Alanine scan mutagenesis of Rad521-212.
A, EMSA.
Alanine point mutants (0, 0.5, 1, 1.5, or 2 μm) of
Rad521-212 were incubated with 15 μm negatively
supercoiled plasmid DNA (pGsat4, 3.2 kilobases), and complexes were
fractionated through an agarose gel. WT, wild type. B,
surface view of the Rad521-212 protomer. The stem and domed cap
regions are colored light brown and light blue,
respectively. Lys-102 and Lys-133 (magenta) are located at the rim of
the stem region, whereas Lys-169, Tyr-171, and Arg-173 (also magenta)
are located at the edge of the domed cap region. The previously identified
ssDNA binding residues (Arg-55, Tyr-65, Lys-152, Arg-153, Arg-156, dark
blue) are clustered at the bottom of the groove formed between the stem
and domed cap regions. C, surface view of the Rad521-212
undecameric ring. All structural figures were created using the PyMOL
program.