DNA Polymerase Delta Exhibits Altered Catalytic Properties on Lysine Acetylation

Abstract

DNA polymerase delta is the primary polymerase that is involved in undamaged nuclear lagging strand DNA replication. Our mass-spectroscopic analysis has revealed that the human DNA polymerase δ is acetylated on subunits p125, p68, and p12. Using substrates that simulate Okazaki fragment intermediates, we studied alterations in the catalytic properties of acetylated polymerase and compared it to the unmodified form. The current data show that the acetylated form of human pol δ displays a higher polymerization activity compared to the unmodified form of the enzyme. Additionally, acetylation enhances the ability of the polymerase to resolve complex structures such as G-quadruplexes and other secondary structures that might be present on the template strand. More importantly, the ability of pol δ to displace a downstream DNA fragment is enhanced upon acetylation. Our current results suggest that acetylation has a profound effect on the activity of pol δ and supports the hypothesis that acetylation may promote higher-fidelity DNA replication.

Keywords: Okazaki fragment maturation; lagging strand replication; lysine acetylation; pol δ.

Figure 1

DNA polymerase δ is acetylated in vitro and in vivo. (A) Purified recombinant pol δ was incubated in the presence of h-p300 and acetyl CoA and then separated on 4–15% SDS-PAGE, as described in the “Section 2.1”. The acetylated subunits of pol δ (lane 3) were then detected by Western blotting using a pan acetyl lysine (AcK) antibody. (B) Whole-cell lysates prepared from HEK293T cells were immunoprecipitated with IgG or AcK antibody. The immunoprecipitated proteins were then separated on 4–15% SDS-PAGE and analyzed by Western blotting using anti POLD1 (p125); 10% of input from the whole-cell lysate (lane 1) served as the control.

Figure 2

Acetylation enhances the synthesis activity of pol δ. (A) The synthesis activity of pol δ was assayed using the 5 nM substrate (U₁:T₁) in the presence of 100 nM of either the unmodified (UM) or acetylated (AC) forms of pol δ. Time-course synthesis assay was monitored at the respective time points (0, 2.5, 5, 7.5, and 10 min) as indicated in the figure and as described in the “Section 2.6”. The substrate is depicted above the gel image, with the asterisk denoting the location of the radiolabel. + indicates the presence and—indicates the absence of the respective forms of pol δ. (B) Graphical plot of the percent of synthesis against time in minutes. Dashed red lines represent full-length synthesis by AC-polδ and dashed black lines represent full-length synthesis by UM-pol δ. Solid red lines represent total synthesis by AC-polδ and solid black lines represent total synthesis by UM-pol δ. The p value (generated by GraphPad Prism) was calculated by an unpaired, two-tailed t-test, * = p < 0.05. (C) Varying concentrations of either acetylated or unmodified pol δ (20, 50, 100, and 200 nM) were assayed for the synthesis activity of both forms of the polymerase. The reactions were set up as described in the “Section 2.6”. The substrate is depicted above each gel image and the asterisk denotes the location of the radiolabel. Exonuclease products (Exo. Prdts.) and synthesis products (Syn. Prdts.) are indicated adjacent to the gel image. The nucleotide sizes are also indicated.

Figure 3

Acetylation stimulates the strand displacement activity of pol δ. The strand displacement activity of pol δ was assayed using the 5 nM substrate at varying concentrations of either unmodified or acetylated pol δ (20, 50, 100, and 200 nM) as described in the “Section 2.6”. (A) Substrate (U₁:D₁: T₁) contains a randomly (RN) generated downstream blocking sequence. (B) Substrate (U₁:D₃: T₃₎ contains an AT-rich downstream blocking (AT) sequence. (C) Substrate (U₁:D₂: T₂₎ contains a GC-rich downstream blocking (GC) sequence. The substrate is depicted above each gel image and the asterisk denotes the location of the radiolabel. Exonuclease products (Exo. Prdts.), synthesis products (Syn. Prdts.), and strand displacement products (Strd. Disp. Prdts.) are indicated adjacent to the gel image. Nucleotide sizes are also indicated.

Figure 4

Acetylation stimulates the structure resolution activity of pol δ. (A) The structure resolution activity of pol δ was assayed using 5 nM of substrate containing a fold-back in the template (U₁:T₄) incubated with the either unmodified (UM) or acetylated (AC) forms of pol δ (20, 50, 100, 200, 400 nM) as indicated in the figure and as described in the “Section 2.6”. The concentration-dependent structure resolution activity of pol δ was assayed using (B) a substrate (U₁:T₅) containing a G-quartet in the template or (C) a substrate (U₁:T₆) containing a G-quadruplex in the template at varying concentrations of either unmodified or acetylated pol δ (20, 50, 100, and 200 nM) as described in the “Section 2.6”. The substrate is depicted above each gel image and the asterisk denotes the location of the radiolabel. Exonuclease products (Exo. Prdts.), synthesis products (Syn. Prdts.), and strand displacement product (Strd. Disp. Prdts.) are indicated adjacent to the gel image. Nucleotide sizes are also indicated.

Figure 5

Accessory proteins stimulate the structure resolution activity of UM and AC-pol δ. The structure resolution activity of pol δ was assayed on 5 nM substrate (U₁:T₆) containing a G-quadraplex in the template in the presence of 10 nM of UM or AC-pol δ. Increasing the concentrations of PCNA (200 and 400 nM), RFC (100 and 300 nM), or RPA (100 and 200 nM) were incubated along with the substrate and both forms of the polymerase. The substrate is depicted above the gel image and the asterisk denotes the location of the radiolabel. Exonuclease products (Exo. Prdts.) and structure resolution product (Strd. Res. Prdts.) are indicated adjacent to the gel image. Nucleotide sizes are also indicated. Gray arrows denotes position of stalling.

Figure 6

AC-pol δ displays higher exonuclease activity. (A) The exonuclease activity of polymerase (100 and 200 nM) was assayed on a 5 nM substrate containing no mismatch (U₂:T₇), or containing mismatch 3 nt (U₃:T₇), 5 nt (U₄:T₇), or 7 nt (U₅:T₇) away from the primer terminus as described in the “Section 2.7”. (B) The synthesis and exonuclease activity was tested on a 5 nM substrate containing a mismatch 5 nt away from the primer terminus (U₁:T₆) in the presence of 10, 50, 100, 200 nM UM or AC-pol δ. The reaction was incubated for 2 mins similar to that described in the “Section 2.7”, with the exception that the polymerase buffer contained 150 µM of dNTPs. The substrate is depicted above the gel image and the asterisk denotes the location of the radiolabel. Exonuclease products (Exo. Prdts.), and synthesis products (Syn. Prdts.) are indicated adjacent to the gel image. Nucleotide sizes are also indicated. Red cross denote position of mismatch.