分子生物学（张海红）10 章.pptVIP

思考题 1.同源重组的特点，功能是什么？ 2.同源重组的两种模型是什么，具体过程如何，二者有什么异同？（看一下动画会更清楚） Holliday模型、DSB模型 3.大肠杆菌的同源重组的蛋白机器（ RecBCD 途径）的作用机制。 4.大肠杆菌和真核生物中在同源重组过程中涉及的关键蛋白有哪些，它们的主要作用是什么？ Change in nuclease activity appears to be inactivation or loss of RecD subunit 3’ overhang could also be coated w/ SSB, interaction of RecBCD and RecA ensures that RecA binds RecBCD pathway Figure 10-5. Steps of DNA processing by RecBCD. Chapter 10 Figure 10-8 Substrates for RecA strand exchange. RecA protein assembles on single-strand DNA and promotes strand invasion Chapter 10 RecA binds to ssDNA and is a strand-exchange protein, catalyzing the pairing of homologous DNA molecules The active form of RecA is a protein-DNA filament – huge, variable size, 100 subunits of RecA, 300 nucleotides RecA filament can bind 1, 2, 3 or 4 strands Coated with RecA make DNA highly extended Polarity of RecA assembly Figure 10-9 Three view of the RecA filament. RecA Filament Chapter 10 RecA binding is cooperative, growing with the addition of subunits to the 3’ side of the first subunit - in the 5’ to 3’ direction = filament ending in a 3’ is most likely to be coated by RecA Polarity of RecA assembly Figure 10-10 Chapter 10 RecA binds to ssDNA RecA “looks” for sequence homology using 2 binding sites. The ssDNA is bound in the primary binding site. dsDNA can occupy the secondary binding site – binding is rapid, weak, transient and seq. independent. RecA moves along dsDNA scanning for homology – base-flipping. Model of two steps in search for homology and DNA strand exchange within the RecA filament Figure 10-11 Chapter 10 Matches of 15+ bp triggers strand exchange RecA promotes formation of stable complex between two DNA molecules = three-stranded joint molecule containing several 100 bp of hybrid DNA The strand in the primary site is paired w/ its complement in the duplex bound in the secondary site - breaking and forming hydrogen bonds Figure 10-11 Chapter 10 Strand-exchange proteins in the RecA family are p