现代分子生物学之同源重组

Holliday junction
Resolution of the Holliday structure----to separate DNA molecules and finish genetic exchange
DNA strands may be cut along either the vertical line or horizontal line
Homologous recombination (HR): a history
early 1900s, certain genes can be inherited together in peas 1911, linked traits could on occasion be inherited separately → linked genes may physically cross over to different chromosomes
• RecA from Eubacteria, • RadA from Archaea, • Rad51 and Dmc1 from Eukaryota, • and the bacteriophage T4 UvsX protein.
Substrates for RecA strand exchange
Chi site: 5'-GCTGGTGG-3'
once every 65 kb, or about 80 times
1009 Chi sites
Presence of Chi
Foreign DNA
DNA activated for recombination
The multiple enzymatic activities of RecBCD provide a means for cells to “determine” whether to recombine with or destroy the foreign DNA molecules
RecA binding to dsDNA is rapid, weak, transient, and independent of DNA sequence
(c) Strand invasion: The cut strands cross and join homologous strands, forming the Holliday structure (or Holliday junction).
(d) Heteroduplex region is formed by branch migration
Homologous recombination in Prokaryotic cells
What will happen, if a piece of foreign DNA enter an E.Coli?
The fate of the foreigh DNA depends on whether it has a specific sequence in it
Mismatch repair
base excision
nucleotide excision
Or by direct chemical reversal of the damage
And nonhomologous end-joining or homologous recombination reseal the double-strand breaks
Homologous Recombination: HR
Models for HR
How it works
Protein Machines for HR (RecBCD pathway)
HR in Eukaryotes (meiosis recombination, proteins)
Examples and its genetic consequences
1931, chromosomal crossover occurs in meiosis, with phenpotype changes, in zea maiz and in drosophila
1936, crossing over occurs in somatic cells in mitosis 1947, bacteria are capable of genetic recombination, like sexual reproduction 1964, a model for recombination in meiosis in fungi (Holliday) 1983, another model(DSBR pathway) explains more During the 1990s, experiments in Drosophila, budding yeast and mammalian cells led to the emergence of other models of homologous recombination----SDSA pathways, which do not always rely on Holliday junctions.
• DNA并非静态存储：
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同源重组是修复方式和减数分Baidu Nhomakorabea之必须；
CSSR与转座是不同DNA间的剪切融合---DNA分子大尺度改变的基础
• 基因表达：DNA序列影响细胞结构功能
• • • RNA的合成：合成工具、基本过程、原核真核异同、后处理 蛋白质的合成：密码、合成工具、基本过程、原核真核异同 基因表达的调控：基本的原则和方法、原核真核异同
The 5' tail is cleaved less frequently than 3' tail, because of its unfavorable position
Ca
Nuclease active site
Recombination is stimulated specifically only on one “side” of the Chi site
or not
DNA destroyed
The nuclease domain of RecB interacts with RecA and load it on the DNA with the 3' tail
Each RecA monomer has more than one DNA-binding site, can hold a single strand and a double helix together: promotes strand invasion.
The SOS system: a form of damage tolerance that allows the cell to trade death for a certain level of mutagenesis Strand-exchange proteins of the RecA family are present in all forms of life.
RecD
RecB accumulates a ssDNA loop during unwinding
Ca2+
RecC recognizes and binds the Chi site
The looped-out ssDNA is pulled back in by RecB, which now is the primary motor The nuclease no longer cleaves the DNA strand with 3'- 5' polarity--the opposite DNA strand is cleaved more frequently.
RecB
RecC
RecD
Using the energy of ATP hydrolysis
>1000 bp/s Ca2+
煽动者
The DNA duplex finally has a 3' single-strand extension with the Chi sequence at the 3' end
• 基因研究技术 问题： 同源重组在分子水平上如何进行？它的生物学意义是什么？
DNA在传代及储存过程中可能遇到的问题及其解决方式
Mistake in DNA replication
Most of the damages to DNA are excised by one of two major DNA repair pathways
Yeast Mating type switch
exchange or conversion of genetic information
Holliday model ( proposed by Robin Holliday, 1964 )
(a) Two homologous DNA molecules line up (exactly or nearly identical for >100 bp). (b) Cuts in one strand of both DNAs.
煽动者
The DNA duplex finally has a 3' single-strand extension with the Chi sequence at the 3' end
The activity of RecBCD is modified when encountering an 8-mer Chi site, so that cleavage of the 3' tail ceases, and cleavage of the 5' tail increases in frequency
strand invasion
The active form of RecA is a protein –DNA filament
RecA protein in Translesion DNA synthesis(TLS)
UV light → synthesis of RecA protein ↑ → join SSB proteins to form proteinDNA filament, as a signal that induce special forms of DNA polymerase.
• 分子生物学的主要脉络：中心法则 • 课程目标：掌握主要脉络、熟悉基本概念、了解细枝末节 • 基因的物质状态：DNA上的一个片段
• • • • DNA本身的结构（为什么可以作为遗传物质） DNA在细胞中的存在形式（染色质、染色体、基因组的特点） DNA的传代方式：复制 DNA在传代及储存过程中可能遇到的问题及其解决方式
Chi : crossover hot spot instigator
Using the energy of ATP hydrolysis
RecBCD (Exo V): helicase /nuclease binds to DNA at the site of a DSB
RecB RecC
>1000 bp/s
DNA breaks are common and can initiate recombination: DSB repair model
DSB repair in replication fork
Prokaryotic and Eukaryotic Factors That Catalyze Recombination Steps
The 5‘--3' helicase activity of RecD and the 3‘--5' helicase activity of RecB drive the trimer forward, with RecC splitting the duplex DNA
RecB
RecC
RecD