The
RuvA Recombination Protein of
Escherichia
coli
David Marcey
© 2006
I.
Introduction
II. RuvA Structure
III. RuvA Tetramer-Holliday Junction
DNA
IV. References
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I.
Introduction
Homologous recombination
is essential for the generation of genetic diversity and for DNA repair
in all organisms. A crucial step in recombination is the resolution
of Holliday junctions, DNA intermediates which form as a result of
strand exchange between two homologous DNA helices:
After the formation
of Holliday intermediates, the length of heteroduplex
DNA is extended through the process of branch migration. Heteroduplex double helical DNA contains two strands that were originally paired with other strands in the original double helices. Branch migration
involves the sequential disruption of DNA base pairs in the two parental
DNA helices, and the formation of new base pairs in the hybrid helices.
Branch migration results in a region with two heteroduplexes and two original duplexes. To visualize this, it is useful to rotate the Holliday
junction (the region near the arrow in the above figure) so that it has no crossing DNA strands and is in a square-planer
configuration:
In
Escherichia coli, branch migration and resolution of Holliday
junctions to produce two, separate, intact DNA helices is accomplished
by a suite of proteins: RuvA, RuvB, and RuvC.
RuvA binds as
a tetramer to the Holliday junction; this binding allows loading of
RuvB, which acts as a molecular motor to drive branch migration. RuvC
then cleaves the junction and resolves the two DNA molecules into
mature recombination products.
At left is shown
an RuvA tetramer
bound to DNA oligonucleotides representing a square-planer
configuration of a Holliday junction. The regions of heteroduplex
DNA (the top and bottom arms of the square planar DNA) correspond to the figures, above.
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II. RuvA Structure
The
RuvA monomer has an "L" shape, composed of three domains:
I, II,
III. The loop connecting domain II
and domain III is not shown.
Domain
I is a six-stranded,
anti-parallel beta barrel, whereas both
domains II and III
consist entirely of alpha helices.
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III. RuvA Tetramer-Holliday
Junction DNA
In
tetrameric RuvA, each monomer is a lobe of the symmetrical tetramer.
The domain I beta barrels of each monomer
are positioned centrally, and domains II
and III are located peripherally.
Examining
the charge distribution of atoms over the surface of the RuvA tetramer,
it is clear that the DNA binding surface is largely positively charged
(basic), whereas the opposite surface is largely negatively charged
(acidic). The yellow,
orange, and red colors represent
relatively negatively charged atoms and the green
and blue colors represent more positively
charged atoms (red and blue
indicate acidic or
basic residues).
The relative positive charge of the DNA binding surface attracts the
negatively charged DNA backbone.
An
exception to the mostly basic surface
of the DNA binding side of the RuvA tetramer is an eight residue acidic
"central pin" containing glutamate55
and aspartate56 from
each monomer. This negatively charged center may repel the negatively
charged oxygens of the DNA backbone, driving the DNA away from the
center of the Holliday junction.
A
number of residues that interact directly
or indirectly (via water molecules) with the DNA backbone line the
channels in which Holliday junction DNA is bound.
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IV.
References
Ariyoshi, M.,
Nishino, T., Iwasaki, H., Shinagawa, H., Morikawa, K.: Crystal Structure
of the Holliday Junction DNA in Complex with a Single Ruva Tetramer.
Proc.Nat.Acad.Sci. 97: 8257-8262 (2000).
Hargreaves, D.,
Rice, D. W., Sedelnikova, S. E., Artymiuk, P. J., Lloyd, R. G., Rafferty,
J. B.: Crystal structure of E.coli RuvA with bound DNA Holliday junction
at 6 A resolution. Nat. Struct. Biol. 5: 441-446 (1998).
Roe, S. M., Barlow,
T., Brown, T., Oram, M., Keeley, A., Tsaneva, I. R., Pearl, L. H.:
Crystal structure of an octameric RuvA-Holliday junction complex.
Mol. Cell 2: 361-372 (1998).
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