Recruitment of HAT complexes by direct activator interactions with the ATM-related Tra1 subunit.

Promoter-specific recruitment of histone acetyltransferase activity is often critical for transcriptional activation. We present a detailed study of the interaction between the histone acetyltransferase complexes SAGA and NuA4, and transcription activators. We demonstrate by affinity chromatography and photo-cross-linking label transfer that acidic activators directly interact with Tra1p, a shared subunit of SAGA and NuA4. Mutations within the COOH-terminus of Tra1p disrupted its interaction with activators and resulted in gene-specific transcriptional defects that correlated with lowered promoter-specific histone acetylation. These data demonstrate that the essential Tra1 protein serves as a common target for activators in both SAGA and NuA4 acetyltransferases.

Engineered cysteine antibodies: an improved antibody-drug conjugate platform with a novel mechanism of drug-linker stability.

Antibody-drug conjugates (ADCs) are fulfilling the promise of targeted therapy with meaningful clinical success. An intense research effort is directed towards improving pharmacokinetic profiles, toxicity and chemical stability of ADCs. The majority of ADCs use amide and thioether chemistry to link potent cytotoxic agents to antibodies via endogenous lysine and cysteine residues. While maleimide-cysteine conjugation is used for many clinical stage ADC programs, maleimides have been shown to exhibit some degree of post-conjugation instability. Previous research with site-directed mutagenic incorporation of cysteine residues for conjugation revealed that the stability of the drug-antibody linkage depends on the site of conjugation. Here we report on a collection of engineered cysteine antibodies (S239C, E269C, K326C and A327C) that can be site-specifically conjugated to potent cytotoxic agents to produce homogenous 2-loaded ADCs. These ADCs confirm that site of conjugation impacts maleimide stability and present a novel mechanism of thioether stabilization, effectively unlinking stability from either local chemical environment or calculated solvent accessibility and expanding the current paradigm for ADC drug-linker stability. These ADCs show potent in vitro and in vivo activity while delivering half of the molar equivalent dose of drug per antibody when compared to an average 4-loaded ADC. In addition, our lead engineered site shields highly hydrophobic drugs, enabling conjugation, formulation and clinical use of otherwise intractable chemotypes.

DNA-DNA interstrand cross-linking by 2,5-bis(1-aziridinyl)-3,6-bis(carbethoxyamino)-1,4-benzoquinone: covalent structure of the dG-to-dG cross-links in calf thymus DNA and a synthetic DNA duplex.

The products of the alkylation reaction of reduced 2,5-bis(1-aziridinyl)-3,6-bis(carbethoxyamino)-1,4-benzoquinone (AZQ, 1a) with duplex DNA were studied using calf thymus DNA and a synthetic oligodeoxynucleotide. Reaction of calf thymus DNA with a mixture of AZQ and ascorbic acid followed by enzymatic digestion of the sugar phosphate backbone afforded numerous AZQ-derived products including substances identified as monoadducts of AZQ with both dG and dA (with the former in greater abundance) and two diadducts, as would be expected for intrastrand or interstrand cross-links, with one containing two dG residues per AZQ and the other one each of dG and dA (with the former adduct in greater abundance). The nucleotide connectivity and covalent structure of the dG-to-dG interstrand cross-links were studied in greater detail using a synthetic DNA duplex containing the nucleotide sequence 5'-d(GGGCCC), where it appeared that the predominant interstrand cross-links bridged dG residues on opposite strands and were separated by two intervening base pairs [5'-d(GNNC)]. The covalent structure of this lesion was tentatively identified as 2b, in which the N7 atoms of two dG residues have been alkylated by the aziridine functions of AZQ, based upon the results of piperidine fragmentation and characterization of the enzymatic and acidic hydrolysates of the cross-linked DNA.

Tracking sliding clamp opening and closing during bacteriophage T4 DNA polymerase holoenzyme assembly.

The bacteriophage T4 DNA polymerase holoenzyme, consisting of the DNA polymerase (gp43), the sliding clamp (gp45), and the clamp loader (gp44/62), is loaded onto DNA in an ATP-dependent, multistep reaction. The trimeric, ring-shaped gp45 is loaded onto DNA such that the DNA passes through the center of the ring. gp43 binds to this complex, thereby forming a topological link with the DNA and increasing its processivity. Using stopped-flow fluorescence-resonance energy transfer, we have investigated opening and closing of the gp45 ring during the holoenzyme assembly process. Two amino acids that lie on opposite sides of the gp45 subunit interface, W91 and V162C labeled with coumarin, were used as the fluorescence donor and acceptor, respectively. Free in solution, gp45 has two closed subunit interfaces with W91 to V162-coumarin distances of 19 A and one open subunit interface with a W91 to V162C-coumarin distance of 40 A. Making the assumption that the distance across the two closed subunit interfaces is unchanged during the holoenzyme assembly process, we have found that the distance across the open subunit interface is first increased to greater than 45 A and is then decreased to 30 A during a 10-step assembly mechanism. The gp45 ring is not completely closed in the holoenzyme complex, consistent with previous evidence suggesting that the C-terminus of gp43 is inserted into the gp45 subunit interface. Unexpectedly, ATP-hydrolysis events are coupled to only a fraction of the total distance change, with conformational changes linked to binding DNA and gp43 coupled to the majority of the total distance change. Using the nonhydrolyzable ATP analogue ATP-gamma-S results in formation of a nonproductive gp45 x gp44/62 complex; however, adding an excess of ATP to this nonproductive complex results in rapid ATP/ATP-gamma-S exchange to yield a productive gp45 x gp44/62 complex within seconds.

Identification and mapping of protein-protein interactions between gp32 and gp59 by cross-linking.

The bacteriophage T4 59 protein (gp59) plays a vital role in recombination and replication by promoting the assembly of the gene 41 helicase (gp41) onto DNA, thus enabling replication as well as strand exchange in recombination. Loading of the helicase onto gp32 (the T4 single strand binding protein)-coated single-stranded DNA requires gp59 to remove gp32 and replace it with gp41. Cross-linking studies between gp32 and gp59 reveal an interaction between Cys-166 of gp32 and Cys-42 of gp59. Since Cys-166 lies in the DNA binding core domain of gp32, this interaction may affect the association of gp32 with DNA. In the presence of gp32 or DNA, gp59 is capable of forming a multimer consisting of at least five gp59 subunits. Kinetics studies suggest that gp59 and gp41 exist in a one-to-one ratio, predicting that gp59 is capable of forming a hexamer (Raney, K. D., Carver, T. E., and Benkovic, S. J. (1996) J. Biol. Chem. 271, 14074-14081). The C-terminal A-domain of gp32 is needed for gp59 oligomer formation. Cross-linking has established that gp59 can interact with gp32-A (a truncated form of gp32 lacking the A-domain) but cannot form higher species. The results support a model in which gp59 binds to gp32 on a replication fork, destabilizing the gp32-single-stranded DNA interaction concomitant with the oligomerization of gp59 that results in a switching of gp41 for gp32 at the replication fork.

DNA-DNA interstrand cross-linking by cis-diamminedichloroplatinum(II): N7(dG)-to-N7(dG) cross-linking at 5'-d(GC) in synthetic oligonucleotides.

The nucleotide sequence specificity of the DNA-DNA interstrand cross-linking reaction of cis-diamminedichloroplatinum(II) (cis-DDP) was studied in synthetic oligonucleotides. Of six self-complementary DNAs tested, only those containing the central sequence 5'-d(GC) formed appreciable interstrand cross-linked product, as assayed by denaturing polyacrylamide gel electrophoresis (DPAGE). The nucleotide connectivity of the interstrand cross-link was defined by sequence random oxidative fragmentation followed by DPAGE, revealing that dG residues on opposite strands at the sequence 5'-d(GC) were connected to one another. The covalent structure of the cross-link was established following hydrolysis of the phosphodiester backbone of a structurally homogeneous sample of a cis-DDP interstrand cross-linked DNA tetradecamer. HPLC analysis of the hydrolysate returned all of the deoxynucleoside residues from the starting DNA except for two deoxyguanosine residues. Also returned was diammine-bis-[N7-(2'- deoxyguanosyl)]platinum(II)2+, identified by a combination of spectroscopic methods, and comparison to a synthetic authentic sample. This study directly establishes that cis-DDP forms interstrand cross-links at the duplex sequence 5'-d(GC), linking deoxyguanosine residues on opposite strands at N7 through a bridging platinum atom. Computer simulation of the interstrand cross-linked product using molecular mechanics energy minimization and molecular dynamics revealed significant structural reorganization at the site of the cross-link including a ca 40 degrees angle between the platinated guaninyl residues, which propagated to adjacent residues by base stacking to yield duplexes bent by some 30 degrees toward the major groove.

Bending of DNA by the mitomycin C-induced, GpG intrastrand cross-link.

Mitomycin C (MC) forms interstrand and intrastrand cross-link adducts and monoalkylation products (monoadducts) with DNA. Each of the three types of adducts was incorporated site-specifically into both a 15-mer and a 21-mer oligodeoxyribonucleotide duplex. The adduct-containing duplexes were 32P-phosphorylated and ligated to form multimers, which were then analyzed for anomalous electrophoretic mobility by nondenaturing polyacrylamide gel electrophoresis, using the method of Koo and Crothers [(1988) Proc. Natl. Acad. Sci. U.S.A. 85, 1763-1767] in order to detect DNA curvature caused by the adducts. The intrastrand cross-link adduct was found to induce a 14.6 +/- 2.0 degrees DNA bend per lesion (minimum value) while no DNA bending was detected for either the interstrand cross-link or the monoadduct. Molecular mechanics modeling indicated that the possible origin of the bend lies in a considerable deviation from parallel of the normals to the best planes of the intrastrand cross-linked guanines, due to a shorter than normal distance between their N2 atoms forced upon them by the cross-link. The observed bending by the MC intrastrand lesion may be the cause of the increased flexibility of MC-modified DNA, localized to distinct regions, as observed in earlier work by hydrodynamic methods and electron microscopy. The MC adduct-caused DNA bend may serve as a recognition site for certain DNA-binding proteins.

The carboxyl terminus of the bacteriophage T4 DNA polymerase contacts its sliding clamp at the subunit interface.

The location of the interaction of the COOH terminus of the bacteriophage T4 DNA polymerase with its trimeric, circular sliding clamp has been established. A peptide corresponding to the COOH terminus of the DNA polymerase was labeled with a fluorophore and fluorescence spectroscopy used to show that it forms a specific complex with the sliding clamp by virtue of its low K(D) value (7.1 +/- 1.0 microM). The same peptide was labeled with a photoaffinity probe and cross-linked to the sliding clamp. Mass spectrometry of tryptic digests determined the sole linkage point to be Ala-159 on the sliding clamp, an amino acid that lies on the subunit interface. These results demonstrate that the COOH terminus of the DNA polymerase is inserted into the subunit interface of its sliding clamp, thereby conferring processivity to the DNA polymerase.

Creating a dynamic picture of the sliding clamp during T4 DNA polymerase holoenzyme assembly by using fluorescence resonance energy transfer.

The coordinated assembly of the DNA polymerase (gp43), the sliding clamp (gp45), and the clamp loader (gp44/62) to form the bacteriophage T4 DNA polymerase holoenzyme is a multistep process. A partially opened toroid-shaped gp45 is loaded around DNA by gp44/62 in an ATP-dependent manner. Gp43 binds to this complex to generate the holoenzyme in which gp45 acts to topologically link gp43 to DNA, effectively increasing the processivity of DNA replication. Stopped-flow fluorescence resonance energy transfer was used to investigate the opening and closing of the gp45 ring during holoenzyme assembly. By using two site-specific mutants of gp45 along with a previously characterized gp45 mutant, we tracked changes in distances across the gp45 subunit interface through seven conformational changes associated with holoenzyme assembly. Initially, gp45 is partially open within the plane of the ring at one of the three subunit interfaces. On addition of gp44/62 and ATP, this interface of gp45 opens further in-plane through the hydrolysis of ATP. Addition of DNA and hydrolysis of ATP close gp45 in an out-of-plane conformation. The final holoenzyme is formed by the addition of gp43, which causes gp45 to close further in plane, leaving the subunit interface open slightly. This open interface of gp45 in the final holoenzyme state is proposed to interact with the C-terminal tail of gp43, providing a point of contact between gp45 and gp43. This study further defines the dynamic process of bacteriophage T4 polymerase holoenzyme assembly.

Sequence-dependent dynamics in duplex DNA.

The submicrosecond bending dynamics of duplex DNA were measured at a single site, using a site-specific electron paramagnetic resonance active spin probe. The observed dynamics are interpreted in terms of the mean squared amplitude of bending relative to the end-to-end vector defined by the weakly bending rod model. The bending dynamics monitored at the single site varied when the length and position of a repeated AT sequence, distant from the spin probe, were changed. As the distance between the probe and the AT sequence was increased, the mean squared amplitude of bending seen by the probe due to that sequence decreased. A model for the sequence-dependent internal flexural motion of duplex DNA, which casts the mean squared bending amplitudes in terms of sequence-dependent bending parameters, has been developed. The best fit of the data to the model occurs when the (AT)(n) basepairs are assumed to be 20% more flexible than the average of the basepairs within the control sequence. These findings provide a quantitative basis for interpreting the kinetics of biological processes that depend on duplex DNA flexibility, such as protein recognition and chromatin packaging.

Sliding clamp of the bacteriophage T4 polymerase has open and closed subunit interfaces in solution.

The sliding clamps of bacteriophage T4 (gp45), Escherichia coli (beta clamp), and yeast (PCNA) are required for processive DNA synthesis by their cognate DNA polymerases. The X-ray crystal structures of all three of these clamps have been shown to be closed, circular complexes. This paper reports investigations of the solution structure of bacteriophage T4 gp45 by analytical ultracentrifugation, fluorescence, and hydrodynamic modeling. Mutants of gp45 with inter- and intrasubunit disulfide bonds were created to alter the solution structure of gp45, with additional mutagenesis used to investigate the importance of the proline-rich loop region found between the two domains of each gp45 monomer. The wild-type gp45 trimer assembles from monomers cooperatively with a dissociation constant of 0.21 microM2 and values between 0.088 and 0. 32 microM2 for the mutants. Velocity ultracentrifugation experiments showed that wild-type gp45 possesses a sedimentation coefficient strongly dependent on concentration, typical of asymmetric or elongated molecules, that when extrapolated to zero concentration yields a sedimentation coefficient of 4.0 S. The loop and the disulfide mutants exhibited sedimentation coefficients with little concentration dependence, typical of symmetric or spherical molecules, that when extrapolated to zero concentration yielded sedimentation coefficients of 4.4-4.8 S. The lower sedimentation coefficient in the former case is consistent with wild-type gp45 being more asymmetric or elongated than the mutant forms. Fluorescence-resonance energy-transfer experiments were used to measure the distance between two amino acids (W91 and V162C-coumarin) on opposite sides of the gp45 subunit interface. For an intrasubunit disulfide mutant, the distance between these two amino acids was determined to be 19 A (14 A in the X-ray crystal structure), consistent with a closed complex. For the mutants without intrasubunit disulfides, the efficiency of fluorescence-resonance energy transfer was in accord with a model of gp45 being an open complex composed of two closed subunit interfaces and a third open interface separated by a distance of 35-38 A. The collective data supplemented with hydrodynamic modeling were consistent with gp45 subunit separation achieved within the plane of the gp45 ring.

Flexibility of duplex DNA on the submicrosecond timescale.

Using a site-specific, Electron Paramagnetic Resonance (EPR)-active spin probe that is more rigidly locked to the DNA than any previously reported, the internal dynamics of duplex DNAs in solution were studied. EPR spectra of linear duplex DNAs containing 14-100 base pairs were acquired and simulated by the stochastic Liouville equation for anisotropic rotational diffusion using the diffusion tensor for a right circular cylinder. Internal motions have previously been assumed to be on a rapid enough time scale that they caused an averaging of the spin interactions. This assumption, however, was found to be inconsistent with the experimental data. The weakly bending rod model is modified to take into account the finite relaxation times of the internal modes and applied to analyze the EPR spectra. With this modification, the dependence of the oscillation amplitude of the probe on position along the DNA was in good agreement with the predictions of the weakly bending rod theory. From the length and position dependence of the internal flexibility of the DNA, a submicrosecond dynamic bending persistence length of around 1500 to 1700 A was found. Schellman and Harvey (Biophys. Chem. 55:95-114, 1995) have estimated that, out of the total persistence length of duplex DNA, believed to be about 500 A, approximately 1500 A is accounted for by static bends and 750 A by fluctuating bends. A measured dynamic persistence length of around 1500 A leads to the suggestion that there are additional conformations of the DNA that relax on a longer time scale than that accessible by linear CW-EPR. These measurements are the first direct determination of the dynamic flexibility of duplex DNA in 0.1 M salt.

Building a replisome solution structure by elucidation of protein-protein interactions in the bacteriophage T4 DNA polymerase holoenzyme.

Assembly of DNA replication systems requires the coordinated actions of many proteins. The multiprotein complexes formed as intermediates on the pathway to the final DNA polymerase holoenzyme have been shown to have distinct structures relative to the ground-state structures of the individual proteins. By using a variety of solution-phase techniques, we have elucidated additional information about the solution structure of the bacteriophage T4 holoenzyme. Photocross-linking and mass spectrometry were used to demonstrate interactions between I107C of the sliding clamp and the DNA polymerase. Fluorescence resonance energy transfer, analytical ultracentrifugation, and isothermal titration calorimetry measurements were used to demonstrate that the C terminus of the DNA polymerase can interact at two distinct locations on the sliding clamp. Both of these binding modes may be used during holoenzyme assembly, but only one of these binding modes is found in the final holoenzyme. Present and previous solution interaction data were used to build a model of the holoenzyme that is consistent with these data.

Sequence-dependent dynamics of duplex DNA: the applicability of a dinucleotide model.

The short-time (submicrosecond) bending dynamics of duplex DNA were measured to determine the effect of sequence on dynamics. All measurements were obtained from a single site on duplex DNA, using a single, site-specific modified base containing a rigidly tethered, electron paramagnetic resonance active spin probe. The observed dynamics are interpreted in terms of single-step sequence-dependent bending force constants, determined from the mean squared amplitude of bending relative to the end-to-end vector using the modified weakly bending rod model. The bending dynamics at a single site are a function of the sequence of the nucleotides constituting the duplex DNA. We developed and examined several dinucleotide-based models for flexibility. The models indicate that the dominant feature of the dynamics is best explained in terms of purine- and pyrimidine-type steps, although distinction is made among all 10 unique steps: It was found that purine-purine steps (which are the same as pyrimidine-pyrimidine steps) were near average in flexibility, but the pyrimidine-purine steps (5' to 3') were nearly twice as flexible, whereas purine-pyrimidine steps were more than half as flexible as average DNA. Therefore, the range of stepwise flexibility is approximately fourfold and is characterized by both the type of base pair step (pyrimidine/purine combination) and the identity of the bases within the pair (G, A, T, or C). All of the four models considered here underscore the complexity of the dependence of dynamics on DNA sequence with certain sequences not satisfactorily explainable in terms of any dinucleotide model. These findings provide a quantitative basis for interpreting the dynamics and kinetics of DNA-sequence-dependent biological processes, including protein recognition and chromatin packaging.