An oxidized abasic lesion inhibits base excision repair leading to DNA strand breaks in a trinucleotide repeat tract
Fig 4
Wild-type pol β crosslinks with DOB and AP, whereas pol β K72A mutant only crosslinks with DOB.
The formation of a pol β•DOB crosslink complex (A) and pol β K72A mutant•DOB crosslink complex (B) was measured using the nicked flap substrates and double-flap substrates containing DOB as described in the “Methods”. Lanes 1 and 7 contain the unphotolyzed substrate only. Lanes 2 and 8 represent reaction mixtures with the unphotolyzed substrates and pol β (1 μM). Lanes 3 and 9 indicate reaction mixtures containing the unphotolyzed substrates and pol β (1 μM) in the presence of 100 mM NaBH4. Lanes 4 and 10 indicate the photolyzed substrate only. Lanes 5 and 11 indicate reaction mixtures with the photolyzed substrates and pol β (1 μM). Lanes 6 and 12 indicate reaction mixtures containing the photolyzed substrates and pol β (1 μM) in the presence of 100 mM NaBH4. (C) The formation of pol β•AP crosslink and pol β K72A mutant•AP crosslink was examined using the nicked flap substrates and double-flap substrates containing a 5’-uracil as described in the “Methods”. Lanes 1 and 6 indicate the substrate only. Lanes 2 and 7 indicate reaction mixtures with 1 μM pol β only. Lanes 3 and 8 indicate reaction mixtures containing 1 μM pol β in the presence of 100 mM NaBH4. Lanes 4 and 9 indicate reaction mixtures with 1 μM pol β K72A mutant only. Lanes 5 and 10 indicate reaction mixtures with 1 μM pol β K72A mutant in the presence of 100 mM NaBH4. Substrates were 32P-labeled at the 3’-end of the downstream strand and are illustrated above each gel. Each experiment was done in triplicate.