication and the repair of damaged DNA. Here we report the ternary structure of the Polε catalytic subunit (Pol2) bound to a nascent G:C base pair (Pol2G:C). Pol2G:C has a typical B-family polymerase fold and embraces the template-primer duplex with the palm, fingers, thumb and exonuclease domains. The overall arrangement of domains is similar to the structure of Pol2T:A reported recently, but there are notable differences in their polymerase and exonuclease active sites. In particular, we observe Ca2+ ions at both positions A and B in the polymerase active site and also observe a Ca2+ at position B of the exonuclease site. We find that the contacts to the nascent G:C base pair in the Pol2G:C structure are maintained in the Pol2T:A structure and reflect the comparable fidelity of Pol2 for nascent purine-pyrimidine and pyrimidine-purine base pairs. We note that unlike that of Pol3, the shape of the nascent base pair binding pocket in Pol2 is modulated from the major grove side by the presence of Tyr431. Together with Pol2T:A, our results provide a framework for understanding the structural basis of high fidelity DNA synthesis by Pol2. [ABSTRACT FROM AUTHOR] more...

Restriction endonuclease BglII completely encircles its target DNA, making contacts to both the major and minor grooves. To allow the DNA to enter and leave the binding cleft, the enzyme dimer has to rearrange. To understand how this occurs, we have solved the structure of the free enzyme at 2.3 Å resolution, as a complement to our earlier work on the BglII?DNA complex. Unexpectedly, the enzyme opens by a dramatic `scissor-like' motion, accompanied by a complete rearrangement of the α-helices at the dimer interface. Moreover, within each monomer, a set of residues ? a ‘lever’ ? lowers or raises to alternately sequester or expose the active site residues. Such an extreme difference in free versus complexed structures has not been reported for other restriction endonucleases. This elegant mechanism for capturing DNA may extend to other enzymes that encircle DNA. [ABSTRACT FROM AUTHOR] more...

Type II restriction enzymes are characterized by their remarkable specificity and simplicity. They require only divalent metals (such as Mg2+ or Mn2+) as cofactors to catalyze the hydrolysis of DNA. However, most of the structural work on endonucleases has been performed in the absence of metals, leaving unanswered questions about their mechanisms of DNA cleavage. Here we report structures of the endonuclease BamHI?DNA complex, determined in the presence of Mn2+ and Ca2+, that describe the enzyme at different stages of catalysis. Overall, the results support a two-metal mechanism of DNA cleavage for BamHI which is distinct from that of EcoRV. [ABSTRACT FROM AUTHOR] more...

Presents research which reported the crystal structure of the interferon regulatory factor (IRF)-1 region bound to the natural positive regulatory domain I (PRD I) DNA element from the interferon-beta promoter. Importance of IRF transcription factors and their DNA-binding region; Details about structure; Suggested basis for occurrence of GAAA repeats within IRF response elements; Assembly of interferon-beta enhancesome. more...

Studies crystallization of restriction endonuclease SfiI in complex with DNA. Method used to study the analysis; Experimental observation of the analysis; X-ray diffraction data collection; Preliminary X-ray data analysis. more...

We have solved the crystal structure of human DNA polymerase-ι (hPolι) bound to a template primer and incoming nucleotide, and shown that hPolι uses Hoogsteen base-pairing for normal DNA synthesis. Jimin Wang questions the space group we used and claims that Hoogsteen base-pairing is not supported by our electron-density maps. We show here that the issue of space group does not affect our conclusions and that our electron-density maps are clear in indicating that Hoogsteen and not Watson–Crick base-pairing occurs in this situation. [ABSTRACT FROM AUTHOR] more...

Summary: Human DNA polymerase-ι (Polι) incorporates correct nucleotides opposite template purines with a much higher efficiency and fidelity than opposite template pyrimidines. In fact, the fidelity opposite template T is so poor that Polι inserts an incorrect dGTP approximately 10 times better than it inserts the correct dATP. We determine here how a template T/U is accommodated in the Polι active site and why a G is incorporated more efficiently than an A. We show that in the absence of incoming dATP or dGTP (binary complex), template T/U exists in both syn and anti conformations, but in the presence of dATP or dGTP (ternary complexes), template T/U is predominantly in the anti conformation. We also show that dATP and dGTP insert differently opposite template T/U, and that the basis of selection of dGTP over dATP is a hydrogen bond between the N2 amino group of dGTP and Gln59 of Polι. [Copyright &y& Elsevier] more...

Summary: Abasic sites are among the most abundant DNA lesions formed in human cells, and they present a strong block to replication. DNA polymerase ι (Polι) is one of the few DNA Pols that does not follow the A-rule opposite an abasic site. We present here three structures of human Polι in complex with DNAs containing an abasic lesion and dGTP, dTTP, or dATP as the incoming nucleotide. The structures reveal a mechanism of translesion synthesis across an abasic lesion that differs from that in other Pols. Both the abasic lesion and the incoming dNTPs are intrahelical and are closely apposed across a constricted active site cleft. The dNTPs partake in distinct networks of hydrogen bonds in the “void” opposite the lesion. These different patterns of hydrogen bonds, as well as stacking interactions, may underlie Polι''s small preference for insertion of dGTP over other nucleotides opposite this common lesion. [Copyright &y& Elsevier] more...

Summary: Acrolein is generated as the end product of lipid peroxidation and is also a ubiquitous environmental pollutant. Its reaction with the N2 of guanine leads to a cyclic γ-HOPdG adduct that presents a block to normal replication. We show here that yeast Rev1 incorporates the correct nucleotide C opposite a permanently ring-closed form of γ-HOPdG (PdG) with nearly the same efficiency as opposite an undamaged G. The structural basis of this action lies in the eviction of the PdG adduct from the Rev1 active site, and the pairing of incoming dCTP with a “surrogate” arginine residue. We also show that yeast Polζ can carry out the subsequent extension reaction. Together, our studies reveal how the exocyclic PdG adduct is accommodated in a DNA polymerase active site, and they show that the combined action of Rev1 and Polζ provides for accurate and efficient synthesis through this potentially carcinogenic DNA lesion. [Copyright &y& Elsevier] more...

Summary: Interferon regulatory factor 3 (IRF-3) is a key transcription factor in the assembly of the mammalian interferon-β (IFN-β) enhanceosome. We present here the structure of IRF-3 DNA binding domain in complex with the complete PRDIII-I regulatory element of the human IFN-β enhancer. We show that four IRF-3 molecules bind in tandem to, variably spaced, consensus and nonconsensus IRF sites on the composite element. The ability of IRF-3 to bind these variable sites derives in part from two nonconserved arginines (Arg78 and Arg86) that partake in alternate protein-DNA contacts. We also show that the protein-DNA contacts are highly overlapped and that all four IRF sites are required for gene activation in vivo. In addition, we show that changing the nonconsensus IRF sites to consensus sites creates a more efficient enhancer in vivo. Together, the structure and accompanying biological data provide insights into the assembly of the IFN-β enhanceosome in mammals. [Copyright &y& Elsevier] more...

Summary: Substrate-induced conformational change of the protein is the linchpin of enzymatic reactions. Replicative DNA polymerases, for example, convert from an open to a closed conformation in response to dNTP binding. Human DNA polymerase-ι (hPolι), a member of the Y family of DNA polymerases, differs strikingly from other polymerases in its much higher proficiency and fidelity for nucleotide incorporation opposite template purines than opposite template pyrimidines. We present here a crystallographic analysis of hPolι binary complexes, which together with the ternary complexes show that, contrary to replicative DNA polymerases, the DNA, and not the polymerase, undergoes the primary substrate-induced conformational change. The incoming dNTP “pushes” templates A and G from the anti to the syn conformation dictated by a rigid hPolι active site. Together, the structures posit a mechanism for template selection wherein dNTP binding induces a conformational switch in template purines for productive Hoogsteen base pairing. [Copyright &y& Elsevier] more...

We present the crystal structure of the catalytic core of human DNA polymerase kappa (hPolκ), the first structure of a human Y-family polymerase. hPolκ is implicated in the proficient extension of mispaired primer termini on undamaged DNAs, and in the extension step of lesion bypass. The structure reveals a stubby “fingers” subdomain, which despite its small size appears to be tightly restrained with respect to a putative templating base. The structure also reveals a novel “thumb” subdomain that provides a basis for the importance of the N-terminal extension unique to hPolκ. And, most surprisingly, the structure reveals the polymerase-associated domain (PAD) juxtaposed on the dorsal side of the “palm” subdomain, as opposed to the fingers subdomain. Together, these properties suggest that the hPolκ active site is constrained at the site of the templating base and incoming nucleotide, but the polymerase is less constrained following translocation of the lesion. [Copyright &y& Elsevier] more...

We report here the structure of BstYI, an “intermediate” type II restriction endonuclease with overlapping sequence specificities to BamHI and BglII. BstYI, a thermophilic endonuclease, recognizes and cleaves the degenerate hexanucleotide sequence 5′-RGATCY-3′ (where R=A or G and Y=C or T), cleaving DNA after the 5′-R on each strand to produce four-base (5′) staggered ends. The crystal structure of free BstYI was solved at 1.85 A˚ resolution by multiwavelength anomalous dispersion (MAD) phasing. Comparison with BamHI and BglII reveals a strong structural consensus between all three enzymes mapping to the α/β core domain and residues involved in catalysis. Unexpectedly, BstYI also contains an additional “arm” substructure outside of the core protein, which enables the enzyme to adopt a more compact, intertwined dimer structure compared with BamHI and BglII. This arm substructure may underlie the thermostability of BstYI. We identify putative DNA recognition residues and speculate as to how this enzyme achieves a “relaxed” DNA specificity. [Copyright &y& Elsevier] more...

Cells counter DNA damage through repair or apoptosis, yet a direct mechanism for this choice has remained elusive. When facing interstrand crosslinks (ICLs), the ICL-repair protein FANCI heterodimerizes with FANCD2 to initiate ICL excision. We found that FANCI alternatively interacts with a pro-apoptotic factor, PIDD1, to enable PIDDosome (PIDD1-RAIDD-caspase-2) formation and apoptotic death. FANCI switches from FANCD2/repair to PIDD1/apoptosis signaling in the event of ICL-repair failure. Specifically, removing key endonucleases downstream of FANCI/FANCD2, increasing ICL levels, or allowing damaged cells into mitosis (when repair is suppressed) all suffice for switching. Reciprocally, apoptosis-committed FANCI reverts from PIDD1 to FANCD2 after a failed attempt to assemble the PIDDosome. Monoubiquitination and deubiquitination at FANCI K523 impact interactor selection. These data unveil a repair-or-apoptosis switch in eukaryotes. Beyond ensuring the removal of unrepaired genomes, the switch's bidirectionality reveals that damaged cells can offset apoptotic defects via de novo attempts at lesion repair. [Display omitted] • FANCI binds either pro-repair FANCD2 or pro-apoptotic PIDD1 in response to ICLs • Interactor selection by FANCI is binary and regulated by deubiquitination • Repair failure triggers PIDD1 recruitment, PIDDosome formation, and apoptotic death • Apoptosis failure triggers reversal to FANCD2 and de novo attempts at ICL repair Shah et al. describe a mechanism by which cells decide their fate, repair or self-removal, after DNA damage. The mechanism relies on the ability of a DNA repair effector, FANCI, to engage an apoptotic device in a manner exclusive with its repair activity. [ABSTRACT FROM AUTHOR] more...