Fig. 2.

Intermediates of uracil processing can be used as MMR initiation sites in substrates containing a uracil residue upstream from a mispair. (A) A covalently closed circular substrate carrying a U/G mispair 54 nucleotides 5′ from a G/T mispair in the AclI site was repaired with high efficiency (lane 1), but this reaction could be inhibited by the addition of Ugi (lane 2). This shows that uracil processing by UNG2 and/or TDG was indispensable for this MMR-catalyzed process. Ugi inhibition could be overcome by the introduction of a nick into the substrate (lanes 3, 4). The nicked G/T substrate was used as a control (lane 5). M, molecular size marker. (B) Left panel shows the schematic representation of the radiolabeled MMR repair tracts starting either at the uracil residue or at the nick. AclI cuts the repaired phagemid into fragments a, b, and c. If the MMR repair tract starts at the uracil residue and continues approximately 150 nucleotides past the AclI site, fragment a should contain a stretch of 54 and fragment b of approximately 150 nucleotides labeled with [³²P]dAMP. If the MMR repair tract starts at the nick and continues approximately 150 nucleotides past the AclI site, fragment a should contain a labeled stretch of 350 and fragment b of approximately 150 nucleotides. Right panel shows an autoradiograph of the repair reactions similar to lanes 1–4 in panel A above, carried out in the presence of [³²P]dATP. (C) A uracil residue opposite adenine can function similarly to that in a U/G mispair. In this experiment, the G/T substrate contained a U/A base pair approximately 50 nucleotides 5′ from the G. Lanes as above. (D) In a covalently closed substrate containing two U/G mispairs, such as would arise through the repeated action of AID, the mispair in the AclI site (lane 1) was repaired to C/G with an efficiency similar to that observed in A above. The higher background in the Ugi-inhibited reaction (lane 2) is due to the fact that AclI partially cleaves DNA containing a U/G mispair in its recognition sequence, as shown in the digest of the untreated supercoiled substrate (lane 6). The numbers under the lanes represent an average of at least 2 independent experiments. M, molecular size marker.