DNA聚合酶β在烷化剂MNNG引起的非定标性突变中的作用
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摘要
化学致癌物能直接在DNA上造成修饰和损伤：如果没有被正确修复，这些损伤将导致突变的形成。
然而机体能主动地对环境的刺激作出反应；突变的生成也并非完全是被动的过程。当受到外源DNA损伤因子的攻击时，大肠杆菌E.coil能启动SOS反应以应对不良环境，提高对致损伤刺激的耐受能力，减少损伤的发生。而在出现SOS反应的细胞中还观察到了在复制过程中的产生的、发生在没有外源损伤DNA上的突变事件，被称为SOS非定标性突变。可诱导的DNA聚合酶在其中起到了关键作用。在UmuD'，RecA和单链结合蛋白SSB的协助下，polV(UmuC)能在单链模板上催化DNA合成并产生高频率的以碱基颠换为主要形式的突变；另一个与非定标性突变有关的易误DNA聚合酶是polⅣ，为dinB基因的编码产物。它可能直接与DNA复制叉相联系而参与了DNA合成，产生—1移码为主的非定标性突变。
但对真核细胞，尤其是哺乳类动物细胞中非定标性突变的情况目前还研究不多。我们和其它实验室的工作证明哺乳类细胞中也存在非定标性突变。我们用携带靶基因SupF tRNA的穿梭质粒pZ189转染在12—24小时前经低
渐江大学博士学位论文 王谷亮
浓度烷化剂 N．甲基N”硝基N．亚硝基皿（MNNG）处理过的细胞并使之在
其中复制。在收回的质粒上我们检测到了延迟发生的、高于对照5倍以上的
突变。由于W在细胞中的半寿期仅为1．！小时；0．20M的MNNG经
洗涤后的极微量残留经过10—20多个半衰期的降解后，己不足攻击转人的
Q
DNA分子，因此这种突变显然是发生在MNNG直接攻击部位以外的正常碱
－基上。这种不依赖于DNA损伤的非定标性突变随后被证明依赖于MN’NG
引起的细胞内基因表达的变化。进一步我们用RTPCR技术证明DNA聚合
酶e（P。lp）的表达在MN’NG处理后显著升高。
Pci6在单碱基切除修复（base excision fpair，BER）起到不可替代的
重要作用，因此认为POIp是参与维持细胞遗传稳定性的细胞内因子。然而
由于以下特征，P。lP表现出很低的DNA复制保真度：它没有3’一5’外切酶
活性因而在＊＊A复制中缺乏校读活性：对底物dN＊P鉴别能力低卜 在长
单链模权上催化DNA复制时散布式的合成方式。最近人们逐渐意识到P。l
p可能作为增变因子参与了遗传不稳定的发生。因此我们在本课题中研究了
P。ID与非定标性突变形成的关系以及MNNG诱导P。lP表达的信号机制。
MNNG处理诱导P。lp的表达：我们用Western印迹方法检测了经
MNNG处理后细胞中的 P。lp蛋白水平的表达情况。MN’NG处理后 12小时，
细胞内P。lp较对照组有轻度升高：在处理组中每单位总蛋白中P。lp的量
为 65．互自定义单位，而对照中为 50．8。处理后 18或 24小时后升高更为明
显。24 ’J’时后处理组细胞中 POIp为 125．5，比对照 66．8高出近 2倍。己经
；发现过表达的 p。ID可以与其它聚合酶竞争，参与核v酸切除修复（NER）、
DNA半保留复制等过程。由于下。ID的低保真度，尤其它在长距离模板上的
保真度更低，我们推测经诱导高表达的P。lp可能冈其低保真度而参与了非
定标性突变的形成。
建立P。lp表达阻断的细胞系：为验证此假设，我们用反义NA技术
3
一
I 浙江大学博士学位论文 王谷亮
1 建立了 Pci D表达下调的转基因细胞系。RT一PCR扩增得到 358 hp的 Pci
lp CDNA片段，并在其上下游引物中分别引人了 h。I和 ie内切酶位点。
l 此片段克隆人TA克隆载体后，经h。I和he消化收回在上下游分别有ho
11和Wb。他性末端的POlpCDNA片段。利用真核纫胞地塞米松（DEX）诱
It
卜 导表达载体pMA力心柏。m叩”多克隆位点中col和Me呐切酶限制性位点的
－位置（he在上游 1545而Xho位于下游 1570），使PolpcDNA片段反向
I 插入到表达载体中，即pMAMneo－p“巾of 6－。插人外源片段中存在1个
卜EcoRI内切酶位点，被用于鉴定重组子中片段的插人与否。扩增纯化的
PNu山山惦o－Hxx－n个。卜p”以及对照空白质粒p MAMllMAMlleo－幼叩”通过磷酸钙沉淀
法转染FL细胞，利用载体携带的抗性基因通过G418筛选转有质粒的细胞。
I 通过有限稀释法共得到16个单细胞来源的转基因细胞系FL．P。l住。经DEX
I 诱导72 ，J’时后，用Western印迹法鉴定各细胞系的反义阻断效果并挑选其
中效果最佳的细胞系以供以后实验之川。?
Chemical carcinogens can cause modifications or damages on DNA directly. If these damages are not repaired properly, they result in mutagenesis and carcinogenesis.
However, organism can react to the stress actively and the mutagenesis is not a passive event. SOS response is employed in DNA damaging agents challenged E. co/i, to cope with abnormal conditions. It has also been demonstrated that SOS response dependent accumulation of mutation during replication of DNA that has not been attacked by exogenous DNA damaging factors can be seen in challenged cells, termed as SOS untargeted mutation. In E coli, DNA polymerases are key enzymes involved in two distinguished pathways contributing to untargeted mutagenesis. Replication of DNA by pol V(UmuC), in the presence of UmuD1, RecA and single strand binding protein (SSB), is highly mutagenic and exhibits a predominant mutation pattern of base transversion. Another error prone polymerase involved in untargeted mutagenesis is pol IV, encoded by dinB gene. SOS induced pol IV increases untargeted mutagenesis not only in A phage, but also in chromosome of E. coli, characterized as -1 frameshift mutation.
Much less is known about untargeted mutagenesis in eukaryotes. We and others have reported evidences of untargeted mutagenesis in mammalian cells. Our previous work identified delayed mutation occurred at target supF tRNA gene in plasmid transfected into cells pretreated with low concentration of alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Direct damage
on supF tRNA gene can be neglected because half-life of MNNG is 1.1 hour and the interval between treatment and transfection was as long as 12-24 hours. Therefore the mutagenesis is not lesion directed. Furthermore, we found the mutagenesis relies on the alteration on gene expression profile induced by MNNG treatment; and also, the expression of DNA polymerase P (Pol P) was proved increased after MNNG treatment.
Pol p is normally regarded as the base excision repair (BER) polymerase. Its role in BER is un-replaceable and thereby is important in mammalian genomic stability. However, it is recently cognized to be associated with genetic instability, due to its error prone features, i.e. lack of 3'?' proofreading activity and low replication fidelity in vitro, distributive manner in DNA synthesis, poor ability to discriminate nucleotides at the level of binding and also the possibility of its expanded roles in long patch BER, NER, DNA replication and recombination at its excess state. Therefore, we set out to explore the possible role of over-expressed Pol p induced by MNNG in untargeted mutagenesis. The signaling pathways involved in mediating Pol P over-expression were also studied.
DNA polymerase p was induced by MNNG treatment. In this research, the expression of DNA polymerase p was examined in MNNG treated cells, where untargeted mutation had been proved arisen. After been treated with MNNG for 2.5 hours, cells were incubated for 12,18 or 24 hours before been harvested. The amounts of Pol P were determined with Western blotting. Results showed a slightly elevated expression of Pol P in MNNG treated cells after 12-hour-incubation (65.1 arbitrary unit in MNNG treated cells and 50.8 in control). The differences were enlarged 6 more hours later (125.5 and 66.8, respectively). Because of its low fidelity, over-expressed error prone Pol P might introduce mutations on both damaged and un-damaged DNA templates by perturbing other polymerases with high fidelity. Thus, we hypothesized that Pol P might be involved in MNNG induced untargeted mutagenesis.
Establishment of Pol p down-regulated cell line: To verify the hypothesis, a human Pol p down-regulated cell line was established using an antisense RNA strategy. A 358 bp Pol ft cDNA fragment framed with introduced restrict sites of
Xho I (upstream) and Nhe I (downstream) was amplified by the method of RT-PCR, and then cloned into a TA-vector. Inserted Pol ft cDNA fragment was recovered from the recombinant vector by Xho I and Nhe I digesting, an
然而机体能主动地对环境的刺激作出反应；突变的生成也并非完全是被动的过程。当受到外源DNA损伤因子的攻击时，大肠杆菌E.coil能启动SOS反应以应对不良环境，提高对致损伤刺激的耐受能力，减少损伤的发生。而在出现SOS反应的细胞中还观察到了在复制过程中的产生的、发生在没有外源损伤DNA上的突变事件，被称为SOS非定标性突变。可诱导的DNA聚合酶在其中起到了关键作用。在UmuD'，RecA和单链结合蛋白SSB的协助下，polV(UmuC)能在单链模板上催化DNA合成并产生高频率的以碱基颠换为主要形式的突变；另一个与非定标性突变有关的易误DNA聚合酶是polⅣ，为dinB基因的编码产物。它可能直接与DNA复制叉相联系而参与了DNA合成，产生—1移码为主的非定标性突变。
但对真核细胞，尤其是哺乳类动物细胞中非定标性突变的情况目前还研究不多。我们和其它实验室的工作证明哺乳类细胞中也存在非定标性突变。我们用携带靶基因SupF tRNA的穿梭质粒pZ189转染在12—24小时前经低
渐江大学博士学位论文 王谷亮
浓度烷化剂 N．甲基N”硝基N．亚硝基皿（MNNG）处理过的细胞并使之在
其中复制。在收回的质粒上我们检测到了延迟发生的、高于对照5倍以上的
突变。由于W在细胞中的半寿期仅为1．！小时；0．20M的MNNG经
洗涤后的极微量残留经过10—20多个半衰期的降解后，己不足攻击转人的
Q
DNA分子，因此这种突变显然是发生在MNNG直接攻击部位以外的正常碱
－基上。这种不依赖于DNA损伤的非定标性突变随后被证明依赖于MN’NG
引起的细胞内基因表达的变化。进一步我们用RTPCR技术证明DNA聚合
酶e（P。lp）的表达在MN’NG处理后显著升高。
Pci6在单碱基切除修复（base excision fpair，BER）起到不可替代的
重要作用，因此认为POIp是参与维持细胞遗传稳定性的细胞内因子。然而
由于以下特征，P。lP表现出很低的DNA复制保真度：它没有3’一5’外切酶
活性因而在＊＊A复制中缺乏校读活性：对底物dN＊P鉴别能力低卜 在长
单链模权上催化DNA复制时散布式的合成方式。最近人们逐渐意识到P。l
p可能作为增变因子参与了遗传不稳定的发生。因此我们在本课题中研究了
P。ID与非定标性突变形成的关系以及MNNG诱导P。lP表达的信号机制。
MNNG处理诱导P。lp的表达：我们用Western印迹方法检测了经
MNNG处理后细胞中的 P。lp蛋白水平的表达情况。MN’NG处理后 12小时，
细胞内P。lp较对照组有轻度升高：在处理组中每单位总蛋白中P。lp的量
为 65．互自定义单位，而对照中为 50．8。处理后 18或 24小时后升高更为明
显。24 ’J’时后处理组细胞中 POIp为 125．5，比对照 66．8高出近 2倍。己经
；发现过表达的 p。ID可以与其它聚合酶竞争，参与核v酸切除修复（NER）、
DNA半保留复制等过程。由于下。ID的低保真度，尤其它在长距离模板上的
保真度更低，我们推测经诱导高表达的P。lp可能冈其低保真度而参与了非
定标性突变的形成。
建立P。lp表达阻断的细胞系：为验证此假设，我们用反义NA技术
3
一
I 浙江大学博士学位论文 王谷亮
1 建立了 Pci D表达下调的转基因细胞系。RT一PCR扩增得到 358 hp的 Pci
lp CDNA片段，并在其上下游引物中分别引人了 h。I和 ie内切酶位点。
l 此片段克隆人TA克隆载体后，经h。I和he消化收回在上下游分别有ho
11和Wb。他性末端的POlpCDNA片段。利用真核纫胞地塞米松（DEX）诱
It
卜 导表达载体pMA力心柏。m叩”多克隆位点中col和Me呐切酶限制性位点的
－位置（he在上游 1545而Xho位于下游 1570），使PolpcDNA片段反向
I 插入到表达载体中，即pMAMneo－p“巾of 6－。插人外源片段中存在1个
卜EcoRI内切酶位点，被用于鉴定重组子中片段的插人与否。扩增纯化的
PNu山山惦o－Hxx－n个。卜p”以及对照空白质粒p MAMllMAMlleo－幼叩”通过磷酸钙沉淀
法转染FL细胞，利用载体携带的抗性基因通过G418筛选转有质粒的细胞。
I 通过有限稀释法共得到16个单细胞来源的转基因细胞系FL．P。l住。经DEX
I 诱导72 ，J’时后，用Western印迹法鉴定各细胞系的反义阻断效果并挑选其
中效果最佳的细胞系以供以后实验之川。?
Chemical carcinogens can cause modifications or damages on DNA directly. If these damages are not repaired properly, they result in mutagenesis and carcinogenesis.
However, organism can react to the stress actively and the mutagenesis is not a passive event. SOS response is employed in DNA damaging agents challenged E. co/i, to cope with abnormal conditions. It has also been demonstrated that SOS response dependent accumulation of mutation during replication of DNA that has not been attacked by exogenous DNA damaging factors can be seen in challenged cells, termed as SOS untargeted mutation. In E coli, DNA polymerases are key enzymes involved in two distinguished pathways contributing to untargeted mutagenesis. Replication of DNA by pol V(UmuC), in the presence of UmuD1, RecA and single strand binding protein (SSB), is highly mutagenic and exhibits a predominant mutation pattern of base transversion. Another error prone polymerase involved in untargeted mutagenesis is pol IV, encoded by dinB gene. SOS induced pol IV increases untargeted mutagenesis not only in A phage, but also in chromosome of E. coli, characterized as -1 frameshift mutation.
Much less is known about untargeted mutagenesis in eukaryotes. We and others have reported evidences of untargeted mutagenesis in mammalian cells. Our previous work identified delayed mutation occurred at target supF tRNA gene in plasmid transfected into cells pretreated with low concentration of alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Direct damage
on supF tRNA gene can be neglected because half-life of MNNG is 1.1 hour and the interval between treatment and transfection was as long as 12-24 hours. Therefore the mutagenesis is not lesion directed. Furthermore, we found the mutagenesis relies on the alteration on gene expression profile induced by MNNG treatment; and also, the expression of DNA polymerase P (Pol P) was proved increased after MNNG treatment.
Pol p is normally regarded as the base excision repair (BER) polymerase. Its role in BER is un-replaceable and thereby is important in mammalian genomic stability. However, it is recently cognized to be associated with genetic instability, due to its error prone features, i.e. lack of 3'?' proofreading activity and low replication fidelity in vitro, distributive manner in DNA synthesis, poor ability to discriminate nucleotides at the level of binding and also the possibility of its expanded roles in long patch BER, NER, DNA replication and recombination at its excess state. Therefore, we set out to explore the possible role of over-expressed Pol p induced by MNNG in untargeted mutagenesis. The signaling pathways involved in mediating Pol P over-expression were also studied.
DNA polymerase p was induced by MNNG treatment. In this research, the expression of DNA polymerase p was examined in MNNG treated cells, where untargeted mutation had been proved arisen. After been treated with MNNG for 2.5 hours, cells were incubated for 12,18 or 24 hours before been harvested. The amounts of Pol P were determined with Western blotting. Results showed a slightly elevated expression of Pol P in MNNG treated cells after 12-hour-incubation (65.1 arbitrary unit in MNNG treated cells and 50.8 in control). The differences were enlarged 6 more hours later (125.5 and 66.8, respectively). Because of its low fidelity, over-expressed error prone Pol P might introduce mutations on both damaged and un-damaged DNA templates by perturbing other polymerases with high fidelity. Thus, we hypothesized that Pol P might be involved in MNNG induced untargeted mutagenesis.
Establishment of Pol p down-regulated cell line: To verify the hypothesis, a human Pol p down-regulated cell line was established using an antisense RNA strategy. A 358 bp Pol ft cDNA fragment framed with introduced restrict sites of
Xho I (upstream) and Nhe I (downstream) was amplified by the method of RT-PCR, and then cloned into a TA-vector. Inserted Pol ft cDNA fragment was recovered from the recombinant vector by Xho I and Nhe I digesting, an
引文
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[51]中国科学院上海生物化学研究所酶结构功能组，上海第二医学院同位素研究室，针麻组．饱和分析法测定3'-5'-磷酸腺苷(cAMP)．生物化
学与生物物理学报，1976，8(2) ：169-177
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[31] Ruiz JF, Domingucz O, Lain-de-Lera T, Garcia-Diaz M, Bernad A, Blanco L. DNA polymerase mu, a candidate hypermutase? Philos-Trans-R-Soc-Lond-B-Biol-Sci. 2001; 356(1405) : 99-109
[32] Zhang Y, Yuan F, Xin H, Wu X, Rajpal DK, Yang D, Wang Z. Human DNA polymerase kappa synthesizes DNA with extraordinarily low fidelity. Nucleic-Acids-Res. 2000; 28(21) : 4147-56
[33] Osheroff WP, Jung HK, Beard WA, et al. The fidelity of DNA polymerase β during distributive and processsive DNA synthesis. J.Biol.Chem, 1999; 274 (6) : 3642-3650．
[34] Canitrot Y, Cazaux C, Frechet M, et al. Overexpression of DNA polymerase beta in cell results in a mutator phenotype and a decreased sensitivity to anticancer drugs. Proc Natl Acad Sci U S A, 1998; 95(21) : 12586．
[35] 冯朝辉，余应年．陈星若．甲基硝基亚硝基胍诱发的遗传不稳定细胞 DNA聚合酶βmRNA表达水平研究．中国病理生理杂志，1999，15： 699-702
[36] Narayan S, Beard WA, Wilson SH. DNA damage induced transcription of a human DNA polymerase β chimeric promoter: recruitment of preinitation complex in vitro by ATF/CREB. Biochemistry 1995; 34:73-80
[37] Chyan YJ, Ackerman S, Shepherd NS, McBride OW, Widen SG Wilson SH; Wood TG The human DNA polymerase β gene structure. Nuclic Acids Res.1994; 22(14) : 2719-25
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[39] Shaulian E, Schreiber M, Piu F, Beeche M, Wagner EF, Karin M. The mammalian UV response: c-Jun induction is required for exit from p53-imposed growth arrest. Cell. 2000; 103(6) : 897-907
[40] Engelberg D, Klein C, Martinetto H, Struhl K, Karin M. The UV response involving the Ras signaling pathway and AP-1 transcription factors is conserved between yeast and mammals. Cell. 1994; 77(3) : 381-90
[41] Bender K, Gottlicher M, Whiteside S. Rahmsdorf HJ, Herrlich P. Sequential DNA damage-independent and-dependent activation of NF-kappaB by UV. EMBO-J. 1998; 17(17) : 5170-81
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[43]Wilhelm D, Bender r K, Knebel A, Angel P. The level of intracellular glutathione is a key regulator for the induction of stress-activated signal transduction pathways including Jun N-terminal protein kinases and p38 kinase by alkylating agents, Mol. Cell. Biol. 1997; 17: 4792-800.
[44]McCalla, DR, Reuvers A. Reaction of N-methyl-N'-nitro-N-nitrosoguanidine with protein: formation of nitroguanido derivations. Can-J-Biochem. 1968, 46(11):1410-5
[45]Devary Y, Rosette C, DiDonato JA, Karin M. NF-kappa B activation by ultraviolet light not dependent on a nucleus signal. Science. 1993;261(5127): 1442-5
[46]Wilhelm D, Bender K, Knebel A, Angel P. The level of intracellular glutathione is a key regulator for the induction of stress-activated signal transduction pathways including Jun N-terminal protein kinases and p38 kinase by alkylating agents, Mol. Cell. Biol. 17 (1997) 4792-800.
[47]Rosette C, Karin M. Ultraviolet light and osmotic stress: activation of the JNK cascade through multiple growth factor and cytokine receptors.Science. 1996; 274(5290): 1194-7
[48]Miller CC, Hale P, Pentland AP. Ultraviolet B injury increases prostaglandin synthesis through a tyrosine kinase-dependent pathway. Evidence for UVB-induced epidermal growth factor receptor activation. J-Biol-Chem.1994; 269(5): 3529-33
[49]萨姆布鲁克 J，弗里奇 EF，曼尼阿蒂斯 T．主编．分子克隆实验指南．第2版．北京：科学出版社，1996．549-564
[50]Wang P, Wu P, Siegel MI, Egan RW, Billah MM. Interleukin (IL)-10 inhibits nuclear factor kappa B (NF kappa B) activation in human monocytes.IL-10 and IL-4 suppress cytokine synthesis by different mechanisms.J-Biol-Chem. 1995; 270(i6): 9558-63
[51]中国科学院上海生物化学研究所酶结构功能组，上海第二医学院同位素研究室，针麻组．饱和分析法测定3'-5'-磷酸腺苷(cAMP)．生物化
学与生物物理学报，1976，8(2) ：169-177
[52] Roos D, Voetman AA, Meerhof LJ, Functional activity of enucleated human polmorphonucleaar leukocytes, Journal of cell biology 1983,97: 368-377
[53] Krokan HE, Nilsen H, Skorpen F, Otterlei M, Slupphaug G Base excision repair of DNA in mammalian cells. FEBS-Lett. 2000; 476(1-2) : 73-7
[54] Idriss HT, Al-Assar O, Wilson SH. DNA polymerase beta. Int J Biochem Cell Biol 2002; 34 (4) : 321-324
[55] Wilson SH. Mammalian base excision repair and DNA polymerase beta, Mutat. Res. 1998; 407: 203-215
[56] Iwanaga A, Ouchida M, Miyazaki K, et al. Functional mutation of DNA polymerase beta found in human gastric cancer-inability of the base excision repair in vitro. Mutat Res, 1999,435(2) : 121-128．
[57] Ochs K, Sobol RW, Wilson SH, Kaina B. Celis deficient in DNA polymerase beta are hypersensitive to alkylating agent-induced apoptosis and chromosomal breakage. Cancer-Res. 1999; 59(7) : 1544-51
[58] Sobol RW, Horton JK, Kuhn R, Gu H, Singhal RK, Prasad R, Rajewsky K, Wilson SH. Requirement of mammalian DN A polymerase-beta in base-excision repair. Nature. 1996; 379(6561) : 183-6
[59] Osheroff WP, Jung HK, Beard WA, et al. The fidelity of DNA polymerase β during distributive and processsive DNA synthesis. J. Biol. Chem; 1999, 274(6) :3642-3650．
[60] Chagovetz AM, Sweasy JB, Preston BD. Increased activity and fidelity of DNA polymerase beta on single-nucleotide gapped DNA. J. Biol. Chem, 1997; 272(44) : 27501-27504．
[61] Wood RD, Shiviji. MKK. Which DNA polymerases are used for DNA-repair in eukaryotes? Carcinogenesis, 1997,18(4) : 605-610．
[62] Blank A, Kim B, Loeb LA. DNA polymerase delta is required for base excision repair of DNA methylation damage in Saccharomyces cerevisiae. Proc Natl Acad Sci USA, 1994,91(19) : 9047．
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