臺灣博碩士論文加值系統

DNA的複製為任何生命體之中樞基本現象，在大腸桿菌中，DNA的複製有一個特定起始位置，命名為oriC，從此處解開雙股螺旋，往兩個相反方向同時進行複製。但是DNA的複製究竟以何種模式進行，目前仍無定論，一般研究根據in vitro data指出DNA的複製為半不連續的模式，然而大部分in vivo data則指出大腸桿菌的DNA合成在兩股皆以不連續性的方式進行。根據本實驗室先前的研究，我們對野生種大腸桿菌在in vivo進行短時間放射性標定觀察其DNA合成模式，顯示大腸桿菌的複製是以不連續性方式。若DNA在前進股的合成是不連續的，則需要有RNA引子的重複合成，因此，領導股勢必擁有一個專門的引子合成複合體，若將可能參與在領導股引子合成複合體之基因突變，是否可使領導股的DNA複製由不連續性轉變為連續性？在本論文，我們測試幾個候選基因（priA, priB, priC及rep）在領導股合成中扮演的角色，藉由放射性短時間標定法分析野生株和突變株複製模式的差異，在短時間標定下，大部分新合成DNA是小於5 kb的，但priA2與priA300其新合成DNA片段卻有往大於5 kb移動的趨勢。為減少在野生種菌株中短時間標定而產生技術上誤差，將priA2與rep等基因送入lig-7及polA4113菌株中進行分析，我們觀察到將priC送入lig-7及polA4113菌株中，並不影響新合成DNA大小分佈，然而在priA2 lig-7, priA2 polA4113或rep polA4113等菌株中，新合成的DNA分子有往大於5 kb移動趨勢，我們的結果顯示著priA或甚至rep可能在大腸桿菌不連續DNA複製模式中扮演重要角色。

Replication of DNA is fundamental to all actively growing cells. In Escherichia coli, DNA replication is initiated at specific site on the genomic DNA, termed oriC, and then proceeds bidirectionally to the terminus. The question of whether discontinuous DNA synthesis operates in only the lagging strand or in both strands at a replication fork remains unresolved. Data from most in vivo studies are clearly in favor of a discontinuous DNA replication model, and the DnaG primase binding sites are known to be abundant on both strands. For discontinuous synthesis to occur in the leading-strand, one would predict that RNA primers are synthesized in the leading-strand, and therefore, there should be a primosome complex for the leading-strand. We hypothesize that inactivation of genes that function only in the leading-strand primosome shall convert the discontinuous synthesis to continuous mode. In this work, we examined several candidate genes ( priA, B, C and rep ) for their possible role in the leading strand DNA synthesis. The size distribution of nascent DNA synthesized from pulse-labeling with 3H-thimidine was analyzed in wild-type, priA, B, C and rep cells. The majority of short pulse-labeled (10 second) DNAs from the wild-type cells are smaller than 5kb, which is consistent with the previous findings by Okazaki et al. A greater percentage of short pulse-labeled DNAs from the priA2,and priA300, but not from priB302, priC303 or rep, mutants was partitioned into high Mr. (eg, greater than 5kb) forms. To minimize the technical problems of analyzing the small amount of DNA synthesized during the very short pulse-labeling, the effects of these mutations on the nascent DNAs synthesized in a lig-7 or polA4113 strain were examined. Introduction of priC303 into a lig-7 or polA4113 strain did not appear to alter the size distribution of nascent DNA synthesized in these mutants, i.e., the bulk of nascent DNA was all Okazaki fragments. However, the nascent DNA synthesized in the priA lig7, priA2 polA4113, or rep polA4113 strains appeared to contain a greater fraction of DNA in high Mr. (eg. greater than 5 kb). Our results indicate that the priA and possibly rep genes may play an important role in the apparent discontinuous DNA replication in E. coli.

指導教授推薦書…………………………………………………………
口試委員會審定書………………………………………………………
長庚大學授權書…………………………………………………………..iii
誌謝………………………………………………………………………..iv
目錄……………………………………………………………………….. v
圖表目錄………………………………………………………………….. vi
中文摘要…………………………………………………………………..vii
英文摘要…………………………………………………………………..ix
第一章 前言…………………………………………………………….. 1
DNA複製模式………………………………………………….. 1
引子合成複合體在複製過程中扮演的角色………………….. 4
參與領導股primosome組成之鑑定………………………….. 7
研究目的……………………………………………………….. 8
第二章 材料與方法…………………………………………………….. 10
第三章 實驗結果……………………………………………………….. 18
1 在野生種大腸桿菌中，探討priA,B,C與rep之單一突變對新合成DNA大小片段影 響…………………………………………………………………………… 18
2 在lig7突變之大腸桿菌中，探討priA,B,C與rep之單一突變對新合成DNA大小片段影響……………………………………………………………………… 19
3 在polA4113突變之大腸桿菌中，探討priA,B,C與rep之單一突變對新合成DNA大小片段影響………………………………………………………………… 21
第四章 討論……………………………………………………………… 23
參考文獻…………………………………………………………………… 49
附錄………………………………………………………………………… 54

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