DNA synthesis catalysed by endogenous templates and DNA-dependent DNA polymerases in spermatogenic cells from rat

The activity levels of DNA polymerases α and β have been measured by autoradiography in squash preparations from rat testis of sexually mature animals. Similar results were obtained with ‘fixed’ samples (dipped in acetone: ethanol for 5 min at 25 °C) or ‘unfixed’ samples (frozen in liquid nitrogen and freeze-dried). The activities of DNA polymerases α and β in situ were distinguished by differential assay conditions and by selective inhibition with compounds such as N-ethylmaleimide and aphidicolin. Using the endogenous chromatin as template, maximal activity for both enzymes was obtained in the presence of all four deoxyribonucleoside triphosphates, MgCl₂ and ethylene glycol. When DNA polymerase activities in several predominant testicular cell types (pre-leptotene primary spermatocytes, pachytene primary spermatocytes, round spermatids and elongated spermatids) were quantitatively compared, on a per cell basis, the following percentage distribution was observed:

     

2.

Changes in RNA synthesis of endogenous "free" and template-engaged RNA polymerases in isolated nuclei of highly synchronized coleopteran embryos   总被引：1，自引：0，他引：1  

J Büning 《Developmental biology》1978,64(1):130-139

     

3.

Towards a structural understanding of RNA synthesis by negative strand RNA viral polymerases     

《Current opinion in structural biology》2016

     

4.

Tagetitoxin inhibits RNA synthesis directed by RNA polymerases from chloroplasts and Escherichia coli.     

D E Mathews  R D Durbin 《The Journal of biological chemistry》1990,265(1):493-498

     

5.

VPg-primed RNA synthesis of norovirus RNA-dependent RNA polymerases by using a novel cell-based assay     

Subba-Reddy CV  Goodfellow I  Kao CC 《Journal of virology》2011,85(24):13027-13037

Molecular studies of human noroviruses (NoV) have been hampered by the lack of a permissive cell culture system. We have developed a sensitive and reliable mammalian cell-based assay for the human NoV GII.4 strain RNA-dependent RNA polymerase (RdRp). The assay is based on the finding that RNAs synthesized by transiently expressed RdRp can stimulate retinoic acid-inducible gene I (RIG-I)-dependent reporter luciferase production via the beta interferon promoter. Comparable activities were observed for the murine norovirus (MNV) RdRp. RdRps with mutations at divalent metal ion binding residues did not activate RIG-I signaling. Furthermore, both NoV and MNV RdRp activities were stimulated by the coexpression of their respective VPg proteins, while mutations in the putative site of nucleotide linkage on VPg abolished most of their stimulatory effects. Sequencing of the RNAs linked to VPg revealed that the cellular trans-Golgi network protein 2 (TGOLN2) mRNA was the template for VPg-primed RNA synthesis. Small interfering RNA knockdown of RNase L abolished the enhancement of signaling that occurred in the presence of VPg. Finally, the coexpression of each of the other NoV proteins revealed that p48 (also known as NS1-2) and VP1 enhanced and that VP2 reduced the RdRp activity. The assay should be useful for the dissection of the requirements for NoV RNA synthesis as well as the identification of inhibitors of the NoV RdRp.     

6.

Requirements for de novo initiation of RNA synthesis by recombinant flaviviral RNA-dependent RNA polymerases   总被引：2，自引：0，他引：2  

Ranjith-Kumar CT  Gutshall L  Kim MJ  Sarisky RT  Kao CC 《Journal of virology》2002,76(24):12526-12536

RNA-dependent RNA polymerases (RdRps) that initiate RNA synthesis by a de novo mechanism should specifically recognize the template initiation nucleotide, T1, and the substrate initiation nucleotide, the NTPi. The RdRps from hepatitis C virus (HCV), bovine viral diarrhea virus (BVDV), and GB virus-B all can initiate RNA synthesis by a de novo mechanism. We used RNAs and GTP analogs, respectively, to examine the use of the T1 nucleotide and the initiation nucleotide (NTPi) during de novo initiation of RNA synthesis. The effects of the metal ions Mg(2+) and Mn(2+) on initiation were also analyzed. All three viral RdRps require correct base pairing between the T1 and NTPi for efficient RNA synthesis. However, each RdRp had some distinct tolerances for modifications in the T1 and NTPi. For example, the HCV RdRp preferred an NTPi lacking one or more phosphates regardless of whether Mn(2+) was present or absent, while the BVDV RdRp efficiently used GDP and GMP for initiation of RNA synthesis only in the presence of Mn(2+). These and other results indicate that although the three RdRps share a common mechanism of de novo initiation, each has distinct preferences.     

7.

RNA synthesis: strategies for the use of bacteriophage RNA polymerases   总被引：14，自引：0，他引：14  

G Krupp 《Gene》1988,72(1-2):75-89

     

8.

Transcription of reiterated and unique DNA sequences by endogenous RNA polymerases in rat liver nucleoplasm.     

T J Beebee  D S Carty 《Biochemical and biophysical research communications》1979,86(1):199-205

     

9.

In situ RNA synthesis in fixed mammalian chromosomes     

M. Tien Kuo  T. C. Hsu  G. F. Saunders 《Experimental cell research》1974,88(2):424-427

     

10.

Properties of RNA synthesis in HeLa nuclei by Micrococcus lysodeikticu or endogenous RNA polymerase   总被引：1，自引：0，他引：1  

W P Summers  G C Mueller 《Biochimica et biophysica acta》1968,169(2):316-326

     

11.

RNA-dependent RNA polymerases in RNA silencing     

Maida Y  Masutomi K 《Biological chemistry》2011,392(4):299-304

     

12.

DNA-dependent RNA polymerases from Schneider 2-L cells of Drosophila. II. Response to infection by an endogenous picorna-like virus     

John P. Phillips  Donald G. Somers  C. Feng 《Biochemical genetics》1982,20(7-8):675-680

     

13.

Resistance of hepatic RNA polymerases to compounds effecting RNA and protein synthesis in vivo   总被引：4，自引：0，他引：4  

B J Benecke  A Ferencz  K H Seifart 《FEBS letters》1973,31(1):53-58

     

14.

Translesion synthesis by RNA polymerases: occurrence and biological implications for transcriptional mutagenesis     

Doetsch PW 《Mutation research》2002,510(1-2):131-140

     

15.

Infidelity of DNA synthesis by themperature-sensitive DNA polymerases from RNA tumor viruses.     

L A Weymouth  L A Loeb 《Biochimica et biophysica acta》1977,478(3):305-315

     

16.

Ehrlich ascites cells DNA-dependent RNA polymerases: effect of amino acids and protein synthesis inhibition   总被引：3，自引：0，他引：3  

S Cereghini  M T Franze-Fernández 《FEBS letters》1974,41(1):161-165

     

17.

Viral RNA polymerases   总被引：13，自引：0，他引：13  

A Ishihama  K Nagata 《CRC critical reviews in biochemistry》1988,23(1):27-76

     

18.

Eukaryotic RNA polymerases   总被引：81，自引：0，他引：81  

A Sentenac 《CRC critical reviews in biochemistry》1985,18(1):31-90

     

19.

Eukaryotic RNA polymerases   总被引：1，自引：0，他引：1  

D G Blair 《Comparative biochemistry and physiology. B, Comparative biochemistry》1988,89(4):647-670

     

20.

Multisubunit RNA polymerases     

Cramer P 《Current opinion in structural biology》2002,12(1):89-97

     

	Pre-leptotene primary spermatocyte %	Pachytene primary spermatocyte %	Round spermatid %	Elongated spermatid %
DNA polymerase α	25	42	30	3
DNA polymerase β	29	34	36	1

VPg-primed RNA synthesis of norovirus RNA-dependent RNA polymerases by using a novel cell-based assay

Molecular studies of human noroviruses (NoV) have been hampered by the lack of a permissive cell culture system. We have developed a sensitive and reliable mammalian cell-based assay for the human NoV GII.4 strain RNA-dependent RNA polymerase (RdRp). The assay is based on the finding that RNAs synthesized by transiently expressed RdRp can stimulate retinoic acid-inducible gene I (RIG-I)-dependent reporter luciferase production via the beta interferon promoter. Comparable activities were observed for the murine norovirus (MNV) RdRp. RdRps with mutations at divalent metal ion binding residues did not activate RIG-I signaling. Furthermore, both NoV and MNV RdRp activities were stimulated by the coexpression of their respective VPg proteins, while mutations in the putative site of nucleotide linkage on VPg abolished most of their stimulatory effects. Sequencing of the RNAs linked to VPg revealed that the cellular trans-Golgi network protein 2 (TGOLN2) mRNA was the template for VPg-primed RNA synthesis. Small interfering RNA knockdown of RNase L abolished the enhancement of signaling that occurred in the presence of VPg. Finally, the coexpression of each of the other NoV proteins revealed that p48 (also known as NS1-2) and VP1 enhanced and that VP2 reduced the RdRp activity. The assay should be useful for the dissection of the requirements for NoV RNA synthesis as well as the identification of inhibitors of the NoV RdRp.     

Requirements for de novo initiation of RNA synthesis by recombinant flaviviral RNA-dependent RNA polymerases

RNA-dependent RNA polymerases (RdRps) that initiate RNA synthesis by a de novo mechanism should specifically recognize the template initiation nucleotide, T1, and the substrate initiation nucleotide, the NTPi. The RdRps from hepatitis C virus (HCV), bovine viral diarrhea virus (BVDV), and GB virus-B all can initiate RNA synthesis by a de novo mechanism. We used RNAs and GTP analogs, respectively, to examine the use of the T1 nucleotide and the initiation nucleotide (NTPi) during de novo initiation of RNA synthesis. The effects of the metal ions Mg(2+) and Mn(2+) on initiation were also analyzed. All three viral RdRps require correct base pairing between the T1 and NTPi for efficient RNA synthesis. However, each RdRp had some distinct tolerances for modifications in the T1 and NTPi. For example, the HCV RdRp preferred an NTPi lacking one or more phosphates regardless of whether Mn(2+) was present or absent, while the BVDV RdRp efficiently used GDP and GMP for initiation of RNA synthesis only in the presence of Mn(2+). These and other results indicate that although the three RdRps share a common mechanism of de novo initiation, each has distinct preferences.