1、Chapter 14,Recombination and Repair,Overview of Recombination,Crossover：當(dāng)兩個DNA分子的股斷裂後，又互相結(jié)合所形成的結(jié)構(gòu)稱為交換。 Recombination：DNA染色體或其他分子的基因信息交換稱為重組作用。,重組作用總覽,在分子的層次中，這種驅(qū)動某段DNA分子互換的機制稱作重組作用 (recombination)。真核生物的有性生殖過程中，減數(shù)分裂的過程讓DNA分子可以在同源染色體間 (homologous chromosomes) 互換，也因為有這樣的現(xiàn)象，增加了子代間的遺傳變異，而這樣的變異可以讓族群在演化篩選過

2、程中，有較大的生存機會。 在所有重組作用中，兩條DNA分子斷裂後，再相互接合在一起；形成一個結(jié)構(gòu)稱作重組點 (crossover) 。一個重組點的形成通常只會造成短暫的雜合分子；兩個重組點的形成，會使某一段DNA片段在兩個DNA分子間轉(zhuǎn)移，這就是重組作用。,Figure 14.01 Two crossovers Result in Recombination,Molecular basis of Homologous Recombination,Heteroduplex：指一個雙股螺旋DNA，其兩條單股各來自兩個不同DNA分子。 Holliday junction：重組時所形成的DNA結(jié)構(gòu)，可

3、在兩個DNA分子結(jié)合的交換點找到。 Homologous recombination：兩個相同或幾乎相同長度的DNA序列之間的重組作用。 Non-homologous recombination：兩個非常不同長度的DNA之間所發(fā)生的重組作用。這會牽涉到能識別各個序列並在序列之間形成交換的蛋白質(zhì)。與定位重組同義。,Patch recombinant：由於暫時性互換而造成DNA雙螺旋帶著異複式短補丁的過程。 Resolution：指兩個DNA分子結(jié)合一起的地方經(jīng)過合體的卵裂後釋放兩個不同的DNA分子。另也表示重組作用所形成的交換及移位所形成的共同合成這兩個的斷裂。 Resolvase：完成DNA分

4、解的酵素。,Figure 14.02 Homologous versus Non-homologous Recombination,Figure 14.03 Formation of a crossover,Figure 14.04 Rearrangement and Resolution of a Holliday Junction,Figure 14.05 Migration of a Holliday Junction,Single-Strand Invasion and Chi Sites,Figure 14.06 RecBCD Recognizes Chi Sites,Figure

5、14.07 Promotes Strand Invasion,What is the process by which DNA can be exchanged between chromosomes? What is a crossover? What are the differences between homologous and non-homologous recombination? What is a Holliday junction? What is a heteroduplex? What are the possible results from the resolut

6、ion of a Holliday junction? What is resolvase? What occurs during single strand invasion? What is needed to complete single strand invasion?,Site-Specific Recombination,Figure 14.08 Integration of Lambda DNA-Overview,Excisionase:酵素的一種，會移除dsDNA，逆轉(zhuǎn)DNA整合作用再密封裂口。特別是切出酶會移除已經(jīng)整合的DNA,Figure 14.09 Integratio

7、n of Lambda DNA-Detail of Crossover,Which kind of recombination takes place when there is little sequence similarity? How does lambda integrate into the E. coli chromosome? What is the function of the attachment sites on the bacterial chromosome and the lambda genome? How does RecA promote single st

8、rand invasion? What is needed for excision of lambda from a bacterial chromosome? When in the eukaryotic cell cycle does recombination take place? Why is it confined to this stage?,Recombination in Higher Organisms Figure 14.10 Timeline of Eukaryotic Recombination in Yeast,Figure 14.11 Spoil Promote

9、s Double Strand Breaks,Overview of DNA Repair,Rad proteins:與酵母菌及動物細(xì)胞的DNA受損重組及修復(fù)有關(guān)的蛋白質(zhì)組。Rad51相對於原核生物的RecA蛋白質(zhì),高等生物的重組作用,高等生物的重組大多發(fā)生於減數(shù)分裂早期。當(dāng)同源染色體發(fā)生互換時，雙股斷裂必定參與其中 一種危險的過程。 雙股斷裂的步驟發(fā)生在真核染色體的第一個減數(shù)分裂時期，也就是細(xì)線期 (leptotene)；而成對的染色體連結(jié)則發(fā)生在下一個時期，就是接合期 (zygotene)，此時期就形成重組所需的雜合接合結(jié)構(gòu) ，就像在細(xì)菌中產(chǎn)生的哈樂得氏接合但細(xì)節(jié)尚不清楚。分割作用發(fā)生在第

10、三時期，稱為粗線期 (pachytene)。最後，重組點分開，重組過後的染色體形成，此時期稱為雙線期 (diplotene)。,像在細(xì)菌中，這單股會被用來侵入另一個DNA的雙股螺璇結(jié)構(gòu)，存於酵母菌的 Rad 蛋白質(zhì) (Rad proteins) 則監(jiān)督這些過程的進(jìn)行。尤其，酵母菌中的 Rad51 蛋白質(zhì)和細(xì)菌中的 RecA 相似，會與自由單股DNA結(jié)合形程螺旋及附著蛋白質(zhì)的細(xì)絲結(jié)構(gòu)。,DNA修復(fù)總覽,若損害沒有修復(fù)或是在複製的過程，經(jīng)過損害區(qū)間仍進(jìn)行和加入不正確的鹼基，這些都會造成突變的發(fā)生。 大部分的細(xì)胞內(nèi)含各樣的控制損害的系統(tǒng)，這種系統(tǒng)可以去修復(fù)損害的DNA。有些不同的修復(fù)系統(tǒng)是為了因應(yīng)處

11、理各樣的損害問題；有些修復(fù)系統(tǒng)只是尋找 DNA 結(jié)構(gòu)的扭曲變形區(qū)，而其他的系統(tǒng)則集中在特定的化學(xué)性缺陷上。,DNA Mismatch Repair System,Excision repair system:一種DNA修復(fù)系統(tǒng)。能察覺DNA雙股螺旋的突起物，移除及取代受損。 Mismatch repair system:一種DNA修補系統(tǒng)，察覺配對錯誤的鹼基，然後剪掉有錯誤鹼基的部份。 DNA adenine methylase (Dam):一種會使GATC序列的腺嘌呤甲基化的細(xì)菌酵素。 DNA cytosine methylase (Dcm):一種會使CCAGG及CCTGG序列的胞嘧啶甲基化

12、細(xì)菌酵素。,Figure 14.12 Principle of Mismatch Repair,Figure 14.13 Methylated Base-Chemical Structure,Figure 14.14 Hemimethylated DNA: Old Strands Versus New,Figure 14.15 The MutSHL Mismatch Repair System,What are the consequences of not repairing DNA damage or the replication mechanism proceeding through

13、 damaged areas? Which repair mechanisms monitor the DNA double helix for damage rather than individual errors? Which mechanism is more sensitive? In mismatch repair how does the repair mechanism know which base is the wrong one?,What kind of sequences do Dam and Dcm methylase recognize? What is spec

14、ial about these sequences? What is hemi-methylatoin and when does it take place? What is the major mismatch repair system in E. coli? The major mismatch repair system in E. coli is made up of 3 proteins; what are they and what is their function?,Figure 14.16 The UvrABC Excision Repair System,What ki

15、nd of damage does the excision repair system fix? What kind of damage does the excision repair system fix? In the UvrAB excision repair system which protein(s) find(s) the mismatches, which protein(s) cut(s) out the damage, and how?,DNA Repair by Excision of Specific Bases,DNA glycosylase:一種酵素，切斷DNA

16、主幹上鹼基與去氧核醣之間的化學(xué)鍵,Specialized DNA Repair Mechanisms,AP endonuclease:一種內(nèi)酸酶，會造成在一個AP位置旁的DNA出現(xiàn)缺口。 AP-site 指少了一鹼基的某個DNA位置(AP=鹼基不見了，P代表嘌呤或嘧啶的意思。) Ung protein:與尿嘧啶-N-醣甘酶同意。 Uracil-N-glycosylase:會從DNA移除尿嘧啶的酵素,Figure 14.17 Removal of Unnatural Bases,Figure 14.18 Dealing with Oxidized Guanine,Figure 14.19 Sui

17、cide Demethylase for O-methyl Bases,Figure 14.20 Ada Play a Dual Role Removing Alkyl Groups,Figure 14.21 Photo-Reactivation Cleaves Pyrimidine Dimers,Figure 14.22 Eukaryotic Transcription-Coupled Excision Repair,RecA,Figure 14.23,Figure 14.24 RecA and LexA Control the SOS System,Figure 14.25 DNA Pol

18、ymerase V is Part of the System,What are DNA glycosylases? Describe the removal of unnatural bases. What is “very short patch repair,” what enzymes are involved and what is their role? What is photo-reactivation and what kind of damage does it repair? How does the transcription coupled excision repair system work?,How does DNA polymerase V