dna-sequence-recognition-dna-primase-using-high-throughput-primase

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. ...

Ben-Gurion University of the Negev

The structural differences between bacterial and human primases render the former an excellent target for drug design. Here we describe a technique for selecting small molecule inhibitors of the activity of T7 DNA primase, an ideal model for bacterial primases due to their common structural and functional features. Using NMR screening, fragment molecules that bind T7 primase were identified and then exploited in virtual filtration to select larger molecules from the ZINC database. The molecules were docked to the primase active site using the available primase crystal structure and ranked based on their predicted binding energies to identify the best candidates for functional and structural investigations. Biochemical assays revealed that some of the molecules inhibit T7 primase-dependent DNA replication. The binding mechanism was delineated via NMR spectroscopy. Our approach, which combines fragment based and virtual screening, is rapid and cost effective and can be applied to other targets.

Identification of DNA primase inhibitors via a combined fragment-based and virtual screening.

Enhanced activation of the signaling pathways that mediate the differentiation of mononuclear monocytes into osteoclasts is an underlying cause of several bone diseases and bone metastasis. In particular, dysregulation and overexpression of macrophage colony-stimulating factor (M-CSF) and its c-FMS tyrosine kinase receptor, proteins that are essential for osteoclast differentiation, are known to promote bone metastasis and osteoporosis, making both the ligand and its receptor attractive targets for therapeutic intervention. With this aim in mind, our starting point was the previously held concept that the potential of the M-CSF mutant as a therapeutic is derived from its inability to dimerize and hence to act as an agonist. The current study showed, however, that dimerization is not abolished in M-CSF and that the protein retains agonistic activity toward osteoclasts. To design an M-CSF mutant with diminished dimerization capabilities, we solved the crystal structure of the M-CSF dimer complex and used structure-based energy calculations to identify the residues responsible for its dimeric form. We then used that analysis to develop M-CSF, a ligand-based, high-affinity antagonist for c-FMS that retained its binding ability but prevented the ligand dimerization that leads to receptor dimerization and activation. The monomeric properties of M-CSF were validated using dynamic light scattering and small-angle X-ray scattering analyses. It was shown that this mutant is a functional inhibitor of M-CSF-dependent c-FMS activation and osteoclast differentiation  Our study, therefore, provided insights into the sequence-structure-function relationships of the M-CSF/c-FMS interaction and of ligand/receptor tyrosine kinase interactions in general.

Engineering a monomeric variant of macrophage colony-stimulating factor (M-CSF) that antagonizes the c-FMS receptor.

Lagging strand DNA synthesis by DNA polymerase requires RNA primers produced by DNA primase. The N-terminal primase domain of the gene 4 protein of phage T7 comprises a zinc-binding domain that recognizes a specific DNA sequence and an RNA polymerase domain that catalyzes RNA polymerization. Based on its crystal structure, the RNA polymerase domain contains two Mg(II) ions. Mn(II) substitution leads to elevated RNA primer synthesis by T7 DNA primase. NMR analysis revealed that upon binding Mn(II), T7 DNA primase undergoes conformational changes near the metal cofactor binding site that are not observed when the enzyme binds Mg(II). A machine-learning algorithm called linear discriminant analysis (LDA) was trained by using the large collection of Mn(II) and Mg(II) binding sites available in the protein data bank (PDB). Application of the model to DNA primase revealed a preference in the enzyme's second metal binding site for Mn(II) over Mg(II), suggesting that T7 DNA primase activity modulation when bound to Mn(II) is based on structural changes in the enzyme.

Modulation of RNA primer formation by Mn(II)-substituted T7 DNA primase.

The bacterial primase-an essential component in the replisome-is a promising but underexploited target for novel antibiotic drugs. Bacterial primases have a markedly different structure than the human primase. Inhibition of primase activity is expected to selectively halt bacterial DNA replication. Evidence is growing that halting DNA replication has a bacteriocidal effect. Therefore, inhibitors of DNA primase could provide antibiotic agents. Compounds that inhibit bacterial DnaG primase have been developed using different approaches. In this paper, we provide an overview of the current literature on DNA primases as novel drug targets and the methods used to find their inhibitors. Although few inhibitors have been identified, there are still challenges to develop inhibitors that can efficiently halt DNA replication and may be applied in a clinical setting.

DnaG Primase-A Target for the Development of Novel Antibacterial Agents.

Primases are key enzymes involved in DNA replication. They act on single-stranded DNA and catalyze the synthesis of short RNA primers used by DNA polymerases. Here, we investigate the DNA binding and activity of the bacteriophage T7 primase using a new workflow called high-throughput primase profiling (HTPP). Using a unique combination of high-throughput binding assays and biochemical analyses, HTPP reveals a complex landscape of binding specificity and functional activity for the T7 primase, determined by sequences flanking the primase recognition site. We identified specific features, such as G/T-rich flanks, which increase primase-DNA binding up to 10-fold and, surprisingly, also increase the length of newly formed RNA (up to 3-fold). To our knowledge, variability in primer length has not been reported for this primase. We expect that applying HTPP to additional enzymes will reveal new insights into the effects of DNA sequence composition on the DNA recognition and functional activity of primases.

Article	Year
Identification of DNA primase inhibitors via a combined fragment-based and virtual screening. Scientific reports\| PubMed ID: 27805033	2016
Engineering a monomeric variant of macrophage colony-stimulating factor (M-CSF) that antagonizes the c-FMS receptor. The Biochemical journal\| PubMed ID: 28655719	2017
Modulation of RNA primer formation by Mn(II)-substituted T7 DNA primase. Scientific reports\| PubMed ID: 28724886	2017
DnaG Primase-A Target for the Development of Novel Antibacterial Agents. Antibiotics (Basel, Switzerland)\| PubMed ID: 30104489	2018
DNA Sequence Context Controls the Binding and Processivity of the T7 DNA Primase. iScience\| PubMed ID: 30428370	2018

Stefan Ilic

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