Molecular Mechanisms of the Whole DNA Repair System: A Comparison of Bacterial and Eukaryotic Systems

DNA is subjected to many endogenous and exogenous damages. All organisms have developed a complex network of DNA repair mechanisms. A variety of different DNA repair pathways have been reported: direct reversal, base excision repair, nucleotide excision repair, mismatch repair, and recombination repair pathways. Recent studies of the fundamental mechanisms for DNA repair processes have revealed a complexity beyond that initially expected, with inter- and intrapathway complementation as well as functional interactions between proteins involved in repair pathways. In this paper we give a broad overview of the whole DNA repair system and focus on the molecular basis of the repair machineries, particularly in Thermus thermophilus HB8

Characterization of DNA polymerase X from Thermus thermophilus HB8 reveals the POLXc and PHP domains are both required for 3′–5′ exonuclease activity

The X-family DNA polymerases (PolXs) comprise a highly conserved DNA polymerase family found in all kingdoms. Mammalian PolXs are known to be involved in several DNA-processing pathways including repair, but the cellular functions of bacterial PolXs are less known. Many bacterial PolXs have a polymerase and histidinol phosphatase (PHP) domain at their C-termini in addition to a PolX core (POLXc) domain, and possess 3′–5′ exonuclease activity. Although both domains are highly conserved in bacteria, their molecular functions, especially for a PHP domain, are unknown. We found Thermus thermophilus HB8 PolX (ttPolX) has Mg2+/Mn2+-dependent DNA/RNA polymerase, Mn2+-dependent 3′–5′ exonuclease and DNA-binding activities. We identified the domains of ttPolX by limited proteolysis and characterized their biochemical activities. The POLXc domain was responsible for the polymerase and DNA-binding activities but exonuclease activity was not detected for either domain. However, the POLXc and PHP domains interacted with each other and a mixture of the two domains had Mn2+-dependent 3′–5′ exonuclease activity. Moreover, site-directed mutagenesis revealed catalytically important residues in the PHP domain for the 3′–5′ exonuclease activity. Our findings provide a molecular insight into the functional domain organization of bacterial PolXs, especially the requirement of the PHP domain for 3′–5′ exonuclease activity

Co-expression of foreign proteins tethered to HIV-1 envelope glycoprotein on the cell surface by introducing an intervening second membrane-spanning domain.

The envelope glycoprotein (Env) of human immunodeficiency virus type I (HIV-1) mediates membrane fusion. To analyze the mechanism of HIV-1 Env-mediated membrane fusion, it is desirable to determine the expression level of Env on the cell surface. However, the quantification of Env by immunological staining is often hampered by the diversity of HIV-1 Env and limited availability of universal antibodies that recognize different Envs with equal efficiency. To overcome this problem, here we linked a tag protein called HaloTag at the C-terminus of HIV-1 Env. To relocate HaloTag to the cell surface, we introduced a second membrane-spanning domain (MSD) between Env and HaloTag. The MSD of transmembrane protease serine 11D, a type II transmembrane protein, successfully relocated HaloTag to the cell surface. The surface level of Env can be estimated indirectly by staining HaloTag with a specific membrane-impermeable fluorescent ligand. This tagging did not compromise the fusogenicity of Env drastically. Furthermore, fusogenicity of Env was preserved even after the labeling with the ligands. We have also found that an additional foreign peptide or protein such as C34 or neutralizing single-chain variable fragment (scFv) can be linked to the C-terminus of the HaloTag protein. Using these constructs, we were able to determine the required length of C34 and critical residues of neutralizing scFv for blocking membrane fusion, respectively

Triphosphate Ion-Selective Electrode Based on Zr-Porphyrin Complex

Ion-selective electrode using zirconium­(IV) complex with octaethylporphin (H₂oep) as a carrier showed high selectivity to triphosphate (TP, H₅tp) against other hydrophilic anions including diphosphate and phosphate. The electroactive species was identified to be [(Zr₄(oep)₄(Htp)₂] (TP/Zr ratio of 0.5) of the unique structure; triphosphates are recognized by one Zr atom through three O atoms on three different P atoms and by another Zr atom through two O atoms on two terminal P atoms and are also involved in complementary intermolecular hydrogen bonding to be surrounded by four porphyrin complexes. In contrast, Zr­(IV) in the other complex with tetraphenylporphin has the higher Lewis acidity, due to the electron-withdrawing property of phenyl rings and, at the higher TP concentration, forms a species having a TP/Zr ratio of unity, which precipitates to lose the electroactivity. The electrode was successfully applied to monitor hydrolysis of TP that provides diphosphate and phosphate

Flow cytometry analysis of Env-expressing cells labeled with Halo ligands or anti-Env monoclonal antibody.

<p>(A–C) Flow cytometry analysis of Env expression level using different staining strategies. 293FT cells were transfected with expression vectors for tethered Env (HXB2-TM11D-Halo, red line; JRFL-TM11D-Halo, blue line), untethered Env (HXB2-WT, red dashed line; JRFL-WT, blue dashed line), or Mock DNA (grey shade). Cells were stained with membrane-impermeable HaloTag AF488 ligand (A), membrane-permeable HaloTag Oregon Green ligand (C) and anti-Env V3 antibody V3-G2-25 (B). Histograms are representative results from three independent experiments. (D) Positive staining rate of HaloTag labeling and anti-Env antibody immunolabeling of cells transfected with HXB2-TM11D-Halo (solid red bar), HXB2-WT (red shade bar), JRFL-TM11D-Halo (solid blue bar), JRFL-WT (blue shade bar) or Mock (solid gray bar). Error bars represent standard deviations of the results from three independent experiments.</p

Connection of HaloTag to Env with the addition of the intervening second MSD.

<p>(A) Upper panel: Design of the Env-TM11D-Halo construct. A CMV promoter drives the expression of the tethered construct in all expression vectors. The 21 aa MSD of TM11D was added to the C terminal of gp41 as an MSD2 to help flipping out the HaloTag. Lower panel: Design of the Env-Halo construct, the control tethered construct without the MSD2 of TM11D. The expected membrane topology of the expressed protein with each construct is depicted schematically on the right. ED: ectodomain of gp41, MSD: membrane-spanning domain of gp41, CT: cytoplasmic tail of gp41. The sequences for Env corresponding to HXB2 or JRFL were used. (B) Confocal microscope analysis of tethered HaloTag in transfected 293FT cells stained with membrane-permeable (TMR, red color) or impermeable (AF488, green color) ligands. The transfected DNA is indicated: Mock, control DNA transfection; HXB2-Halo, Halo directly connects with gp41 (without the MSD of TM11D); HXB2-TM11D-Halo, a construct with the MSD of TM11D added between Env and Halo. Scale bar = 20 µm. (C) Effect of Halo ligands on the fusogenicity of HaloTag attached Env by DSP assay. Halo TMR and AF488 ligands were used to label the HXB2-TM11D-Halo fusion protein. The effect on the fusion activity was measured by DSP assay, which measures pore formation during cell-cell fusion by split <i>Renilla luciferase</i> (RL) reporter proteins. DSP activities for the ligand-labeled Env were compared with the HXB2-TM11D-Halo and non-tethered HXB2 Env protein without labeling (only add DMEM culture medium, and the value of HXB2-TM11D-Halo was set at 100%). Error bars represent standard deviations of the results of triplicate experiments. Student’s <i>t</i>-test was used to determine the statistical significance of the measured variables for each differently labeled (open column) and the non-labeled (solid column). ns = nonsignificant.</p

Effect of tethered neutralizing antibodies evaluated by syncytia formation and DSP assay.

<p>(A) Immunoblotting analysis of tethered fusion protein expression in 293FT cells with anti-gp120 (upper panel), anti-Flag (middle panel) or Chessie 8 anti-gp41 antibodies (lower panel). The expression vector used is indicated above the lane and the position of different fusion proteins is shown to the right. The anti-gp120 antibody detected the precursor form of tethered proteins and processed gp120 band; the anti-Flag antibody detected the tethered precursor and processed gp41-TM11D-scFv band; and the Chessie 8 anti-gp41antibody detected the processed gp41 (including gp41-TM11D-Halo and gp41-TM11D-scFv) bands. (B) The MFI of different constructs determined by flow cytometry. HaloTag Alexa Fluor 488 ligand was used to stain proteins expressed on the cell surface of 293FT cells transfected with different tethered constructs. The Tac-Halo vector (Halo is expressed in the cytoplasm) was used as a negative control for surface staining. Data was acquired with a BD FACSCalibur system and at least 12,000 events were collected and analyzed using FlowJo software. The MFI of HXB2-TM11D-Halo was set at 100%. Error bars represent standard deviations of the results of triplicate experiments. Student’s <i>t</i> test was used for the statistical analysis of the measured variables between individual construct (open column) and control (solid column). Significance was reported with p<0.05(*). ns = nonsignificant. (C) Fusion activity measured by DSP assay. The relative fusion activity was measured by DSP assay. DSP activities for each construct were compared with that of Env tethered with TM11D-MSD and HaloTag (HXB2-TM11D-Halo was set at 100%). Error bars represent standard deviations of the results of triplicate experiments. Student’s <i>t</i>-test was used to determine the statistical significance of the measured variables for each construct (open column) and control (solid column). Statistical significance was indicated when p<0.05(*), p<0.001 (***). ns = nonsignificant. (D) Normalization of DSP activity with surface expression level of Env. The DSP activities of each tethered construct shown in panel C were normalized by respective surface expression level defined by MFI measured by flow cytometry shown in panel B. Student’s <i>t</i>-test was used to determine the statistical significance of the calculated variables for each construct (open column) and HXB2-TM11D-13H11 (solid grey column). Statistical significance was indicated when p<0.001 (***).</p