Fig. 2: Rad24 interaction with the 5′-recessed DNA junction.
From: DNA is loaded through the 9-1-1 DNA checkpoint clamp in the opposite direction of the PCNA clamp
a, Top: structure of Rad24 bound to the DNA in cartoon view. The 5′-strand is orange and the 3′-strand is cyan. Residues 81-HKRK-84 of the AAA+ ___domain are in dark red. Areas marked by three dashed red rectangles are shown as close-up views in b–d, respectively. Bottom: electrostatic surface view showing a contiguous DNA-binding basic patch on the top of the AAA+ ___domain. LL: long linker between AAA+ and the collar domains that enables the formation of a large groove to accommodate the DNA. b, Top: interaction between the AAA+ ___domain and DNA. The four tandem basic residues are shown as cyan sticks. His-81 and Arg-83 insert into the DNA minor groove, and Lys-84 forms two hydrogen bonds with the DNA T28 phosphate of the 3′-strand. Asn-269 of the AAA+ ___domain and Thr-271 of the AAA+ ___domain form three hydrogen bonds with the DNA phosphates of C-27 and T-26. Bottom: sequence alignment of the Rad24 four tandem basic residues. S.c., Saccharomyces cerevisiae; H.s., Homo sapiens; M.m., Mus musculus; D.m., Drosophila melanogaster. c, Top: DNA lies on the Rad24 AAA+ ___domain. Phe-340 and His-341 of the collar ___domain stabilize the 5′-recessed DNA junction and mimic a nucleotide base to form a hydrophobic stack with the last base pair at the 5′-junction; they each rotate 32° with respect to the interacting base, resembling the 36° rotation of a normal base. His-341, Lys-345 and His-351 surround the 5′-OH of the 5′-strand and prevent a 5′-overhang from binding there. The 5′-OH and the A-3 phosphate form hydrogen bonds with Gly-349 and His-438, respectively. Bottom: sequence alignment of Rad24 base-mimicking residues Phe-340 and His-341, and the 5′-OH blocking Lys-345, His-351. d, Residues guiding 3′-overhang ssDNA into the Rad24-RFC chamber. Tyr-442 is positioned at the 5′-recessed DNA junction resembling a separation pin.
