HBV Polymerase Residues Asp429 and Asp551, Invariant at Motifs A and C are Essential to DNA Binding

Jung, Guhung,Kim, Younhee,Hong, Young Bin The Korea Science and Technology Center 1998 BMB Reports Vol.31 No.5

HBV polymerase shares several regions of amino acid homology with other DNA-directed and RNA-directed polymerases. The amino acid residues Asp429, Gly518, Asp551, Lys585, and Gly641 in the conserved motifs A, B', C, D, and E in the polymerase domain of HBV polymerase were mutated to alanine or histidine by in vitro site-directed mutagenesis. Those mutants were overexpressed, purified, and analyzed against DNA-dependent DNA polymerase activity and affinity for DNA binding. All those mutants did not show DNA-dependent DNA polymerase activities indicating that those five amino acid residues are all critical in DNA polymerase activity. South-Western analysis shows that amino acid residues Asp429 and AsP551 are essential to DNA binding, and Gly518 and Lys585 also affect DNA binding to a certain extent.

Nucleotide Insertion Fidelity of Human Hepatitis B Viral Polymerase

Jung,Guhung,Kim,Younhee,Suh,Se Won,Hong,Young Bin The Korea Science and Technology Center 2000 BMB Reports Vol.33 No.2

The hepadnaviruses replicate their nucleic acid through a reverse transcription step. The MBP-fused HBV polymerase was expressed in E. coli and purified by using amylose affinity column chromatography. The purified protein represented DNA-dependent DNA polymerase activity. In this report, the MBP-HBV polymerase was shown to lack 3’→5’exonuclease activity, like other retroviral RTs. The ratio of the insertion efficiency for the wrong versus right base pairs indicates the misinsertion frequency(f). The nucleotide insertion fidelity (1/f), observed with the MBP-HBV polymerse and HIV-1 RT, was between 60 and 54,000, between 50 and 73,000, respectively, showing that they are in close range. A relatively efficient nucleotide incorporation by the MBP-HBV polymerase was observed with a specificity of three groups: (1) A:T, T:A>C:G, G:C (matched pairs), (2) A:C, C:A>G:T, T:G (purine-pyrimidine and pyrimidine-purine mispairs), and (3) C:C, A:A, G:G, T:T>T:C, C:T>A:G, G:A (purine-purine or pyrimidine-pyrimidine mispairs), and their order is (1)>(2)>(3). The data from the nucleotide insertion fidelity by the MBP-HBV polymerase suggest that the HBV polymerase may be as error-prone as HIV-1 RT.

Structure-function analysis of PRDI DNA polymerase

Jung, Guhung The Microbiological Society of Korea 1990 微生物과 産業 Vol.16 No.3

PRDI DNA polymerase is the smallest member of the family B DNA polymerase (Jung et al., 1987). This DNA polyerase is specified by bacteriophage PRDI which infects a wide variety of gram-negative bacteria(Mindich and Bamford, 1988). Because PRDI is highly amenable to genetic and biochemical manipulation, it is a convenient model system with which to study structure-function relationships of DNA polymerase molecules. To determine the functional roles of the highly conserved regions of the family B DNA polymerases, we have initiated site-directed mutagenesis with PRD1 DNA polymerase, and our results show that mutations at the conserved regions within PRD1 DNA polymerase inactivate polymerase complementing activity and catalytic activity.

DNA Polymerase의 구조-기능 관계

이선민,정구흥 한국생화학분자생물학회 1990 생화학분자생물학회 소식 Vol.10 No.4

Since DNA contains the genetic information of an organism, accurate DNA replication is one of the most important events of the life cycle of an organism. DNA polymerases are key enzymes catalyzing the accurate replication of DNA. DNA polymerases have been classified, based on their amino acid sequence similarities, into two major groups: family A and family B. The family B DNA polymerases share regions of highly conserved amino acid sequcences. These conserved regions occur in the Same order in all family B DNA polymerases molecules. Therefore, it is likely that these sequence conservations are a consequence of their contribution to the DNA polymerase function and structure. However, little is known at present about functional roles of these highly conserved regions. To determine the functional roles of the highly conserved regions of the family B DNA polymerases, amino acid change generated in conserved regions by the site-directed mutagenesis. The results show that mutations at the conserved regions within PRDI DNA polymerase inactvate polymerasc complementing activity and catalytic activity. A new conserved region between DNA polymerase family A and B in the N-terminal portion has been identified which contains three highly conserved amino acids known to be involved in the 3'-5'exonuclease active site of Klenow fragment of E. aoli polymerase I.

Expression and Purification of Fusion Proteins of Yeast ARS - Binding Factor I / Escherichia coli β - Galactosidase

In Seop So,Guhung Jung,Hyune Mo Rho,Jiyoung Kim 한국미생물생명공학회(구 한국산업미생물학회) 1993 한국미생물생명공학회 학술발표초록집 Vol.1993 No.1

Expression and Purification of Fusion Proteins of Yeast ARS - Binding Factor Ⅰwith Escherichia coli β- Galactosidase

In Seop So,Guhung Jung,Hyune Mo Rho,Jiyoung Kim 생화학분자생물학회 1993 BMB Reports Vol.26 No.5

Yeast autonomously replicating sequence binding factor Ⅰ (ABF-1) is a multifunctional phosphoprotein which plays a role in DNA replication and transcription. We fused two different portion of yeast ABFI gene to the β-galactosidase gene and expressed the fusion proteins in E. coli cells. Two kinds of fusion proteins, ABFI(55-104)-lacZ and ABFI(55-367)-lacZ were produced under the control of E. coli tac promoter at a level of 35∼40% and 10∼15%, respectively, of total bacterial proteins. The fusion proteins retained the β-galactosidase activity. The larger fusion protein was expressed in soluble forms, while the smaller fusion protein was expressed in an 80% soluble form. The two fusion proteins were purified by β-galactosidase affinity chromatography. The fusion proteins contained the zinc finger motif derived from ABF-I protein but did not bind to the ABF Ⅰ binding site of ARS1. The results imply a possibility that if the zinc finger motif serves as DNA binding motif, other additional regions may be required for ABF Ⅰ to bind to its recognition sequence.

Downregulation of catalase by reactive oxygen species via hypermethylation of CpG island II on the catalase promoter

Min, Ji Young,Lim, Seung-Oe,Jung, Guhung Elsevier 2010 FEBS letters Vol.584 No.11

<P><B>Abstract</B></P><P>Catalase, which decomposes reactive oxygen species (ROS), is reduced in hepatocellular carcinoma (HCC); however, the reasons are poorly defined. In this study, it is demonstrated that prolonged exposure to ROS induced methylation of CpG island II on the catalase promoter and downregulated catalase expression at the transcriptional level in HCC cell lines. In addition, hypermethylation of CpG island II was also observed in tumor tissues, together with a decrease in catalase mRNA and protein expression levels when compared to non-tumor tissues. From these data, we suggest that ROS may downregulate catalase through the methylation of promoter during the development of HCC.</P>

Phosphorylation of hepatitis B virus core C-terminally truncated protein (Cp149) by PKC increases capsid assembly and stability

Kang, Hang,Yu, Jaehoon,Jung, Guhung Portland Press 2008 Biochemical journal Vol.416 No.1

<P>The HBV (hepatitis B virus) core is a phosphoprotein whose assembly, replication, encapsidation and localization are regulated by phosphorylation. It is known that PKC (protein kinase C) regulates pgRNA (pregenomic RNA) encapsidation by phosphorylation of the C-terminus of core, which is a component packaged into capsid. Neither the N-terminal residue phosphorylated by PKC nor the role of the C-terminal phosphorylation have been cleary defined. In the present study we found that HBV Cp149 (core protein C-terminally truncated at amino acid 149) expressed in Escherichia coli was phosphorylated by PKC at Ser106. PKC-mediated phosphorylation increased core affinity, as well as assembly and capsid stability. In vitro phosphorylation with core mutants (S26A, T70A, S106A and T114A) revealed that the Ser106 mutation inhibited phosphorylation of core by PKC. CD analysis also revealed that PKC-mediated phosphorylation stabilized the secondary structure of capsid. When either pCMV/FLAG-Cp149[WT (wild-type)] or pCMV/FLAG-S106A Cp149 was transfected into Huh7 human hepatoma cells, mutant capsid level was decreased by 2.06-fold with the S106A mutant when compared with WT, although the same level of total protein was expressed in both cases. In addition, when pUC1.2x and pUC1.2x/S106A were transfected, mutant virus titre was decreased 2.31-fold compared with WT virus titre. In conclusion, PKC-mediated phosphorylation increased capsid assembly, stability and structural stability.</P>

p53 inhibits tumor cell invasion via the degradation of snail protein in hepatocellular carcinoma

Lim, Seung-Oe,Kim, Hongtae,Jung, Guhung Elsevier 2010 FEBS letters Vol.584 No.11

<P><B>Abstract</B></P><P>The tumor suppressor protein p53 is a key regulator of cell cycle arrest and apoptosis. Snail protein regulates cancer-associated malignancies. However, the relationship between p53 and Snail proteins in hepatocellular carcinoma (HCC) has not been completely understood. To determine whether Snail and p53 contribute to hepatocarcinogenesis, we analyzed the expression of Snail proteins in p53-overexpressing HCC cells. We found that p53 wild-type (WT) induced the degradation of Snail protein via murine double minute 2-mediated ubiquitination, whereas p53 mutant did not induce Snail degradation. As we expected, only p53WT induced endogenous Snail protein degradation and inhibited tumor cell invasion. These findings contribute to a better understanding of the role of p53 mutation and Snail overexpression as a late event in hepatocarcinogenesis.</P><P><B>Structured summary</B></P><P>MINT-7718917: <I>p53</I> (uniprotkb:P04637) <I>physically interacts</I> (MI:0915) with <I>Snai1</I> (uniprotkb:O95863) by <I>anti bait coimmunoprecipitation</I> (MI:0006)</P><P>MINT-7719877: <I>Snai1</I> (uniprotkb:O95863) <I>physically interacts</I> (MI:0915) with <I>ubiquitin</I> (uniprotkb:P62988) by <I>anti tag coimmunoprecipitation</I> (MI:0007)</P><P>MINT-7718928: <I>Snai1</I> (uniprotkb:O95863) <I>physically interacts</I> (MI:0915) with <I>p53</I> (uniprotkb:P04637) by <I>anti tag coimmunoprecipitation</I> (MI:0007)</P><P>MINT-7718939: <I>Snai1</I> (uniprotkb:O95863) <I>physically interacts</I> (MI:0915) with <I>MDM2</I> (uniprotkb:Q00987) by <I>anti tag coimmunoprecipitation</I> (MI:0007)</P>

Heat shock protein 70 and heat shock protein 90 synergistically increase hepatitis B viral capsid assembly

Seo, Hyun Wook,Seo, Joon Pyung,Jung, Guhung Elsevier 2018 Biochemical and biophysical research communication Vol.503 No.4

<P><B>Abstract</B></P> <P>Hepatitis B virus (HBV) infection can cause chronic liver diseases, cirrhosis, and hepatocellular carcinoma (HCC). Heat shock proteins (Hsps) are important factors in the formation of the HBV capsid and in genome replication during the viral life cycle. Hsp90 is known to promote capsid assembly. However, the functional roles of Hsp70 in HBV capsid assembly with Hsp90 have not been studied so far. Using microscale thermophoresis analyses and <I>in vitro</I> nucleocapsid formation assays, we found that Hsp70 bound to a HBV core protein dimer and facilitated HBV capsid assembly. Inhibition of Hsp70 by methylene blue (MB) led to a decrease in capsid assembly. Moreover, Hsp70 inhibition reduced intracellular capsid formation and HBV virus particle number in HepG2.2.15 cells. Furthermore, we examined synergism between Hsp70 and Hsp90 on HBV capsid formation <I>in vitro.</I> Our results clarify the role of Hsp70 in HBV capsid formation via an interaction with core dimers and in synergistically promoting capsid assembly with Hsp90.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Heat shock protein 70 (Hsp70) interacts with Cp149 dimer. </LI> <LI> Hsp70 synergistically increases formation of HBV capsid with Hsp90. </LI> <LI> Inhibition of Hsp70 reduces HBV biogenesis through suppression of HBV capsid assembly. </LI> </UL> </P>