Sergey Korolev

Crystal structures of open and closed forms of binary and ternary complexes of the large fragment of Thermus aquaticus DNA polymerase I: structural basis for nucleotide incorporation.

The crystal structures of two ternary complexes of the large fragment of Thermus aquaticus DNA polymerase I (Klentaq1) with a primer/template DNA and dideoxycytidine triphosphate, and that of a binary complex of the same enzyme with a primer/template DNA, were determined to a resolution of 2.3, 2.3 and 2.5 Å, respectively. One ternary complex structure differs markedly from the two other structures by a large reorientation of the tip of the fingers domain. This structure, designated ‘closed’, represents the ternary polymerase complex caught in the act of incorporating a nucleotide. In the two other structures, the tip of the fingers domain is rotated outward by 46° (‘open’) in an orientation similar to that of the apo form of Klentaq1. These structures provide the first direct evidence in DNA polymerase I enzymes of a large conformational change responsible for assembling an active ternary complex.

Major Domain Swiveling Revealed by the Crystal Structures of Complexes of E. coli Rep Helicase Bound to Single-Stranded DNA and ADP

Crystal structures of binary and ternary complexes of the E. coli Rep helicase bound to single-stranded (ss) DNA or ssDNA and ADP were determined to a resolution of 3.0 A and 3.2 A, respectively. The asymmetric unit in the crystals contains two Rep monomers differing from each other by a large reorientation of one of the domains, corresponding to a swiveling of 130 degrees about a hinge region. Such domain movements are sufficiently large to suggest that these may be coupled to translocation of the Rep dimer along DNA. The ssDNA binding site involves the helicase motifs Ia, III, and V, whereas the ADP binding site involves helicase motifs I and IV. Residues in motifs II and VI may function to transduce the allosteric effects of nucleotides on DNA binding. These structures represent the first view of a DNA helicase bound to DNA.

Mutations in Cohesin Complex Members SMC3 and SMC1A Cause a Mild Variant of Cornelia de Lange Syndrome with Predominant Mental Retardation

Mutations in the cohesin regulators NIPBL and ESCO2 are causative of the Cornelia de Lange syndrome (CdLS) and Roberts or SC phocomelia syndrome, respectively. Recently, mutations in the cohesin complex structural component SMC1A have been identified in two probands with features of CdLS. Here, we report the identification of a mutation in the gene encoding the complementary subunit of the cohesin heterodimer, SMC3, and 14 additional SMC1A mutations. All mutations are predicted to retain an open reading frame, and no truncating mutations were identified. Structural analysis of the mutant SMC3 and SMC1A proteins indicate that all are likely to produce functional cohesin complexes, but we posit that they may alter their chromosome binding dynamics. Our data indicate that SMC3 and SMC1A mutations (1) contribute to approximately 5% of cases of CdLS, (2) result in a consistently mild phenotype with absence of major structural anomalies typically associated with CdLS, and (3) in some instances, result in a phenotype that approaches that of apparently nonsyndromic mental retardation.

Structure of the major single‐stranded DNA‐binding domain of replication protein A suggests a dynamic mechanism for DNA binding

Although structures of single‐stranded (ss)DNA‐binding proteins (SSBs) have been reported with and without ssDNA, the mechanism of ssDNA binding in eukarya remains speculative. Here we report a 2.5 Å structure of the ssDNA‐binding domain of human replication protein A (RPA) (eukaryotic SSB), for which we previously reported a structure in complex with ssDNA. A comparison of free and bound forms of RPA revealed that ssDNA binding is associated with a major reorientation between, and significant conformational changes within, the structural modules—OB‐folds—which comprise the DNA‐binding domain. Two OB‐folds, whose tandem orientation was stabilized by the presence of DNA, adopted multiple orientations in its absence. Within the OB‐folds, extended loops implicated in DNA binding significantly changed conformation in the absence of DNA. Analysis of intermolecular contacts suggested the possibility that other RPA molecules and/or other proteins could compete with DNA for the same binding site. Using this mechanism, protein–protein interactions can regulate, and/or be regulated by DNA binding. Combined with available biochemical data, this structure also suggested a dynamic model for the DNA‐binding mechanism.

The crystal structure of spermidine synthase with a multisubstrate adduct inhibitor.

Polyamines are essential in all branches of life. Spermidine synthase (putrescine aminopropyltransferase, PAPT) catalyzes the biosynthesis of spermidine, a ubiquitous polyamine. The crystal structure of the PAPT from Thermotoga maritima (TmPAPT) has been solved to 1.5 Å resolution in the presence and absence of AdoDATO (S-adenosyl-1,8-diamino-3-thiooctane), a compound containing both substrate and product moieties. This, the first structure of an aminopropyltransferase, reveals deep cavities for binding substrate and cofactor, and a loop that envelops the active site. The AdoDATO binding site is lined with residues conserved in PAPT enzymes from bacteria to humans, suggesting a universal catalytic mechanism. Other conserved residues act sterically to provide a structural basis for polyamine specificity. The enzyme is tetrameric; each monomer consists of a C-terminal domain with a Rossmann-like fold and an N-terminal β-stranded domain. The tetramer is assembled using a novel barrel-type oligomerization motif.

Structure of N-myristoyltransferase with bound myristoylCoA and peptide substrate analogs.

N-myristoyltransferase (Nmt) attaches myristate to the N-terminal glycine of many important eukaryotic and viral proteins. It is a target for anti-fungal and anti-viral therapy. We have determined the structure, to 2.9 Å resolution, of a ternary complex of Saccharomyces cerevisiae Nmt1p with bound myristoylCoA and peptide substrate analogs. The model reveals structural features that define the enzymes substrate specificities and regulate the ordered binding and release of substrates and products. A novel catalytic mechanism is proposed involving deprotonation of the N-terminal ammonium of a peptide substrate by the enzymes C-terminal backbone carboxylate.

Anchoring Notch Genetics and Biochemistry: Structural Analysis of the Ankyrin Domain Sheds Light on Existing Data

Notch signaling is important in development and in human disease. Notch receptors regulate transcription through direct interactions with several proteins at the promoter regions of target genes. To understand the mechanism of Notch signaling, numerous deletion and mutagenesis studies have been carried out to identify functional domains in Notch, but domain definition and their role during the assembly of the transcriptionally active complex remains controversial. Recently reported biophysical and structural studies of the Notch ANK domain permit us to reevaluate the existing domain assignments and their predicted functional role, thereby providing further insight into the mechanism of Notch signaling.

Mechanism of RecO recruitment to DNA by single-stranded DNA binding protein

RecO is a recombination mediator protein (RMP) important for homologous recombination, replication repair and DNA annealing in bacteria. In all pathways, the single-stranded (ss) DNA binding protein, SSB, plays an inhibitory role by protecting ssDNA from annealing and recombinase binding. Conversely, SSB may stimulate each reaction through direct interaction with RecO. We present a crystal structure of Escherichia coli RecO bound to the conserved SSB C-terminus (SSB-Ct). SSB-Ct binds the hydrophobic pocket of RecO in a conformation similar to that observed in the ExoI/SSB-Ct complex. Hydrophobic interactions facilitate binding of SSB-Ct to RecO and RecO/RecR complex in both low and moderate ionic strength solutions. In contrast, RecO interaction with DNA is inhibited by an elevated salt concentration. The SSB mutant lacking SSB-Ct also inhibits RecO-mediated DNA annealing activity in a salt-dependent manner. Neither RecO nor RecOR dissociates SSB from ssDNA. Therefore, in E. coli, SSB recruits RMPs to ssDNA through SSB-Ct, and RMPs are likely to alter the conformation of SSB-bound ssDNA without SSB dissociation to initiate annealing or recombination. Intriguingly, Deinococcus radiodurans RecO does not bind SSB-Ct and weakly interacts with the peptide in the presence of RecR, suggesting the diverse mechanisms of DNA repair pathways mediated by RecO in different organisms.

The 2B domain of the Escherichia coli Rep protein is not required for DNA helicase activity

The Escherichia coli Rep protein is a 3′ to 5′ SF1 DNA helicase required for replication of bacteriophage φX174 in E. coli, and is structurally homologous to the E. coli UvrD helicase and the Bacillus stearothermophilus PcrA helicase. Previous crystallographic studies of Rep protein bound to single-stranded DNA revealed that it can undergo a large conformational change consisting of an ≈130° rotation of its 2B subdomain about a hinge region connected to the 2A subdomain. Based on crystallographic studies of PcrA, its 2B subdomain has been proposed to form part of its duplex DNA binding site and to play a role in duplex destabilization. To test the role of the 2B subdomain in Rep-catalyzed duplex DNA unwinding, we have deleted its 2B subdomain, replacing it with three glycines, to form the RepΔ2B protein. This RepΔ2B protein can support φX174 replication in a rep− E. coli strain, although the growth rate of E. coli containing the repΔ2B gene is ≈1.5-fold slower than with the wild-type rep gene. Pre-steady-state, single-turnover DNA unwinding kinetics experiments show that purified RepΔ2B protein has DNA helicase activity in vitro and unwinds an 18-bp DNA duplex with rates at least as fast as wild-type Rep, and with higher extents of unwinding and higher affinity for the DNA substrate. These studies show that the 2B domain of Rep is not required for DNA helicase activity in vivo or in vitro, and that it does not facilitate DNA unwinding in vitro.

Structural conservation of RecF and Rad50: implications for DNA recognition and RecF function

RecF, together with RecO and RecR, belongs to a ubiquitous group of recombination mediators (RMs) that includes eukaryotic proteins such as Rad52 and BRCA2. RMs help maintain genome stability in the presence of DNA damage by loading RecA‐like recombinases and displacing single‐stranded DNA‐binding proteins. Here, we present the crystal structure of RecF from Deinococcus radiodurans. RecF exhibits a high degree of structural similarity with the head domain of Rad50, but lacks its long coiled‐coil region. The structural homology between RecF and Rad50 is extensive, encompassing the ATPase subdomain and the so‐called ‘Lobe II’ subdomain of Rad50. The pronounced structural conservation between bacterial RecF and evolutionarily diverged eukaryotic Rad50 implies a conserved mechanism of DNA binding and recognition of the boundaries of double‐stranded DNA regions. The RecF structure, mutagenesis of conserved motifs and ATP‐dependent dimerization of RecF are discussed with respect to its role in promoting presynaptic complex formation at DNA damage sites.