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研究生:吳俊逸
研究生(外文):Wu, Juin-Yi
論文名稱:大腸桿菌甲基指引修復系統對未配對寡核酸環修復之試管中研究
論文名稱(外文):Methyl-directed Repair of Mismatched Small Heterologous Sequences in Cell Extracts From Escherichia coli
指導教授:方偉宏方偉宏引用關係
指導教授(外文):Fang Woei-Horng
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:醫事技術學系
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:1997
畢業學年度:85
語文別:中文
論文頁數:80
中文關鍵詞:甲基指引修復寡核酸環
外文關鍵詞:methyl-directed repairsmall heterologous sequences
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大腸桿菌核酸複製透過甲基指引核酸配對錯誤修復系統之監控,可使其忠
誠度提高一百至一千倍。當核酸複製時,修復反應專一性地發生於新合成
之一股,此專一性來自新合成之一股其 d(GATC) 序列有一段時間尚未被
dam methylase 所甲基化,而指引修復反應發生於此股。除了 dam
methylase 外,大腸桿菌這套修復系統還需要 mutH, mutL, mutS 和 uvrD
等基因產物一起參與反應,且這套系統修復反應之機制及修復單一鹼基配
對錯誤之專一性已有完整之研究。這套修復系統除了可以修復單一鹼基配
對錯誤外,尚可修復小量鹼基嵌入及刪除之未配對寡核酸環,且亦發現這套
修復系統因括人類細胞之各種生物體,有高度地功能上之相似性。另外,當
大腸桿菌於 mutH, mutL, mutS 和 uvrD 等基因有缺陷時,大腸桿菌不僅
容易出現核酸之轉變突變 (transition mutation),亦常出現核酸之股架
位移突變 (frame shift mutation)。這項事實與許多人類疾病有病人同
時出現核酸配對錯誤修復系統缺陷與微衛星 (microsatellite) 不穩定之
現象類似。因此我們想重新對大腸桿菌這套核酸配對錯誤修復系統,修復
小量鹼基嵌入/刪除之寡核酸環的能力及專一性,作進一步之研究。

為了解鹼基嵌入/刪除之寡核酸環被大腸桿菌甲基指引修復系統修復之情
形,我們設計了一系列不同鹼基數目之未配對寡核酸環異雙股核酸,鹼基數
目從 1-8 和 22,且包括了鹼基嵌入與刪除兩種形式,在試管中與大腸桿菌
之細胞萃取物進行修復反應後分析修復情形。我們之研究結果顯示,與鹼
基配對錯誤被修復之情形類似, 寡核酸環異雙股核酸在試管中可被大腸桿
菌細胞萃取物修復,同樣具甲基指引之股專一性,而且這樣之修復能力同樣
需 mutH, mutL, mutS 和 uvrD 等基因產物之參與。另外,我們還發現寡
核酸環被修復之效率會受其環內鹼基組成影響,但不受寡核酸環形式為嵌
或刪除而影響。此外,我們亦觀察到寡核酸環被修復之效率有隨著寡核酸
環之鹼基數目增加而下降之趨勢,但鹼基數目多至 22 時,仍然呈現小量之
甲基指引修復效率。



In Escherichia coli, the fidelity of DNA replication is
increased 100-1000 fold by postreplication, methyl-directed DNA
repair. The repair is directed on the newly replicated strand
that is transiently unmethylated at d(GATC) sequences, which are
subsequently methylated by the dam gene product. In addition to
dam methylation, the products of mutS, mutL, mutH and uvrD gene
are required for this repair system. The mechanism and repair
specificity of base-base mismatches of this system are well
ocumented. Thisystem can repair base-base mispair as well as
small nucleotide insertion/deletion mismatches. Similar repair
pathways have been found in a variety of organisms, including in
human. Defective mismatch repair have been implicated in several
human diseases. In addition, microsatellite instability has also
been observed in these diseases. In E.coli, the mutator effects
observed in E.coli mutH, mutL, mutS, and uvrD mismatch repair
deficient mutant, are primarily transition and frame shift
mutation. Elucidation the biochemical mechanism of insertion/
deletion repair using bacterial system may provide useful
information for mutation prevention.

In order to understand the repair efficiency of small nucleotide
heterologies by methyl directed repair system, we constructed a
series of M13mp18 hemimethylated heteroduplex substrates
containing 1-8 and 22 unpaired bases. Repair efficiency of
thesesubstrates were determined by using an in vitro assay.
Our results showed that the repair of small nucleotide
heterologies in E.coli extracts was similarto base- of base
mismatch repair, being strand specific and highly biased to the
unmethylated strand.The in vitro activity was dependent on
products of mutH, mutL, mutS, and uvrD loci, and was equeally
efficient on nucleotide insertions amposition of the
heterologies. However, the extent of repair of
heteroduplexescontaining small heterologies sequences was found
to decrease with increasingnumber of unpaired bases, though
heteroduplexes containing extra nucleotides as large as 22 bases
provoked low level of methyl-directed repair.

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