Posttranslational Regulation of Human DNA Polymerase ι

Human DNA polymerases (pols) η and ι are Y-family DNA polymerase paralogs that facilitate translesion synthesis past damaged DNA. Both polη and polι can be monoubiquitinated in vivo. Polη has been shown to be ubiquitinated at one primary site. When this site is unavailable, three nearby lysines may become ubiquitinated. In contrast, mass spectrometry analysis of monoubiquitinated polι revealed that it is ubiquitinated at over 27 unique sites. Many of these sites are localized in different functional domains of the protein, including the catalytic polymerase domain, the proliferating cell nuclear antigen-interacting region, the Rev1-interacting region, and its ubiquitin binding motifs UBM1 and UBM2. Polι monoubiquitination remains unchanged after cells are exposed to DNA-damaging agents such as UV light (generating UV photoproducts), ethyl methanesulfonate (generating alkylation damage), mitomycin C (generating interstrand cross-links), or potassium bromate (generating direct oxidative DNA damage). However, when exposed to naphthoquinones, such as menadione and plumbagin, which cause indirect oxidative damage through mitochondrial dysfunction, polι becomes transiently polyubiquitinated via Lys¹¹- and Lys⁴⁸-linked chains of ubiquitin and subsequently targeted for degradation. Polyubiquitination does not occur as a direct result of the perturbation of the redox cycle as no polyubiquitination was observed after treatment with rotenone or antimycin A, which both inhibit mitochondrial electron transport. Interestingly, polyubiquitination was observed after the inhibition of the lysine acetyltransferase KATB3/p300. We hypothesize that the formation of polyubiquitination chains attached to polι occurs via the interplay between lysine acetylation and ubiquitination of ubiquitin itself at Lys¹¹ and Lys⁴⁸ rather than oxidative damage per se.     

A DEVD-Inhibited Caspase Other than CPP32 Is Involved in the Commitment of Cerebellar Granule Neurons to Apoptosis Induced by K+ Deprivation

Abstract: Cultured cerebellar granule neurons undergo apoptosis when switched from a medium containing depolarizing levels of K⁺ (25 mM KCI) to medium containing lower levels of K⁺ (5 mM KCI). We used this paradigm to investigate the role of caspases in the death process. Two broad-spectrum caspase inhibitors, tert-butoxycarbonyl-Asp·(O-methyl)·fluoromethyl ketone and benzyloxycarbonyl-Val-Ala-Asp·fluoromethyl ketone, significantly reduced cell death (90 and 60%, respectively) at relatively low concentrations (10–25 µM), suggesting that caspase activation is involved in the apoptotic process. DNA fragmentation, a hallmark of apoptosis, was also reduced by these caspase inhibitors, suggesting that caspase activation occurred upstream of DNA cleavage in the sequence of events leading to cell death. As a step toward identifying the caspase(s) involved, the effects of N-acetyl Tyr-Val-Ala-Asp·chloromethyl ketone (YVAD·cmk), an interleukin-1β converting enzyme-preferring inhibitor, and N-acetyl Asp-Glu-Val-Asp·fluoromethyl ketone (DEVD·fmk), a CPP32-preferring inhibitor, were also evaluated. YVAD·cmk provided only modest (<20%) protection and only at the highest concentration (100 µM) tested, suggesting that interleukin-1β converting enzyme and/or closely related caspases were not involved. In comparison, DEVD·fmk inhibited cell death by up to 50%. Western blot analyses, however, failed to detect an increase in processing/activation of CPP32 or in the proteolysis of a CPP32 substrate, poly(ADP-ribose) polymerase, during the induction of apoptosis in granule neurons. Similarly, the levels of Nedd2, a caspase that is highly expressed in the brain and that is partially inhibited by DEVD·fmk, also remained unaffected in apoptotic neurons undergoing apoptosis. These results suggest that a DEVD-sensitive caspase other than CPP32 or Nedd2 mediates the induction of apoptosis in K⁺-deprived granule neurons.     

Significant impact of divalent metal ions on the fidelity,sugar selectivity,and drug incorporation efficiency of human PrimPol

Human PrimPol is a recently discovered bifunctional enzyme that displays DNA template-directed primase and polymerase activities. PrimPol has been implicated in nuclear and mitochondrial DNA replication fork progression and restart as well as DNA lesion bypass. Published evidence suggests that PrimPol is a Mn²⁺-dependent enzyme as it shows significantly improved primase and polymerase activities when binding Mn²⁺, rather than Mg²⁺, as a divalent metal ion cofactor. Consistently, our fluorescence anisotropy assays determined that PrimPol binds to a primer/template DNA substrate with affinities of 29 and 979 nM in the presence of Mn²⁺ and Mg²⁺, respectively. Our pre-steady-state kinetic analysis revealed that PrimPol incorporates correct dNTPs with 100-fold higher efficiency with Mn²⁺ than with Mg²⁺. Notably, the substitution fidelity of PrimPol in the presence of Mn²⁺ was determined to be in the range of 3.4 × 10⁻² to 3.8 × 10⁻¹, indicating that PrimPol is an error-prone polymerase. Furthermore, we kinetically determined the sugar selectivity of PrimPol to be 57–1800 with Mn²⁺ and 150–4500 with Mg²⁺, and found that PrimPol was able to incorporate the triphosphates of two anticancer drugs (cytarabine and gemcitabine), but not two antiviral drugs (emtricitabine and lamivudine).     

The Detection of Hamster Connexins: A Comparison of Expression Profiles with Wild-Type Mouse and the Cancer-Prone Min Mouse

The open reading frames of 17 connexins from Syrian hamster (using tissues) and 16 connexins from the Chinese hamster cell line V79, were fully (Cx30, Cx31, Cx37, Cx43 and Cx45) or partially sequenced. We have also detected, and partially sequenced, seven rat connexins that previously were unavailable. The expression of connexin genes was examined in some hamster organs and cultured hamster cells, and compared with wild-type mouse and the cancer-prone Min mouse. Although the expression patterns were similar for most organs and connexins in hamster and mouse, there were also some prominent differences (Cx29 and 30.3 in testis; Cx31.1 and 32 in eye; Cx46 in brain, kidney and testis; Cx47 in kidney). This suggests that some connexins have species-specific expression profiles. In contrast, there were minimal differences in expression profiles between wild type and Min mice. Species-specific expression profiles should be considered in attempts to make animal models of human connexin-associated diseases.     

Regeneration of Triticum aestivum apical explants after microinjection of germ line progenitor cells with DNA

A highly efficient method of regenerating fertile, phenotypically normal plants from shoot apex cultures of T. aestivum was developed. The hypodermal layer (L2) of the vegetative apex containing germ line precursor cells could be located with bright field microscopy and targeted for microinjection. Fluorescently labelled dextrans were used as markers to develop a microinjection procedure which did not disrupt nuclear or cytoplasmic structure. This procedure was used to inject plasmid DNA into L2 cells. Capillary microinjection did not shear the plasmid DNA and delivery of DNA was confirmed by polymerase chain reaction analysis of DNA isolated from injected apices. The significance of these findings in relation to the development of cereal transformation systems will be discussed.     

Heat stress in the presence of low RNA polymerase activity increases chromosome copy number of Escherichia coli

Summary A temperature shift-up accompanied by a reduction in RNA polymerase activity in Escherichia coli causes an increased rate of initiation leading to a 1.7- to 2.2-fold increase in chromosome copy number. A temperature shift-up without a reduction in polymerase activity induces only a transient non-scheduled initiation of chromosome replication caused by heat shock with no detectable effect on chromosome copy number.     

Docking and binding mode analysis of aryl diketoacids (ADK) at the active site of HCV RNA-dependent RNA polymerase

The pharmacophore-guided docking study of aryl diketoacid (ADK) analogues revealed two distinctive hydrophobic binding sites (a pocket and a groove) around the UTP-binding site of hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp). Interestingly, the hydrophobic binding sites have appropriate shape and size to specifically substituted aromatic rings, which suggests the specific role of substituents on the aromatic ring in determining the binding affinity of the ADK analogue to the active site of the target enzyme. Binding mode analysis of ADK analogues with potent antiviral activity shows highly substituted aromatic rings map well onto the hydrophobic binding sites. For less active compounds, their lack of aromatic substitution and thereby insufficient size can be primarily ascribed to their inability to bind to the hydrophobic binding site. The characteristic binding mode of ADK analogues proposed in this study provides a useful tool in designing a structure–activity relationship study of novel ADK analogues based on various aromatic substituents.     

DNA Sequences from Formalin-Fixed Nematodes: Integrating Molecular and Morphological Approaches to Taxonomy

To effectively integrate DNA sequence analysis and classical nematode taxonomy, we must be able to obtain DNA sequences from formalin-fixed specimens. Microdissected sections of nematodes were removed from specimens fixed in formalin, using standard protocols and without destroying morphological features. The fixed sections provided sufficient template for multiple polymerase chain reaction-based DNA sequence analyses.     

The Schizosaccharomyces pombe cdc6 gene encodes the catalytic subunit of DNA polymerase δ

The cdc6 mutants of Schizosaccharomyces pombe have been classified as being defective in progression through the G2 phase of the cell cycle. We cloned an S. pombe gene that could complement the temperature-sensitive growth of the cdc6-23 mutant. Unexpectedly, the cloned gene was allelic to pol3, which encodes the catalytic subunit of DNA polymerase δ. Integration mapping confirmed that cdc6 and pol3 are identical. The cdc6-23 mutant carries one amino acid substitution in the conserved N3 region of Pol3. Received: 17 October 1996 / Accepted: 19 November 1996