Journal: Molecular and Cellular Biology

Article Title: DNA Polymerase Beta Participates in Mitochondrial DNA Repair

doi: 10.1128/MCB.00237-17

Figure Lengend Snippet: The N terminus of Polβ interacts with mitochondrial transport and mtDNA maintenance components. (A) HEK293T cells overexpressing Polβ (see Fig. S2A to C in the supplemental material) were incubated with GST-tagged N- or C-terminal Polβ recombinant fragments attached to glutathione-Sepharose beads. The bound proteins from the mitochondrial extracts were eluted after washing (see diagram). (B) Silver staining of gel used for subsequent mass spectrometry (also see Table 1 and Fig. S2D). (C) Immunoblotting (IB) of the GST pulldown used in mass spectrometry detected mitochondrial proteins HSPA9 and TFAM. Mitochondrial import proteins TOM70 and TIM50 and accessory mtDNA repair proteins TWINKLE and POLDIP2 were also detected. In all cases, mitochondrial proteins bound only to the N-terminal Polβ fragment. (D) GST-TFAM fragments were purified to establish which region of the TFAM protein bound to mitochondrial Polβ. (E) Identification of Polβ in the GST-TFAM-bound fraction using antibody detection. Polβ bound to the N-terminal fragment of TFAM containing the HMG1 domain. DNA maintenance proteins POLDIP2, PARP1, and OGG1 also bound to the HMG1-containing fragment.

Article Snippet: Antibodies used in binding analysis were to OGG1 (SC-12076; Santa Cruz), PARP1 (SC-8007; Santa Cruz), Polβ (ab26343; Abcam), TFAM (7495S; Cell Signaling Technology, Danvers, MA), Tim50 (SC-393678; Santa Cruz), Tom70 (SC-26495; Santa Cruz), and TWINKLE (SC-155418; Santa Cruz).

Techniques: Incubation, Recombinant, Silver Staining, Mass Spectrometry, Western Blot, Purification