Form Of Pol Research Articles

Biochemical Analysis of DNA Polymerase η Fidelity in the Presence of Replication Protein A

DNA polymerase η (pol η) synthesizes across from damaged DNA templates in order to prevent deleterious consequences like replication fork collapse and double-strand breaks. This process, termed translesion synthesis (TLS), is an overall positive for the cell, as cells deficient in pol η display higher mutation rates. This outcome occurs despite the fact that the in vitro fidelity of bypass by pol η alone is moderate to low, depending on the lesion being copied. One possible means of increasing the fidelity of pol η is interaction with replication accessory proteins present at the replication fork. We have previously utilized a bacteriophage based screening system to measure the fidelity of bypass using purified proteins. Here we report on the fidelity effects of a single stranded binding protein, replication protein A (RPA), when copying the oxidative lesion 7,8-dihydro-8-oxo-guanine(8-oxoG) and the UV-induced cis-syn thymine-thymine cyclobutane pyrimidine dimer (T-T CPD). We observed no change in fidelity dependent on RPA when copying these damaged templates. This result is consistent in multiple position contexts. We previously identified single amino acid substitution mutants of pol η that have specific effects on fidelity when copying both damaged and undamaged templates. In order to confirm our results, we examined the Q38A and Y52E mutants in the same full-length construct. We again observed no difference when RPA was added to the bypass reaction, with the mutant forms of pol η displaying similar fidelity regardless of RPA status. We do, however, observe some slight effects when copying undamaged DNA, similar to those we have described previously. Our results indicate that RPA by itself does not affect pol η dependent lesion bypass fidelity when copying either 8-oxoG or T-T CPD lesions.

Do nuclear bodies in oocytes of Tenebrio molitor (Coleoptera: Polyphaga, Tenebrionidae) contain two forms of RNA polymerase II?

Late vitellogenic oocytes of the mealworm beetle, Tenebrio molitor, which are transcriptionally inert, contain numerous fibrogranular nuclear bodies (NBs). Previously, we have shown that these NBs contain both unphosphorylated and phosphorylated forms of RNA polymerase II (pol II) [Tissue Cell 33 (2001) 549]. The conclusion on the presence of phosphorylated pol II was based on our immunoelectron experiments with monoclonal antibody (mAb) H5 against the phosphorylated serine-2 of the carboxy-terminal domain (CTD) of pol II. Because the specificity of mAb H5 was recently questioned by demonstration of its cross-reaction with SR-proteins [J. Struct. Biol. 140 (2002) 154], we re-examined here the occurence of pol II in T. molitor oocyte NBs using other appropriate antibodies. We confirm the presence of phosphorylated pol II in NBs using the affinity-purified polyclonal antibody against the phosphorylated CTD. Using double immunogold labeling with this antibody plus mAb 8WG16 against the unphosphorylated CTD, we confirm the presence of two forms of pol II in NBs. Additionally, the presence of pol II in NBs was verified here using mAb ARNA3 against the epitope outside CTD. We suggest that at the transcriptionally inactive stage, T. molitor oocyte NBs represent storage domains for pol II disengaged from the transcription.

Immunogold localization of RNA polymerase II and pre-mRNA splicing factors in Tenebrio molitor oocyte nuclei with special emphasis on karyosphere development

Ultrastructural and immunomorphological characteristics of the developing karyosphere and extrachromosomal nuclear bodies (NBs) in Tenebrio molitor oocytes are presented. Three consecutive stages of karyosphere development were identified: reticular, compact and ring-shaped. At the beginning of the karyosphere development (reticular and compact stages), condensed chromosomes are associated with a fibrogranular material (FGM). The successive karyosphere development is accompanied by the reorganization of FGM into fibrogranular NBs. Special attention was given to the nuclear distribution of hyperphosphorylated and non-phosphorylated forms of RNA polymerase II (pol II) and pre-mRNA splicing factors (snRNPs and SC35 protein) during karyosphere development and NB formation. The immunoelectron microscopy revealed that two forms of pol II and splicing factors being assembled in FGM are deposited in appropriate NBs. Some NBs were also shown to contain coilin, a marker protein for Cajal (coiled) bodies. We suggest that different types of NBs appearing in T. molitor oocyte nuclei along with the cessation of transcriptional activity during the karyosphere development represent storage domains for inactive RNA transcription/processing machinery to later usage in early embryogenesis.

DNA polymerase α in the fission yeast Schizosaccharomyces pombe: Identification and tracing of the catalytic subunit during the cell cycle

A recombinant protein was obtained in Escherichia coli by subcloning part of the Schizosaccharomyces pombe POL1 gene at the 3′-end of lacZ. Antibodies raised against this protein were used to identify the POL1 gene product in extracts of exponentially growing S. pombe cells. A major 170-kDa protein, whose structure and properties were typical of the catalytic subunit of eukaryotic DNA polymerases α (pol α), was detected. The same antibodies were used to trace pol α and to quantify its level during the S. pombe cell cycle. We found that pol α was present at all stages of the cycle and that its cellular pool was subject to limited (threefold) increase in G 1 and S phases, with a decline to the initial level soon after. In addition, we found that a second form of pol α with slightly lower molecular weight (165 kDa) existed only during late G 1 and S phases. Moreover, absence of initiation or perturbations in the course of DNA replication induced overproduction of the 165-kDa form.