Bacillus subtilis tau subunit of DNA polymerase III interacts with bacteriophage SPP1 replicative DNA helicase G40P

Genetic evidence suggests that the Bacillus subtilis dnaX gene only encodes for the τ subunit of both DNA polymerases III (Pol IIIs). The B.subtilis full-length protein and their mutant derivatives τ(373– 563) (lacking the N-terminal, domains I–III or amino acid residues 1–372) and τ(1–372) (lacking the C-terminal region or amino acids 373–563) have been purified. The τ protein forms tetramers, τ(373– 563) forms dimers, whereas τ(1–372), depending on the ionic strength, forms trimers or tetramers in solution. In the absence of single-stranded (ss) DNA and a nucleotide cofactor, τ interacts with the SPP1 hexameric replicative G40P DNA helicase in solution or with G40P-ATP bound to ssDNA, with a 1:1 stoichiometry. G40P(109–442), lacking the N-terminal amino acid residues 1–108, interacts with the C-terminal moiety of τ. The data indicate that the interaction of G40P with the τ subunit of Pol III, is relevant for the loading of the Pol IIIs into the SPP1 G38P-promoted open complex.     

The recognition domain of the BpuJI restriction endonuclease in complex with cognate DNA at 1.3-A resolution

Type IIS restriction endonucleases recognize asymmetric DNA sequences and cleave both DNA strands at fixed positions downstream of the recognition site. The restriction endonuclease BpuJI recognizes the asymmetric sequence 5′-CCCGT; however, it cuts at multiple sites in the vicinity of the target sequence. BpuJI consists of two physically separate domains, with catalytic and dimerization functions in the C-terminal domain and DNA recognition functions in the N-terminal domain. Here we report the crystal structure of the BpuJI recognition domain bound to cognate DNA at 1.3-Å resolution. This region folds into two winged-helix subdomains, D1 and D2, interspaced by the DL subdomain. The D1 and D2 subdomains of BpuJI share structural similarity with the similar subdomains of the FokI DNA-binding domain; however, their orientations in protein-DNA complexes are different. Recognition of the 5′-CCCGT target sequence is achieved by BpuJI through the major groove contacts of amino acid residues located on both the helix-turn-helix motifs and the N-terminal arm. The role of these interactions in DNA recognition is also corroborated by mutational analysis.     

Crystal structure of the C-terminal domain of human DNA primase large subunit: Implications for the mechanism of the primase—polymerase α switch

DNA polymerases cannot synthesize DNA without a primer, and DNA primase is the only specialized enzyme capable of de novo synthesis of short RNA primers. In eukaryotes, primase functions within a heterotetrameric complex in concert with a tightly bound DNA polymerase α (Pol α). In humans, the Pol α part is comprised of a catalytic subunit (p180) and an accessory subunit B (p70), and the primase part consists of a small catalytic subunit (p49) and a large essential subunit (p58). The latter subunit participates in primer synthesis, counts the number of nucleotides in a primer, assists the release of the primer-template from primase and transfers it to the Pol α active site. Recently reported crystal structures of the C-terminal domains of the yeast and human enzymes'' large subunits provided critical information related to their structure, possible sites for binding of nucleotides and template DNA, as well as the overall organization of eukaryotic primases. However, the structures also revealed a difference in the folding of their proposed DNA-binding fragments, raising the possibility that yeast and human proteins are functionally different. Here we report new structure of the C-terminal domain of the human primase p58 subunit. This structure exhibits a fold similar to a fold reported for the yeast protein but different than a fold reported for the human protein. Based on a comparative analysis of all three C-terminal domain structures, we propose a mechanism of RNA primer length counting and dissociation of the primer-template from primase by a switch in conformation of the ssDNA-binding region of p58.Key words: DNA primase, prim1, prim2, replication, 4Fe-4S cluster, crystal structure, DNA polymerase α     

Junctional Adhesion Molecule A Interacts with Afadin and PDZ-GEF2 to Activate Rap1A, Regulate β1 Integrin Levels, and Enhance Cell Migration

Junctional adhesion molecule-A (JAM-A) is a transmembrane tight junction protein that has been shown to regulate barrier function and cell migration through incompletely understood mechanisms. We have previously demonstrated that JAM-A regulates cell migration by dimerization of the membrane-distal immunoglobulin-like loop and a C-terminal postsynaptic density 95/disc-large/zona occludens (PDZ) binding motif. Disruption of dimerization resulted in decreased epithelial cell migration secondary to diminished levels of β1 integrin and active Rap1. Here, we report that JAM-A is physically and functionally associated with the PDZ domain-containing molecules Afadin and PDZ-guanine nucleotide exchange factor (GEF) 2, but not zonula occludens (ZO)-1, in epithelial cells, and these interactions mediate outside-in signaling events. Both Afadin and PDZ-GEF2 colocalized and coimmunoprecipitated with JAM-A. Furthermore, association of PDZ-GEF2 with Afadin was dependent on the expression of JAM-A. Loss of JAM-A, Afadin, or PDZ-GEF2, but not ZO-1 or PDZ-GEF1, similarly decreased cellular levels of activated Rap1, β1 integrin protein, and epithelial cell migration. The functional effects observed were secondary to decreased levels of Rap1A because knockdown of Rap1A, but not Rap1B, resulted in decreased β1 integrin levels and reduced cell migration. These findings suggest that JAM-A dimerization facilitates formation of a complex with Afadin and PDZ-GEF2 that activates Rap1A, which regulates β1 integrin levels and cell migration.     

A Substitution in the Fingers Domain of DNA Polymerase δ Reduces Fidelity by Altering Nucleotide Discrimination in the Catalytic Site

DNA polymerase δ (Pol δ) is one of the major replicative DNA polymerases in eukaryotic cells, catalyzing lagging strand synthesis as well as playing a role in many DNA repair pathways. The catalytic site for polymerization consists of a palm domain and mobile fingers domain that opens and closes each catalytic cycle. We explored the effect of amino acid substitutions in a region of the highly conserved sequence motif B in the fingers domain on replication fidelity. A novel substitution, A699Q, results in a marked increase in mutation rate at the yeast CAN1 locus, and is synthetic lethal with both proofreading deficiency and mismatch repair deficiency. Modeling the A699Q mutation onto the crystal structure of Saccharomyces cerevisiae Pol δ template reveals four potential contacts for A699Q but not for A699. We substituted alanine for each of these residues and determined that an interaction with multiple residues of the N-terminal domain is responsible for the mutator phenotype. The corresponding mutation in purified human Pol δ results in a similar 30-fold increase in mutation frequency when copying gapped DNA templates. Sequence analysis indicates that the most characteristic mutation is a guanine-to-adenine (G to A) transition. The increase in deoxythymidine 5′-triphosphate-G mispairs was confirmed by performing steady state single nucleotide addition studies. Our combined data support a model in which the Ala-to-Gln substitution in the fingers domain of Pol δ results in an interaction with the N-terminal domain that affects the base selectivity of the enzyme.     

Conformational and thermodynamic changes of the repressor/DNA operator complex upon monomerization shed new light on regulation mechanisms of bacterial resistance against beta-lactam antibiotics

In absence of β-lactam antibiotics, BlaI and MecI homodimeric repressors negatively control the expression of genes involved in β-lactam resistance in Bacillus licheniformis and in Staphylococcus aureus. Subsequently to β-lactam presence, BlaI/MecI is inactivated by a single-point proteolysis that separates its N-terminal DNA-binding domain to its C-terminal domain responsible for its dimerization. Concomitantly to this proteolysis, the truncated repressor acquires a low affinity for its DNA target that explains the expression of the structural gene for resistance. To understand the loss of the high DNA affinity of the truncated repressor, we have determined the different dissociation constants of the system and solved the solution structure of the B. licheniformis monomeric repressor complexed to the semi-operating sequence OP₁ of blaP (1/2OP₁blaP) by using a de novo docking approach based on inter-molecular nuclear Overhauser effects and chemical-shift differences measured on each macromolecular partner. Although the N-terminal domain of the repressor is not subject to internal structural rearrangements upon DNA binding, the molecules adopt a tertiary conformation different from the crystallographic operator–repressor dimer complex, leading to a 30° rotation of the monomer with respect to a central axis extended across the DNA.

These results open new insights for the repression and induction mechanisms of bacterial resistance to β-lactams.

     

Crystal Structure of the Human Pol α B Subunit in Complex with the C-terminal Domain of the Catalytic Subunit

In eukaryotic DNA replication, short RNA-DNA hybrid primers synthesized by primase-DNA polymerase α (Prim-Pol α) are needed to start DNA replication by the replicative DNA polymerases, Pol δ and Pol ϵ. The C terminus of the Pol α catalytic subunit (p180_C) in complex with the B subunit (p70) regulates the RNA priming and DNA polymerizing activities of Prim-Pol α. It tethers Pol α and primase, facilitating RNA primer handover from primase to Pol α. To understand these regulatory mechanisms and to reveal the details of human Pol α organization, we determined the crystal structure of p70 in complex with p180_C. The structured portion of p70 includes a phosphodiesterase (PDE) domain and an oligonucleotide/oligosaccharide binding (OB) domain. The N-terminal domain and the linker connecting it to the PDE domain are disordered in the reported crystal structure. The p180_C adopts an elongated asymmetric saddle shape, with a three-helix bundle in the middle and zinc-binding modules (Zn1 and Zn2) on each side. The extensive p180_C-p70 interactions involve 20 hydrogen bonds and a number of hydrophobic interactions resulting in an extended buried surface of 4080 Ų. Importantly, in the structure of the p180_C-p70 complex with full-length p70, the residues from the N-terminal to the OB domain contribute to interactions with p180_C. The comparative structural analysis revealed both the conserved features and the differences between the human and yeast Pol α complexes.     

The DNA polymerase III holoenzyme contains γ and is not a trimeric polymerase

There is widespread agreement that the clamp loader of the Escherichia coli replicase has the composition DnaX₃δδ’χψ. Two DnaX proteins exist in E. coli, full length τ and a truncated γ that is created by ribosomal frameshifting. τ binds DNA polymerase III tightly; γ does not. There is a controversy as to whether or not DNA polymerase III holoenzyme (Pol III HE) contains γ. A three-τ form of Pol III HE would contain three Pol IIIs. Proponents of the three-τ hypothesis have claimed that γ found in Pol III HE might be a proteolysis product of τ. To resolve this controversy, we constructed a strain that expressed only τ from a mutated chromosomal dnaX. γ containing a C-terminal biotinylation tag (γ-C_tag) was provided in trans at physiological levels from a plasmid. A 2000-fold purification of Pol III* (all Pol III HE subunits except β) from this strain contained one molecule of γ-C_tag per Pol III* assembly, indicating that the dominant form of Pol III* in cells is Pol III₂τ₂ γδδ’χψ. Revealing a role for γ in cells, mutants that express only τ display sensitivity to ultraviolet light and reduction in DNA Pol IV-dependent mutagenesis associated with double-strand-break repair, and impaired maintenance of an F’ episome.     

Solution structures of 2 : 1 and 1 : 1 DNA polymerase-DNA complexes probed by ultracentrifugation and small-angle X-ray scattering

We report small-angle X-ray scattering (SAXS) and sedimentation velocity (SV) studies on the enzyme–DNA complexes of rat DNA polymerase β (Pol β) and African swine fever virus DNA polymerase X (ASFV Pol X) with one-nucleotide gapped DNA. The results indicated formation of a 2 : 1 Pol β–DNA complex, whereas only 1 : 1 Pol X–DNA complex was observed. Three-dimensional structural models for the 2 : 1 Pol β–DNA and 1 : 1 Pol X–DNA complexes were generated from the SAXS experimental data to correlate with the functions of the DNA polymerases. The former indicates interactions of the 8 kDa 5′-dRP lyase domain of the second Pol β molecule with the active site of the 1 : 1 Pol β–DNA complex, while the latter demonstrates how ASFV Pol X binds DNA in the absence of DNA-binding motif(s). As ASFV Pol X has no 5′-dRP lyase domain, it is reasonable not to form a 2 : 1 complex. Based on the enhanced activities of the 2 : 1 complex and the observation that the 8 kDa domain is not in an optimal configuration for the 5′-dRP lyase reaction in the crystal structures of the closed ternary enzyme–DNA–dNTP complexes, we propose that the asymmetric 2 : 1 Pol β–DNA complex enhances the function of Pol β.     

DNA structure and the Werner protein modulate human DNA polymerase delta-dependent replication dynamics within the common fragile site FRA16D

Common fragile sites (CFS) are chromosomal regions that exhibit instability during DNA replication stress. Although the mechanism of CFS expression has not been fully elucidated, one known feature is a severely delayed S-phase. We used an in vitro primer extension assay to examine the progression of DNA synthesis through various sequences within FRA16D by the replicative human DNA polymerases δ and α, and with human cell-free extracts. We found that specific cis-acting sequence elements perturb DNA elongation, causing inconsistent DNA synthesis rates between regions on the same strand and complementary strands. Pol δ was significantly inhibited in regions containing hairpins and microsatellites, [AT/TA]₂₄ and [A/T]_19–28, compared with a control region with minimal secondary structure. Pol δ processivity was enhanced by full length Werner Syndrome protein (WRN) and by WRN fragments containing either the helicase domain or DNA-binding C-terminal domain. In cell-free extracts, stalling was eliminated at smaller hairpins, but persisted in larger hairpins and microsatellites. Our data support a model whereby CFS expression during cellular stress is due to a combination of factors—density of specific DNA secondary-structures within a genomic region and asymmetric rates of strand synthesis.     

3D architecture of DNA Pol α reveals the functional core of multi-subunit replicative polymerases

Eukaryotic DNA replication requires the coordinated activity of the multi-subunit DNA polymerases: Pol α, Pol δ and Pol . The conserved catalytic and regulatory B subunits associate in a constitutive heterodimer that represents the functional core of all three replicative polymerases. Here, we combine X-ray crystallography and electron microscopy (EM) to describe subunit interaction and 3D architecture of heterodimeric yeast Pol α. The crystal structure of the C-terminal domain (CTD) of the catalytic subunit bound to the B subunit illustrates a conserved mechanism of accessory factor recruitment by replicative polymerases. The EM reconstructions of Pol α reveal a bilobal shape with separate catalytic and regulatory modules. Docking of the B–CTD complex in the EM reconstruction shows that the B subunit is tethered to the polymerase domain through a structured but flexible linker. Our combined findings provide a structural template for the common functional architecture of the three major replicative DNA polymerases.     

X-ray Crystal Structure of Escherichia coli RNA Polymerase σ70 Holoenzyme

Escherichia coli RNA polymerase (RNAP) is the most studied bacterial RNAP and has been used as the model RNAP for screening and evaluating potential RNAP-targeting antibiotics. However, the x-ray crystal structure of E. coli RNAP has been limited to individual domains. Here, I report the x-ray structure of the E. coli RNAP σ⁷⁰ holoenzyme, which shows σ region 1.1 (σ_1.1) and the α subunit C-terminal domain for the first time in the context of an intact RNAP. σ_1.1 is positioned at the RNAP DNA-binding channel and completely blocks DNA entry to the RNAP active site. The structure reveals that σ_1.1 contains a basic patch on its surface, which may play an important role in DNA interaction to facilitate open promoter complex formation. The α subunit C-terminal domain is positioned next to σ domain 4 with a fully stretched linker between the N- and C-terminal domains. E. coli RNAP crystals can be prepared from a convenient overexpression system, allowing further structural studies of bacterial RNAP mutants, including functionally deficient and antibiotic-resistant RNAPs.     

Talin1 and Rap1 Are Critical for Osteoclast Function

To determine talin1''s role in osteoclasts, we mated TLN1^fl/fl mice with those expressing cathepsin K-Cre (CtsK-TLN1) to delete the gene in mature osteoclasts or with lysozyme M-Cre (LysM-TLN1) mice to delete TLN1 in all osteoclast lineage cells. Absence of TLN1 impairs macrophage colony-stimulating factor (M-CSF)-stimulated inside-out integrin activation and cytoskeleton organization in mature osteoclasts. Talin1-deficient precursors normally express osteoclast differentiation markers when exposed to M-CSF and receptor activator of nuclear factor κB (RANK) ligand but attach to substrate and migrate poorly, arresting their development into mature resorptive cells. In keeping with inhibited resorption, CtsK-TLN1 mice exhibit an ∼5-fold increase in bone mass. Osteoclast-specific deletion of Rap1 (CtsK-Rap1), which promotes talin/β integrin recognition, yields similar osteopetrotic mice. The fact that the osteopetrosis of CtsK-TLN1 and CtsK-Rap1 mice is substantially more severe than that of those lacking αvβ3 is likely due to added failed activation of β1 integrins. In keeping with osteoclast dysfunction, mice in whom talin is deleted late in the course of osteoclastogenesis are substantially protected from ovariectomy-induced osteoporosis and the periarticular osteolysis attending inflammatory arthritis. Thus, talin1 and Rap1 are critical for resorptive function, and their selective inhibition in mature osteoclasts retards pathological bone loss.     

Structure of the DNA-binding domain of NgTRF1 reveals unique features of plant telomere-binding proteins

Telomeres are protein–DNA elements that are located at the ends of linear eukaryotic chromosomes. In concert with various telomere-binding proteins, they play an essential role in genome stability. We determined the structure of the DNA-binding domain of NgTRF1, a double-stranded telomere-binding protein of tobacco, using multidimensional NMR spectroscopy and X-ray crystallography. The DNA-binding domain of NgTRF1 contained the Myb-like domain and C-terminal Myb-extension that is characteristic of plant double-stranded telomere-binding proteins. It encompassed amino acids 561–681 (NgTRF1^561–681), and was composed of 4 α-helices. We also determined the structure of NgTRF1^561–681 bound to plant telomeric DNA. We identified several amino acid residues that interacted directly with DNA, and confirmed their role in the binding of NgTRF1 to telomere using site-directed mutagenesis. Based on a structural comparison of the DNA-binding domains of NgTRF1 and human TRF1 (hTRF1), NgTRF1 has both common and unique DNA-binding properties. Interaction of Myb-like domain with telomeric sequences is almost identical in NgTRF1^561–681 with the DNA-binding domain of hTRF1. The interaction of Arg-638 with the telomeric DNA, which is unique in NgTRF1^561–681, may provide the structural explanation for the specificity of NgTRF1 to the plant telomere sequences, (TTTAGGG)_n.     

Identification of the Functional Domains of the Telomere Protein Rap1 in Schizosaccharomyces pombe

The telomere at the end of a linear chromosome plays crucial roles in genome stability. In the fission yeast Schizosaccharomyces pombe, the Rap1 protein, one of the central players at the telomeres, associates with multiple proteins to regulate various telomere functions, such as the maintenance of telomere DNA length, telomere end protection, maintenance of telomere heterochromatin, and telomere clustering in meiosis. The molecular bases of the interactions between Rap1 and its partners, however, remain largely unknown. Here, we describe the identification of the interaction domains of Rap1 with its partners. The Bqt1/Bqt2 complex, which is required for normal meiotic progression, Poz1, which is required for telomere length control, and Taz1, which is required for the recruitment of Rap1 to telomeres, bind to distinct domains in the C-terminal half of Rap1. Intriguingly, analyses of a series of deletion mutants for rap1 ⁺ have revealed that the long N-terminal region (1–456 a.a. [amino acids]) of Rap1 (full length: 693 a.a.) is not required for telomere DNA length control, telomere end protection, and telomere gene silencing, whereas the C-terminal region (457–693 a.a.) containing Poz1- and Taz1-binding domains plays important roles in those functions. Furthermore, the Bqt1/Bqt2- and Taz1-binding domains are essential for normal spore formation after meiosis. Our results suggest that the C-terminal half of Rap1 is critical for the primary telomere functions, whereas the N-terminal region containing the BRCT (BRCA1 C-terminus) and Myb domains, which are evolutionally conserved among the Rap1 family proteins, does not play a major role at the telomeres.     

Role of accessory DNA polymerases in DNA replication in Escherichia coli: analysis of the dnaX36 mutator mutant

The dnaX36(TS) mutant of Escherichia coli confers a distinct mutator phenotype characterized by enhancement of transversion base substitutions and certain (−1) frameshift mutations. Here, we have further investigated the possible mechanism(s) underlying this mutator effect, focusing in particular on the role of the various E. coli DNA polymerases. The dnaX gene encodes the τ subunit of DNA polymerase III (Pol III) holoenzyme, the enzyme responsible for replication of the bacterial chromosome. The dnaX36 defect resides in the C-terminal domain V of τ, essential for interaction of τ with the α (polymerase) subunit, suggesting that the mutator phenotype is caused by an impaired or altered α-τ interaction. We previously proposed that the mutator activity results from aberrant processing of terminal mismatches created by Pol III insertion errors. The present results, including lack of interaction of dnaX36 with mutM, mutY, and recA defects, support our assumption that dnaX36-mediated mutations originate as errors of replication rather than DNA damage-related events. Second, an important role is described for DNA Pol II and Pol IV in preventing and producing, respectively, the mutations. In the system used, a high fraction of the mutations is dependent on the action of Pol IV in a (dinB) gene dosage-dependent manner. However, an even larger but opposing role is deduced for Pol II, revealing Pol II to be a major editor of Pol III mediated replication errors. Overall, the results provide insight into the interplay of the various DNA polymerases, and of τ subunit, in securing a high fidelity of replication.