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. 2013 May 9;50(3):323–332. doi: 10.1016/j.molcel.2013.03.019

Figure 1.

Figure 1

A Single Ribonucleotide in a DNA Heteroduplex Acts as an Initiation Site for MMR

(A) Schematic representation of the in vitro MMR assay. In the absence of a nick, very little repair of the T/G mismatch takes place, and digestion of the phagemid DNA with SalI and DraI gives rise to 2,484 (a + b), 694 (c), and 19 bp fragments (the smallest fragment is not detectable on these 1% agarose gels stained with GelRed). Upon introduction of a nick at the Nt.BstNBI site, T/G-to-C/G repair restores the SalI site, such that the phagemid DNA is cut into 1,324 (a), 1,160 (b), 694 (c), and 19 bp fragments.

(B) The presence of a single cytidine (rC) 54 nucleotides 5′ from the mispaired T made the T/G substrate susceptible to MMR even in the absence of a Nt.BstNBI nick. The substrates were incubated with MutSα-deficient LoVo nuclear extracts (supplemented or not with recombinant MutSα), and repair efficiency was quantitated by estimating the percentage of DNA in bands a and b. Autoradiograph of the same gel (32P) showed that [α-32P]dCMP was incorporated preferentially into the repaired fragments a and b. The figure shows a representative result. The experiment was carried out in triplicate.

(C) Kinetic analysis performed with the supercoiled T/G and rC-T/G substrates in MutSα-complemented LoVo extracts.

(D) As in (B), but the substrates were incubated with MutLα-deficient extracts of 293TLα− cells supplemented or not with recombinant MutLα.

(E) Kinetic analysis performed with the supercoiled T/G and rC-T/G substrates in MutLα-complemented 293TLα− extracts.

(F) Position and distance of the ribocytidine does not affect MMR efficiency. Supercoiled (sc), Nt.BstNBI-nicked (oc) T/G phagemids, or T/G-rC substrates containing a single rC residue 60 or 308 nucleotides 3′ from the mispaired T, were incubated with 293TLα− cells supplemented or not with recombinant MutLα. Repair efficiency was quantitated by estimating the percentage of DNA in bands a and b. Autoradiograph of the same gel (32P) showed that [α-32P]dCMP was incorporated preferentially into the repaired fragments a and b. The figure shows a representative result.

(G) Band-shift assay (Cejka et al., 2005) using recombinant MutSα and oligonucleotides C/G, T/G, or rC/G. The figure shows an autoradiograph of a 6% native polyacrylamide gel. M, size marker (1 kb ladder, New England BioLabs).