Answers to this one and the next
From 2006.igem.org
Recombinase Enhancer R.C. Johnson and M.F. Bruist. 1989. Intermediates in Hin-mediated DNA inversion: a role for Fis and the recombinational enhancer in the strand exchange reaction [1]
- RE sequence must be present
- w/o RE, Hin can still assemble to with the two Hix’s however, Hin can’t generate inversion
- Prevention of Fis binding causes loss of enhancer function
- 60 bp in length
- Binds two Fis proteins (98 AA’s)
- Enhancer cannot function when located too close to a recombination site
- HU facilitates the required bending of the DNA to create the inversion interaction
- HU stimulation is dependent upon location of RE
- Dependence upon HU is lesser when distance between RE and recombination site is greater
- Required to induce the bending of the shorter, yet stiffer, segment of DNA
- When RE is located >650 bp for the recombination site the dependence upon HU to form the complex is lost
- RE combined w/ supercoiling mediate the rotation of DNA strands subsequent to double-stranded cleavage by Hin within two recombination sites
Haykinson and Johnson. 1993. DNA looping and the helical repeat in vitro and in vivo: effect of HU protein and enhancer location on Hin invertasome assembly [2]
- The two recombination sites bound by Hin are assembled together at the Fis-bound RE
- w/o HU invertasome assembly is very inefficient
- “HU is the only protein in E. coli that can promote invertasome formation with short DNA lengths between RE and recombination sites
- HU binds non-specifically to DNA between the enhancer and recombination site to facilitate DNA looping
- “The RE can function at great distances !!! >4 kb !!! and on either side of a recombination site on a plasmid to stimulate Hin-mediated DNA inversion”
- RE is not functional at a position 32 bp minimum
- Due to restricted looping
- Enhancer Structure: AGTCAACAATTGACC – 33 bp – GTGCAAATTGTGACC; proximal Fis-binding site of the left and the distal Fis-binding site on the right
