Effects of 3 - Aminobenzamide on Polymerase α - dependent DNA Repair

박종근,변미경,박상대 ( Jong Kun Park,Mee Kyeong Byeon,Sang Dai Pak ) 한국유전학회 1986 Genes & Genomics Vol.8 No.1

The action of 3-aminobenzamide (3AB), an inhibitor of poly (ADP-ribose) polymerase, synergistically enhanced the amount of unscheduled DNA synthesis and DNA strand breaks in asynchronous CHO (Chinese hamster ovay)-K 1 cells exposed to methylmethane sulfonate (MMS). In this system, the time dependent decrease in the strand breaks was inhibited by 3AB. In synchronous CHO-K 1 cells, however, the sensitivity of exonuclease II on the repair patches blocked by aphidicolin, the inhibitor of DNA polymerase showed nonsignificant difference between normal-and 3AB-chased groups. Similarly, the rate of removal of exo II-sensitive sites was not altered by 3AB in WIL-2 (human lymphoid) cells. These results, therefore, suggest that poly (ADP-ribose) polymerization does not exert any significant role in the ligation of polymerase-dependent repair synthesis induced by MMS.

알킬화제에 의한 DNA 단사절단 , 절제회복 및 자매염색분체 교환에 미치는 Poly ( ADP - ribose ) polymerase 저해제의 상승효과

김철근,박종군,박상대 ( Chul Geun Kim,Jong Kun Park,Sang Dai Park ) 생화학분자생물학회 1983 BMB Reports Vol.16 No.2

Benzamide and 3-aminobenzamide, potent inhibitors of poly (ADP-ribose) polymerase, increased MMS or MNNG-induced DNA strand breaks and significantly retarded the rejoining of strand breaks in CHO cells. These inhibitors also synergistically enhanced the frequencies of unscheduled DNA synthesis (UDS) and sister chromatid exchanges (SCE) induced by MNNG. Amounts of UDS and strand breaks induced by the combined treatment with MMS and MNNG were found to be additive but these amount were enhanced in the presence of inhibitors. These results suggest that poly (ADP-ribose) functions a regulatory role in the repair of DNA damage by virtue of stabilizing chromatin structure whenever strand breaks occur in DNA.

Enhancing Effects of Inhibitors of Poly(ADP-ribose) Polymerase on Alkylating Agents-Induced DNA Strand Breaks, Excision Repair and sister Chromatid Exchanges

김철근,박종군,박상대,Kim, Chul-Geun,Park, Jong-Kun,Park, Sang-Dai 생화학분자생물학회 1983 한국생화학회지 Vol.16 No.2

CHO 세포에 있어서 강력한 poly(ADP-ribose) polymerase 저해제인 benzamide와 3-aminobenzmide는 MMS와 MNNG에 의한 DNA 단사절단을 증가시켰으며 단사절단의 재결합을 유의성 있게 억제하였다. 이들 저해제는 또한 MNNG에 의한 비주기성 DNA 합성(UDS)과 자매염색분체 교환(SCE)에 상승 효과를 나타내었다. MMS와 MNNG의 복합처리에 의한 UDS와 단사절단은 상가효과를 보이나 저해제 처리에 의해 이의 효과는 상승되었다. 이들 결과로 보아 poly(ADP-ribose)는 DNA에 단사절단이 일어날때마다 염색사구초를 안정화 시킴으로서 DNA 손상에 대한 회복에 조절작용을 나타낸다고 사료된다. Benzamide and 3-aminobenzamide, potent inhibitors of poly (ADP-ribose) polymerase, increased MMS or MNNG-induced DNA strand breaks and significantly retarded the rejoining of strand breaks in CHO cells. These inhibitors also synergistically enhanced the frequencies of unscheduled DNA synthesis (UDS) and sister chromatid exchanges (SCE) induced by MNNG. Amounts of UDS and strand breaks induced by the combined treatment with MMS and MNNG were found to be additive but these amount were enhanced in the presence of inhibitors. These results suggest that poly (ADP-ribose) functions a regulatory role in the repair of DNA damage by virtue of stabilizing chromatin structure whenever strand breaks occur in DNA.

Ribonucleotide Reductase Activity of Schizosaccbarornyces pombe Is Inhibited by Escherichia coli RecA Antibody

이정섭,천민석,김옥봉,박종군,김시욱,박열,양영기,홍승환,박상대,Lee, Jung-Sup,Chun, Min-Suck,Kim, Ok-Bong,Park, Jong-Kun,Kim, Si-Wouk,Park, Yeal,Yang, Young-Ki,Hong, Seung-Hwan,Park, Sang-Dai The Korean Society for Integrative Biology 1995 동물학회지 Vol.38 No.4

분열형 호모인 Schizosaccbarornyces pombe에서 RecA 유사 단백질은 hydroxyurea (HU)나 methotrexate (MTX)와 같은 nucleotide pool 형성에 관여하는 효소의 저해제에 의해 유도발현된다. 본 연구에서는 대장균의 RecA 단백질과 S. pombe의 ribonucleotide reductase가 구조적으로 유사한지를 RecA 항체를 이용한 면역침전법과 [5-$^3$H]CDP를 기질로하는 thin layerchromatography방법을 통하여 알아보고자 하였다. S. pombe의 rinonucleotide reductase활성은 100mM HU에 의해 대조군에 비해 26.3% 저해되었으며, RecA 항체를 이용한 면역침전에 의해서도 43.3% 저해되었다. 이와 같은 결과는 S. pombe의 rinonucleotide reductase가 대장균의 RecA 단백질과 구조적으로 유사함을 시사하는 것이다. We have previoosly demonstrated that the RecA-like protein of Schizosaccharomyces pombe (S. pombe) is immunologically related to Escherichia coil (E. coil) RecA protein and that the cellular level of the protein is significantly increased by inhibitors of nucleotide pool-forming enzymes such as hydroxyurea (HU) and methotrexate (MTX) (lee and Park, 1994; lee et al., 1994). In this study, we report that the ribonudeotide redudase activity of S. pombe is inhibited by E. coil RecA antibody, as determined by thin layer chromatography using [5-$^3$H]CDP as a substrate. The relative activity of ribonucleotide reductase was dramatically inhibited by 100 mM of flu (26.4% reduction) in in vitro assay, compared to that of non-treated control. The ribonucleotide reductase activity was also inhibited by immunoprecipitation with E. coil RecA antibody (43.3% reduction). These results indicate that the strudure of S. pombe ribonucleotide reductase is in part similar to that of E. coil RecA protein.

홍삼추출물에 의한 유전독성 감소 효과(Ⅰ) - 배양 NIH3T3 세포에서 자외선에 의한 유전독성의 감소에 미치는 홍삼추출물 처리효과

김완주 ( Wan Ju Kim ),유병수 ( Byung Soo Ryu ),전병훈 ( Byung Hoon Chun ),이정섭 ( Jung Sup Lee ),박상대 ( Sang Dai Park ),김인호 ( In Ho Kim ),최수봉 ( Soo Bong Choi ),박종군 ( Jong Kun Park ) 대한화장품학회 1998 대한화장품학회지 Vol.24 No.1

자외선에 의한 유전독성의 감소에 미치는 홍삼추출물의 영향을 배양 NIH3T3 세포계에서 분석하였다. 자외선을 조사한 후 정상 배지에서 배양한 시간간격에 따라 세포의 생존률은 증가하였는데 홍삼추출물이 함유된 배지에서 배양한 경우는 약 15%정도 증가한 생존률을 보였다. 자외선을 조사한 후 감소된 DNA복제가 정상배지 배양시간에 따라 증가하는 정도도 홍삼추출물을 후처리할 경우 현저한 중가를 보였다. 자외선 상해를 회복하기 위한 절제회복능은 홍삼추출물을 처리할 경우 유의미한 증가를 보였다. 이러한 절제회복과정중 효소에 의한 절제단계가 홍삼추출물 처리에 의해 활성화됨을 단사절단 분석을 통하여 규명하였다. 이상의 결과는 홍삼추출물이 자외선 상해의 절제 회복에 유의미한 증가를 보이며 따라서 유전독성을 감소시키는 항노화제로써 사용할 수 있음을 시사한다. We have studied the effects of red ginseng root extract on the decrease of UV-induced genotoxicity in cultured NIH3T3 cells. The increase in survival and the recovery from DNA synthesis inhibition in UV-irradiated cells as a funtion of normal medium incubation time was potentiated by the presence of the ginseng extract. The extract also increased the UV-induced excision repair as determined by unscheduled DNA synthesis. The amount of UV-induced DNA single strand breaks that are accumulated by polymerase inhibitors was signifcantly increased by the presence of the extract. These results suggest that the red ginseng extract activates the incision/excision step of UV-induced repair and could be used as a reagent for protecting UV-induced genotoxicity and cytotoxicity.

DNA Strand Breaks and Replication Inhibition by Benzo(a)pyrene in Mammalian Cells

박상대,박종군,최인순 한국유전학회 1984 Genes & Genomics Vol.6 No.3

DNA strand breaks and replication inhibition by benzo(a)pyrene (BP) were determined in the presence or absence of metabolic activation in excision defective and proficient mammalian cells. The DNA single strand breaks were induced in a small extent by BP in CHO-K1 cells, but not in XP2OS cells. However, single strand breaks were effectively induced by BP with S-15 fraction or by coculture with mouse embryonic fibroblasts. The production of strand breaks was remarkably increased by post-incubation with hydroxyurea and ara-C in a dose dependent manner, which was not shown in XP2OS cells. Rates of DNA synthesis were reduced by BP in a dose dependent manner in all cell lines. At low concentration of BP, the replicon initiation was specifically inhibited, but at high concentration the chain elongation appeared to be inhibited in both CHO-K1 and XP2OS cells.

색소 건피증 세포의 절제 회복능 복구에 미치는 세포융합 및 추출물의 영향

박상대,박종군,이명애 한국유전학회 1990 Genes & Genomics Vol.12 No.4

The treatments of fusion-elution and permeabilization-cell extracts were used to study the characteristics of xeroderma pigmentosum (XP) factors that complement the defects of XP cells in the repair of DNA damages induced by UV-irradiation. Unscheduled DNA synthesis in XP1EH (complementation group A) cells exposed to 10J/㎡ UV-light, permeabilized and repair-labeled with ^3H-dTTP, was remarkably increased by the treatment with cell extracts of HeLa or XP3KA (complementation group C) cells. Single strand breaks of XP A or C cells, fused with HeLa cells, was produced to a similar extent of those in HeLa cells irradiated with the same dose of UV-light. These results indicate that XP A and C cells are defective in different genes, and that the XP A and XP C factors are present in at least two fold excess for the maximal activity of excision repair in HeLa cells. The rate of single strand break of DNA in XP and HeLa cells was measured as a function of time after UV-irradiation. The maximum elution rate in all repair-defective XP cell lines appeared at 1-2 hr and then gradually reduced as the time lapsed, while those of HeLa cells appeared at 0-1 hr. The above results strongly suggest that the defects of XP cells might be underlain on the problems in the factors provoking conformational changes for the accessibility of the repair enzyme to the damaged sites.

포유동물세포의 DNA 사절단과 절제회복에 미치는 4NQO 와 MMS 의 상호작용

박상대,박종군,이정섭 한국유전학회 1984 Genes & Genomics Vol.6 No.1

The rates of unscheduled DNA synthesis induced by the combined treatment with 4NQO and MMS in CHO cells were less than the sum of those induced by each agent independently. These results were remarkable in MMS·post-treatment, but these initial discrepancies were removed as time lapsed with fluctuations at one hour time point. The alkaline elution rates in the combined treatment with 4NQO and MMS were higher than those of single 4NQO treatment, but were lower than those of MMS treatment. These abnormal profiles were reversed by incubation with proteinase K or treatment with hydroxyurea and ara-C. However, the amounts of DNA single strand breaks induced by MMS-posttreatment were less than those induced by MMS-pretreatment which were almost equal to additivity. The strand break frequencies in the MMS-posttreatment were lower than those in MMS treatment alone. These results indicate that MMS may have common steps in the repair of 4NQO-induced DNA damages but exerts an inhibitory effect on 4NQO-induced DNA repair in the MMS-posttreatment.

하등 진핵세포계에서 Poly(ADP - Ribose)Polymerase 유전자의 비교 분석

박상대,박종군,김완주 한국유전학회 1997 Genes & Genomics Vol.19 No.4

Poly(ADP-ribose) polymerase (PARP) is known to be activated by DNA strand breaks resulted from various cellular processes including DNA repair, replication and recombination. In the present study, the presence of PARP gene was investigated in insect and lower eukaryotes such as yeast and fungus. PCR was directed to genomic DNAs of an insect Tenebrio molitor and yeast S. pombe. By Southern hybridization analysis, the PCR products hybridized with flesh fly Sarcophaga peregrina PARP cDNA used as a probe. Genomic DNAs of T. molitor, ant C. japonicus and fungus A. nidulans also hybridized with Sarcophaga PARP probe. Interestingly, genomic DNA of S. pombe hybridized with a plant Arabidopsis PARP probe. Therefore, we screened genomic DNA library of S. pombe with Arabidopsis PARP cDNA and positive hybridization signals were obtained in more than fifteen clones. The amino acid sequence of PARP gene from S. pombe showed lower similarity with other functional genes previously reported except the "FSCF" and "EKRMKL" amino acid sequence of DNA binding domain and automodification domain, respectively. From all these results, the presence of PARP gene in T. molitor and C. japonicus was demonstrated, and the possible existence of PARP gene in lower eukaryotes including S. pombe and A. nidulans was especially suggested in this study.

대사활성계 존재하의 포유동물 배양세포에 있어 DNA 사 절단과 복제에 미치는 벤조피렌의 영향

박상대,박종군 한국유전학회 1988 Genes & Genomics Vol.10 No.4

DNA single strand breaks were induced by BP treatment in a small extent in CHO-K1 cells, but none in XP20S cells (complementation group A). By coculturing with mouse embryo fibroblast cells, however, CHO-K1 cells showed a significant formation of strand breaks and their accumulation by post-incubation with hydroxyurea and ara-C in a dose dependant manner, which was not seen in XP20S cells, indicating the UV-mimetic nature of BP-induced DNA damages. The pattern of DNA replication in CHO-K1 cells was different from that in XP20S cells by 24hr treatment of BP according to their repair abilities. The dose dependant inhibition of replicon initiation at 12hr exposure of CHO-K1 cells to BP changed to the blocking of chain elongation by 24hr exposure period. Inhibition of different modes of DNA replication was demonstrated when metabolic activation system was introduced into XP20S cells. All these results suggested that lower doses and shorter times were required for both strand break formation and inhibition of replication by the presence of metabolic activation system than by the absence of it during the exposure to BP, and that BP-DNA adducts were efficiently removed in CHO-K1 cells but not in XP20S cells resulting in differences in the single strand breaks or in the mode of inhibition of DNA replication.