Characterization of DNA Binding by the Isolated N‑Terminal Domain of Vaccinia Virus DNA Topoisomerase IB

Vaccinia TopIB (vTopIB), a 314-amino acid eukaryal-type IB topoisomerase, recognizes and transesterifies at the DNA sequence 5′-(T/C)­CCTT↓, leading to the formation of a covalent DNA–(3′-phosphotyrosyl²⁷⁴)–enzyme intermediate in the supercoil relaxation reaction. The C-terminal segment of vTopIB (amino acids 81–314), which engages the DNA minor groove at the scissile phosphodiester, comprises an autonomous catalytic domain that retains cleavage specificity, albeit with a cleavage site affinity lower than that of the full-length enzyme. The N-terminal domain (amino acids 1–80) engages the major groove on the DNA face opposite the scissile phosphodiester. Whereas DNA contacts of the N-terminal domain have been implicated in the DNA site affinity of vTopIB, it was not known whether the N-terminal domain per se could bind DNA. Here, using isothermal titration calorimetry, we demonstrate the ability of the isolated N-terminal domain to bind a CCCTT-containing 24-mer duplex with an apparent affinity that is ∼2.2-fold higher than that for an otherwise identical duplex in which the pentapyrimidine sequence is changed to ACGTG. Analyses of the interactions of the isolated N-terminal domain with duplex DNA via solution nuclear magnetic resonance methods are consistent with its DNA contacts observed in DNA-bound crystal structures of full-length vTopIB. The chemical shift perturbations and changes in hydrodynamic properties triggered by CCCTT DNA versus non-CCCTT DNA suggest differences in DNA binding dynamics. The importance of key N-terminal domain contacts in the context of full-length vTopIB is underscored by assessing the effects of double-alanine mutations on DNA transesterification and its sensitivity to ionic strength

Characterization of <i>Mycobacterium smegmatis</i> PolD2 and PolD1 as RNA/DNA Polymerases Homologous to the POL Domain of Bacterial DNA Ligase D

Mycobacteria exploit nonhomologous end-joining (NHEJ) to repair DNA double-strand breaks. The core NHEJ machinery comprises the homodimeric DNA end-binding protein Ku and DNA ligase D (LigD), a modular enzyme composed of a C-terminal ATP-dependent ligase domain (LIG), a central 3′-phosphoesterase domain (PE), and an N-terminal polymerase domain (POL). LigD POL is proficient at adding templated and nontemplated deoxynucleotides and ribonucleotides to DNA ends in vitro and is the catalyst in vivo of unfaithful NHEJ events involving nontemplated single-nucleotide additions to blunt DSB ends. Here, we identify two mycobacterial proteins, PolD1 and PolD2, as stand-alone homologues of the LigD POL domain. Biochemical characterization of PolD1 and PolD2 shows that they resemble LigD POL in their monomeric quaternary structures, their ability to add templated and nontemplated nucleotides to primer-templates and blunt ends, and their preference for rNTPs versus dNTPs. Deletion of <i>polD1</i>, <i>polD2</i>, or both from a <i>Mycobacterium smegmatis</i> strain carrying an inactivating mutation in LigD POL failed to reveal a role for PolD1 or PolD2 in templated nucleotide additions during NHEJ of 5′-overhang DSBs or in clastogen resistance. Whereas our results document the existence and characteristics of new stand-alone members of the LigD POL family of RNA/DNA polymerases, they imply that other polymerases can perform fill-in synthesis during mycobacterial NHEJ

The PAF Complex and Prf1/Rtf1 Delineate Distinct Cdk9-Dependent Pathways Regulating Transcription Elongation in Fission Yeast

<div><p>Cyclin-dependent kinase 9 (Cdk9) promotes elongation by RNA polymerase II (RNAPII), mRNA processing, and co-transcriptional histone modification. Cdk9 phosphorylates multiple targets, including the conserved RNAPII elongation factor Spt5 and RNAPII itself, but how these different modifications mediate Cdk9 functions is not known. Here we describe two Cdk9-dependent pathways in the fission yeast <i>Schizosaccharomyces pombe</i> that involve distinct targets and elicit distinct biological outcomes. Phosphorylation of Spt5 by Cdk9 creates a direct binding site for Prf1/Rtf1, a transcription regulator with functional and physical links to the Polymerase Associated Factor (PAF) complex. PAF association with chromatin is also dependent on Cdk9 but involves alternate phosphoacceptor targets. Prf1 and PAF are biochemically separate in cell extracts, and genetic analyses show that Prf1 and PAF are functionally distinct and exert opposing effects on the RNAPII elongation complex. We propose that this opposition constitutes a Cdk9 auto-regulatory mechanism, such that a positive effect on elongation, driven by the PAF pathway, is kept in check by a negative effect of Prf1/Rtf1 and downstream mono-ubiquitylation of histone H2B. Thus, optimal RNAPII elongation may require balanced action of functionally distinct Cdk9 pathways.</p></div

Prf1 does not stably associate with the PAF complex.

<p>(<b>A</b>) TAP purification was carried out using whole cell extracts from the indicated strains and purified material was analyzed by SDS-PAGE and silver staining. The proteins contained in the labeled bands were identified by mass spectrometry (as indicated on the right). “CBP” denotes the residual fusion to the calmodulin binding peptide resulting from the TAP procedure. Leo1 was detected in two bands that also contained either Paf1-CBP (in the <i>paf1-TAP</i> lane) or Paf1 (in the <i>tpr1-TAP</i> lane). Molecular weight standards (in kD) are denoted on the left. (<b>B</b>) Single-step TAP purifications were performed using extracts from the indicated strains. 5% of input fractions (“input”) and 50% of bead-bound fractions (“beads”) were analyzed by SDS-PAGE and western blotting.</p

Model depicting the roles of the Prf1/Rtf1 and PAF pathways in RNAPII elongation.

<p>The Prf1 pathway, involving direct association of Prf1 with phosphorylated Spt5 CTD, is labeled “1.” The PAF pathway, involving multiple Cdk9 targets, is labeled “2.” Potential crosstalk between the two pathways is indicated by the broken double arrow. See text for details.</p

Prf1 and PAF pathways have opposing biological effects.

<p>(<b>A</b>) Quantification of the abnormal septation patterns in the indicated strains. Strains carrying the <i>cdk9^as</i> allele were cultured in DMSO (−) or 2 µM 3-MB-PP1 (+) for 15 hours prior to fixation for microscopy. Error bars denote standard deviations from 3 independent experiments. (<b>B</b>) ChIP of RNAPII was carried out in the indicated strains and quantified by qPCR using primers specific to the <i>nup189</i>⁺ gene. Values were normalized to that for primer pair 1. Error bars denote standard deviations from three independent experiments. Significant differences from wild-type values (unpaired t-test) are indicated.</p

Prf1 and PAF have shared roles in histone H2B ubiquitylation but are functionally distinct.

<p>(<b>A</b>) Whole-cell extracts from strains of the indicated genotypes were analyzed by SDS-PAGE and western blotting with the indicated antibodies. (<b>B</b>) Cells of the indicated genotypes were fixed, stained with DAPI/calcofluor, and visualized by microscopy. For each strain fluorescent images (denoting DAPI/calcofluor staining) are shown on the left; bright-field images are shown on the right. (<b>C</b>) Quantification of the abnormal septation patterns in the indicated strains. “Septa” refers to cells with at least one visible division septum, “multi-sep” refers to cells with twinned septa or multiple septa between two nuclei, and “chains” refers to unseparated chains of cells. Error bars denote standard deviations from 3 independent experiments.</p

Prf1 and PAF are recruited to chromatin via alternate mechanisms.

<p>(<b>A</b>) Strains used for ChIP with IgG resin (recognizing the TAP tag) are indicated at the bottom. All strains also harbored the <i>cdk9^as</i> allele and were treated with either DMSO (−) or 20 µM 3-MB-PP1 (+) for two hours before crosslinking for ChIP (also indicated by “3MB” at the far right). Assays were quantified by qPCR using primers specific for the <i>act1</i>⁺ (left) or <i>adh1</i>⁺ (right) genes. Lengths of the gene coding regions (in base pairs) and positions of PCR amplicons are indicated at the top. Numbering of the datasets for the corresponding PCR amplicons shown for <i>act1</i>⁺ (left) is used throughout the figure. Error bars denote standard deviations from 2–3 independent experiments. (<b>B and C</b>) Strains of genotypes indicated at the bottom were used for ChIP of either Prf1-TAP (left) or Tpr1-TAP (right). Cultures were treated with DMSO or 3-MB-PP1 as in (A).</p

Modified Vaccinia Virus Ankara Triggers Type I IFN Production in Murine Conventional Dendritic Cells via a cGAS/STING-Mediated Cytosolic DNA-Sensing Pathway

<div><p>Modified vaccinia virus Ankara (MVA) is an attenuated poxvirus that has been engineered as a vaccine against infectious agents and cancers. Our goal is to understand how MVA modulates innate immunity in dendritic cells (DCs), which can provide insights to vaccine design. In this study, using murine bone marrow-derived dendritic cells, we assessed type I interferon (IFN) gene induction and protein secretion in response to MVA infection. We report that MVA infection elicits the production of type I IFN in murine conventional dendritic cells (cDCs), but not in plasmacytoid dendritic cells (pDCs). Transcription factors IRF3 (IFN regulatory factor 3) and IRF7, and the positive feedback loop mediated by IFNAR1 (IFN alpha/beta receptor 1), are required for the induction. MVA induction of type I IFN is fully dependent on STING (stimulator of IFN genes) and the newly discovered cytosolic DNA sensor cGAS (cyclic guanosine monophosphate-adenosine monophosphate synthase). MVA infection of cDCs triggers phosphorylation of TBK1 (Tank-binding kinase 1) and IRF3, which is abolished in the absence of cGAS and STING. Furthermore, intravenous delivery of MVA induces type I IFN in wild-type mice, but not in mice lacking STING or IRF3. Treatment of cDCs with inhibitors of endosomal and lysosomal acidification or the lysosomal enzyme Cathepsin B attenuated MVA-induced type I IFN production, indicating that lysosomal enzymatic processing of virions is important for MVA sensing. Taken together, our results demonstrate a critical role of the cGAS/STING-mediated cytosolic DNA-sensing pathway for type I IFN induction in cDCs by MVA. We present evidence that vaccinia virulence factors E3 and N1 inhibit the activation of IRF3 and the induction of IFNB gene in MVA-infected cDCs.</p></div

MVA induces type I IFN production in conventional dendritic cells (cDCs).

<p>(A) Murine pDCs and cDCs were purified from Flt3L-BMDCs using FACS. 2×10⁵ pDCs (CD11c⁺B220⁺PDCA-1⁺) and 1×10⁶ cDCs (CD11c⁺B220⁻PDCA-1⁻) were either stimulated with CpG (at a final concentration of 10 µg/ml) or infected with either WT VAC or MVA at a MOI of 10. Supernatants were collected at 22 h post infection. The concentrations of IFN-α and IFN-β were determined by ELISA. Data are means ± SEM (n = 6). A representative experiment is shown, repeated at least twice. (B) GM-CSF-BMDCs (1×10⁶) were infected with WT VAC or MVA at a MOI of 10. Supernatants were collected at 1, 4, 8, 14, and 22 h post infection. The concentrations of IFN-α and IFN-β were determined by ELISA. Data are means ± SEM (n = 3). A representative experiment is shown, repeated once. (C) GM-CSF-BMDCs (1×10⁶) were infected with MVA at a MOI of 0.25, 0.5, 1, 5, or 10. Supernatants were collected at 22 h post infection. The concentrations of IFN-α and IFN-β were determined by ELISA. Data are means ± SEM (n = 3). A representative experiment is shown, repeated once. (D) GM-CSF-BMDCs (1×10⁶) were infected with MVA or WT VAC at a MOI of 10. Cells were collected at 6 h post infection. Real-time PCR analysis of IFNA4 and IFNB mRNAs were performed. Data are means ± SEM (n = 3). A representative experiment is shown, repeated twice. ***, <i>p</i><0.001; comparisons were made between MVA and WT VAC infected cells. (E) GM-CSF-BMDCs (1×10⁶) were infected with MVA or WT VAC at a MOI of 10. Cells were collected at 1, 2, 4, and 8 h post infection. Western blot analysis was performed using anti-phospho-TBK1, anti-TBK1, anti-phosphoserine-396 of IRF3, and anti-IRF3. Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) was used as a loading control. “hpi”, hours post infection. “M”, mock infection control.</p