Journal: Nucleic Acids Research

Article Title: DNA–protein cross-links between abasic DNA damage and mitochondrial transcription factor A (TFAM)

doi: 10.1093/nar/gkac1214

Figure Lengend Snippet: Elucidation of the terminal structures of SSB in TFAM-mediated reactions. (A) Proposed reaction mechanisms by which AP-DNA undergoes strand cleavage reactions catalyzed by TFAM, NaOH, endo III, or endo IV glycosylase. The resulting 3′ pUA-SSB can be trapped by thiol-containing chemicals, such as βMe or GSH. Product 1 is 3′pUA-SSB; 2 is 3′p-SSB, 3 is 3′OH-SSB; 4 is 3′dR-SSB; 5 is thiol-conjugates after reactions with βMe. ( B ) Gel electrophoretic analysis of reaction products of AP(5′) with NaOH, TFAM, endo III, or endo IV glycosylase. Reactions with asterisks indicate the presence of 5 mM βMe during the reaction for trapping 3′pUA in situ ; reactions without asterisks indicate products were derivatized with βMe after TFAM-mediated reactions. All reactions were quenched with NaBH 4 before denaturing PAGE analysis. ( C ) Reaction time courses of AP(5′) with TFAM (left), in the presence of 10 mM βMe (middle), or 10 mM GSH (right). The resulting DPCs were converted to peptide-DNA cross-links via proteolytic digestion to facilitate their migration. ( D ) Quantification of formation of DPC and SSB and disappearance of AP(5′).

Article Snippet: Endo III digestion was with 4 μM AP-DNA at 65°C for 1 h in the presence of 30 units of Thermotoga maritima ( Tma ) endo III (NEB, M0291S), 10 mM HEPES pH 7.4, 10 mM MgCl 2 .

Techniques: In Situ, Migration