Lecture 11
Lecture 11 February-25-14 10:14 AM
Recombination: nicking of DNA, single stranded overhang attacks the duplex, strand displacement, further synthesis of DNA, intermediate junction E. coli RecBCD Pathway of Homologous Recombination I Sometimes RecABCD -recA helps in homology search Repair is the initial condition that drove evolution
BCD - encodes for different subunits Under conditions of high magnesium, the activity is higher Acts as endonuclease, and degrades and unwinds DNA, until it reaches a Chi site (a consensus). Chi sites serve as initiator for replication. Overhang gets coded by recA protein. 30 bp is not enough, if over this, the D-loop will form (displacement-formation), after which recA is released ATP is needed to unload (no ATP no recombination!) HOLLIDAY junction - the polynucleotide switch between duplex - They are heteroduplex which contain potential mismatches (contain both blue and red from different parents) - Holliday junction migration - A diffusion process, it is neutral, slow process, less enzymes are Lecture Page 1
- Holliday junction migration - A diffusion process, it is neutral, slow process, less enzymes are involved G and H resolutions are two possible resolutions - equally likely to happen RuvC - enzyme that drives the resolution H is the parental resolution (heteroduplexes: non-crossover recombinants)- blue with blue, red with red G is another resolution (crossover recombinants) - red with blue
Binding of RecA to single-stranded DNA
Single stranded areas are much thicker than double stranded areas - protein are bound to DNA (they are huge) At the right pic, same thing, the single stranded DNA are fat The left pic is a nicer experiment than the right one because there's no control for the double stranded This can be done without ATP recA requires ATP Synapsis - A binding to the outside of DNA There is actual binding between red and blue? How to test? We can do a melt curve, if binding is superficial it will come apart at low temp It is superficial alignment Synapsis depends on recA
Lecture Page 2
Synapsis depends on recA
RecA dependent synapsis
Areas of synapsis where red meets blue If there is no sequence similarity and no rec A, there is no synapsis Only stable at low temperature, a not strong association Conditions for having synapsis
Important - ATP (gamma) S RecBCD Appears to Nick DNA Near Chi (χ) sites to Initiate Recombination Lecture Page 3
RecBCD Appears to Nick DNA Near Chi (χ) sites to Initiate Recombination Steps a and b of E. coli Rec BCD Pathway for Homologous Recombination
Nicking of DNA by RecBCD Near to the Chi site
Label 3' overhang allow We want to look for liberation of the 3' end due to nicking Why do we want to boil? Causes DNA to melt - making DNA single stranded - It doesn’t make a difference recBCD is necessary 0 means no chi site, + has true chi site Only a portion was transformed Synthetic Holliday structure
Lecture Page 4
Synthetic Holliday structure
Annealing happens instantaneously Will junction migrate? No, because there is unfavourable base pairing Ruv proteins - ABC - a different set of proteins
There is sequential loading of proteins, the junction moves Why feed with ATP(gamma)S? - Necessary to see the result All lanes have hydrolyzed ATP It can't be hydrolyzed, block ATP hydrolysis cycle, necessary to see intermediates Artificial junction is hoped to be a natural, natural ones are too unpredictable
Lecture Page 5
Recombination: nicking of DNA, single stranded overhang attacks the duplex, strand displacement, further synthesis of DNA, intermediate junction E. coli RecBCD Pathway of Homologous Recombination I Sometimes RecABCD -recA helps in homology search Repair is the initial condition that drove evolution
BCD - encodes for different subunits Under conditions of high magnesium, the activity is higher Acts as endonuclease, and degrades and unwinds DNA, until it reaches a Chi site (a consensus). Chi sites serve as initiator for replication. Overhang gets coded by recA protein. 30 bp is not enough, if over this, the D-loop will form (displacement-formation), after which recA is released ATP is needed to unload (no ATP no recombination!) HOLLIDAY junction - the polynucleotide switch between duplex - They are heteroduplex which contain potential mismatches (contain both blue and red from different parents) - Holliday junction migration - A diffusion process, it is neutral, slow process, less enzymes are Lecture Page 1
- Holliday junction migration - A diffusion process, it is neutral, slow process, less enzymes are involved G and H resolutions are two possible resolutions - equally likely to happen RuvC - enzyme that drives the resolution H is the parental resolution (heteroduplexes: non-crossover recombinants)- blue with blue, red with red G is another resolution (crossover recombinants) - red with blue
Binding of RecA to single-stranded DNA
Single stranded areas are much thicker than double stranded areas - protein are bound to DNA (they are huge) At the right pic, same thing, the single stranded DNA are fat The left pic is a nicer experiment than the right one because there's no control for the double stranded This can be done without ATP recA requires ATP Synapsis - A binding to the outside of DNA There is actual binding between red and blue? How to test? We can do a melt curve, if binding is superficial it will come apart at low temp It is superficial alignment Synapsis depends on recA
Lecture Page 2
Synapsis depends on recA
RecA dependent synapsis
Areas of synapsis where red meets blue If there is no sequence similarity and no rec A, there is no synapsis Only stable at low temperature, a not strong association Conditions for having synapsis
Important - ATP (gamma) S RecBCD Appears to Nick DNA Near Chi (χ) sites to Initiate Recombination Lecture Page 3
RecBCD Appears to Nick DNA Near Chi (χ) sites to Initiate Recombination Steps a and b of E. coli Rec BCD Pathway for Homologous Recombination
Nicking of DNA by RecBCD Near to the Chi site
Label 3' overhang allow We want to look for liberation of the 3' end due to nicking Why do we want to boil? Causes DNA to melt - making DNA single stranded - It doesn’t make a difference recBCD is necessary 0 means no chi site, + has true chi site Only a portion was transformed Synthetic Holliday structure
Lecture Page 4
Synthetic Holliday structure
Annealing happens instantaneously Will junction migrate? No, because there is unfavourable base pairing Ruv proteins - ABC - a different set of proteins
There is sequential loading of proteins, the junction moves Why feed with ATP(gamma)S? - Necessary to see the result All lanes have hydrolyzed ATP It can't be hydrolyzed, block ATP hydrolysis cycle, necessary to see intermediates Artificial junction is hoped to be a natural, natural ones are too unpredictable
Lecture Page 5
