生物化学原理课件（英文）：Chapter35 DNA recombination

1、Chapter35 DNA recombination Outline *Biological Roles *Recombination Types 1. Homologous recombination 2. Site-specific recombination 3. Transposition recombination *Mechanism 1. “Cut and paste” mechanism 2. “Copy and paste” mechanism Biological Roles for Recombination CGenerating new gene/allele co

2、mbinations (crossing over during meiosis) CGenerating new genes (e.g., IgG rearrangement) CIntegration of a specific DNA element CDNA repair Practical Uses of Recombination CUsed to map genes on chromosomes (recombination frequency proportional to distance between genes) CMaking transgenic cells and

3、 organisms Types of Recombination 1.Homologous - occurs between sequences that are nearly identical (e.g., during meiosis) 2.Site-Specific - occurs between sequences with a limited stretch of similarity; involves specific sites 3.Transposition DNA element moves from one site to another, usually litt

4、le sequence similarity involved Examples of Recombination Fig. 22.1 Homologous genetic recombination *Also known as general recombination or general homologous recombination *this is the exchange of genetic material between two molecules that share a large degree of identity with one another. *This

5、is the type of recombination that is required during meiotic crossing over, for bacteriophage recombination, for recombination following bacterial conjugation, and during the formation of plasmid multimers The Holliday Model of Genetic Recombination * This model of recombination was first proposed b

6、y Robin Holliday in 1964 and re-established by David Dressler and Huntington Potter in 1976 who demonstrated that the proposed physical intermediates existed. The Holliday model of general recombination The 3D structure of the Holliday junction DSB Repair Model *DSB is initiating event *Cut duplex i

7、s donor of genetic information *Enzymes are not sequence specific *First stable intermediate between two chromatids is a three stranded structure Recombination Proteins in E. coli *RecA *RecBCD *RuvA *RuvB *RuvC RecA Protein * The RecA protein is a multifunctional powerhouse! It has strand-exchange,

8、 ATPase and co-protease activities all packed into a compact 352 amino-acid, 38 kDa structure. It is required for all recombination pathways in E. coli. * The RecA protein will bind cooperatively to a ssDNA molecule with each monomer of RecA binding to a span of 4-6 nucleotides. Assembly of the nucl

9、eoprotein complex proceeds in a 5 - 3 direction. The complex is both fairly stable (half-life is 30 min) and is the active species that will promote strand exchange RecA will promote strand exchange between DNA molecules as long as the following conditions apply * One of the two molecules must have

10、a ssDNA region to which RecA can bind. * The two molecules must share a region of homologous (i.e. nearly identical) DNA sequence - a minimum of 50 bp is required. * There must be a free end within this region of homology which can initiate the strand exchange. The strand exchange reaction * RecA bi

11、nds to the ssDNA partner. * The two molecules are aligned possible through the formation of a triple-stranded intermediate. * Displacement of one of the old strands. This requires concurrent migration of the RecA nucleoprotein filament along the molecule - which proceeds in one direction only (5 - 3

12、) - and consequent winding/unwinding. ATP hydrolysis takes place during this step Model of a RecADNA complex A model for RecA-mediated pairing Model for RecA-mediated strand exchange RecBCD Protein * The recB, recC a helicase activity; an endonuclease activity; an ATPase activity; and, an ssDNA exon

13、uclease activity. * The RecBCD helicase activity can unwind DNA faster than it rewinds. Thus as it travels along a DNA molecule, it can generate ssDNA loops. * The RecBCD complex functions as a DNA exonuclease. It will bind to double-stranded breaks in DNA and degrade both strands simultaneously. Ho

14、wever, when RecBCD encounters a Chi sequence, its activity changes. The RecD subunit is released and the RecBC proteins act as a helicase to unwind the DNA in an ATP dependent reaction. This generates a ssDNA region that can serve (along with RecA) to initiate strand exchange and a recombination rea

15、ction. The generation of a 3-ending single-strand DNA segment by RecBCD to initiate recombination RuvA and RuvB * DNA helicase that catalyzes branch migration * RuvA tetramer binds to HJ (each DNA helix between subunits) * RuvB is a hexamer ring, has helicase and an autobiography at the Nobel Prize

16、site) spent many years patiently studying the behavior of unusual genetic elements in maize. She concluded that these elements were, in fact, mobile. Her work, all the more amazing because much of it was carried out before the structure of DNA was solved, was largely ignored until the mid 1970s when

17、 similar elements were discovered in bacteria. Transposable Genetic Elements in Bacteria *Insertion Sequences *Simple transposons *Composite Transposons *Bacteriophage Elements (Mu) Insertion Sequences *Insertion Sequences or IS elements are the simplest mobile element. They consist of a fairly shor

18、t (700 - 1500 bp) DNA segment flanked by a 10 - 40 bp inverted repeat sequence. The segment codes for the protein (transposase) that catalyses the transposition event: Transposable Genetic Elements in Bacteria Simple transposons *Are similar to IS elements. They contain DNA segments flanked by short

19、 inverted repeat sequences. The DNA segments, however, usually code for a number of gene products. In addition to a transposase, they may also code for a resolvase (this will depend on their mechanism of transposition) and they may contain for one or more antibiotic resistance genes: *Composite tran

20、sposons are DNA segments that are flanked by an IS element at either end. In other words, instead of each IS element moving independently, they now act in concert and move together along with the intervening DNA. *Each IS element is a typical IS element although only one of the two elements typicall

21、y retains a functional transposase activity. The IS elements may be in the same or in the opposite orientation with respect to one another. *The intervening segment often carries the genetic determinants for a number of antibiotic or other toxin resistances Composite Transposons Mechanisms of Transposition * Simple transposition the Cut the P elements in Drosophila Retrotransposon (the copia element in Drosophila and the Ty element in yeast) 1.Retroviral-like retrotransposon-These elements contain genes resembling the gag