1. Volume 10, Issue 5, Pages 1213-1222 (November 2002)
Acetylation Regulates the DNA End-Trimming Activity of DNA Polymerase β 
Sameez Hasan, Nazim El-Andaloussi, Ulrike Hardeland, Paul O. Hassa, Christine Bürki, Ralph Imhof, Primo Schär, Michael O. Hottiger 
Molecular Cell 
Volume 10, Issue 5, Pages (November 2002)
DOI: /S (02)

2. Figure 1 DNA Polymerase Activity Is Associated with p300
(A) p300 was immunoprecipitated from 150 μg HeLa nuclear extracts, and the immunocomplexes were subjected to a DNA-polymerase assay using poly dA/oligo dT as a template.
(B) p300 forms a complex with Polβ. p300 was immunoprecipitated from 150 μg HeLa nuclear extracts. Coimmunoprecipitated proteins were visualized by Western blot analysis using an anti-p300 antibody (top) or an anti-Polβ antibody (bottom).
(C) Colocalization of Polβ (upper left panel, green) and p300 (upper right, red) in vivo. The lower left panel shows the overlay signal displaying colocalization of Polβ and p300 in yellow. The same cells stained with DAPI overlaid to the phase-contrast picture (lower right). The arrows point at speckled structures within the nucleus.
Molecular Cell  , DOI: ( /S (02) )

3. Figure 2 Mapping of the DNA Polymerase Interaction Domain within p300
(A) (Upper panel) Schematic representation of p300 and its interaction domains: netted box, cysteine-histidine rich region 1; dotted box, cysteine-histidine rich region 2; gray box, histone acetyltransferase (HAT) domain; and square patterned box, cysteine-histidine rich region 3. (Lower panel) GST pull-down experiments for DNA polymerases with bacterially expressed p300 fragments 1–5. Glutathione sepharose purified GST-p300 fragments were incubated with HeLa nuclear extracts (NE) and subjected to a DNA polymerase assay.
(B and C) GST pull-down experiments for Polβ. The bound p300 fragments were incubated with 500 ng of purified Polβ and subjected to a DNA polymerase assay (B) or to a Western blot analysis using a Polβ-specific antibody (C).
Molecular Cell  , DOI: ( /S (02) )

4. Figure 3 Mapping of the p300 Interaction Domain within Polβ
(A) Schematic representation of Polβ and deletion mutants (top panel). GST pull-down experiments for p300 using C-terminal deletion mutants of Polβ. Interacting p300 was monitored by Western blot analysis using a p300-specific antibody (lower panel).
(B) Schematic representation of Polβ and deletion mutants (top panel). GST pull-down experiments for Polβ using p300 fragments 3, 4, and 5, and GST as control. Interacting Polβ was monitored by Western blot analysis using a Polβ-specific antibody.
Molecular Cell  , DOI: ( /S (02) )

5. Figure 4 Polβ Is Acetylated In Vitro and In Vivo
(A) GST-p300-HAT and p300 full-length were bacterially (left panel) or baculovirus (right panel) expressed, respectively, and subsequently used in an acetylation assay with 5 μg Polβ. The lower panel shows a Coomassie-stained gel after the acetylation assay, which was subsequently analyzed using autoradiography (top panel).
(B) Myc-tagged Polβ was transfected into human cells, and the cells were labeled with [3H]-acetate. After total extract preparation, myc Polβ was immunoprecipitated using a myc-specific antibody. The complex was resolved on SDS-PAGE and was analyzed either by Western blot analysis (left) or autoradiography (right).
Molecular Cell  , DOI: ( /S (02) )

6. Figure 5
Acetylation of Polβ Selectively Affects Its dRP-Lyase Function in Partially Reconstituted BER
(A) A BER intermediate relevant for Polβ activity (upper panel). Incubation of the dRP intermediate with different nonacetylated or acetylated Polβ and different amounts (as indicated, lower left- and right-hand panels, respectively). Ligation efficiencies were quantified and normalized to the reactions missing T4 DNA ligase.
(B) A BER intermediate containing a 1 nucleotide gap with 3′ OH and 5′ P ends (upper panel). Incubation of the intermediate with different nonacetylated or acetylated Polβ and different amounts (as indicated, lower left- and right-hand panels, respectively). Ligation efficiencies were quantified and normalized to the reactions missing T4 DNA ligase.
(C) A BER intermediate relevant for dRP-lyase activity of Polβ (upper panel). Incubation with 1 pmol of Polβ efficiently converted the 5′ dRP intermediate into a product with a 5′-phosphate end, while acetylated Polβ was not able to do so with a comparable rate (lower right- and left-hand panels).
(D) A DNA intermediate relevant for AP-lyase activity of Polβ (upper panel). Incubation with 1 pmol of Polβ (nonacetylated or acetylated, left- and right-hand panels, respectively) efficiently converted the AP-intermediate into a product of 24 nucleotide length.
Molecular Cell  , DOI: ( /S (02) )

7. Figure 6 Identification of the Acetylation Site in Polβ
Analysis of different Polβ mutants for the ability to be acetylated by GST-HAT of p300. Polβ mutants with the mutations K72A, K81A, K84A, or the wild-type protein were acetylated by GST-HAT p300. Proteins were separated by SDS-PAGE and analyzed by exposing the gel to an X-ray film.
Molecular Cell  , DOI: ( /S (02) )