Functions of alternative replication protein A in initiation and elongation

Abstract

Replication protein A (RPA) is a single-stranded DNA-binding complex that is essential for DNA replication, repair, and recombination in eukaryotic cells. In addition to this canonical complex, we have recently characterized an alternative replication protein A complex (aRPA) that is unique to primates. aRPA is composed of three subunits: RPA1 and RPA3, also present in canonical RPA, and a primate-specific subunit RPA4, homologous to canonical RPA2. aRPA has biochemical properties similar to those of the canonical RPA complex but does not support DNA replication. We describe studies that aimed to identify what properties of aRPA prevent it from functioning in DNA replication. We show aRPA has weakened interaction with DNA polymerase alpha (pol alpha) and that aRPA is not able to efficiently stimulate DNA synthesis by pol alpha on aRPA-coated DNA. Additionally, we show that aRPA is unable to support de novo priming by pol alpha. Because pol alpha activity is essential for both initiation and Okazaki strand synthesis, we conclude that the inability of aRPA to support pol alpha loading causes aRPA to be defective in DNA replication. We also show that aRPA stimulates synthesis by DNA polymerase alpha in the presence of PCNA and RFC. This indicates that aRPA can support extension of DNA strands by DNA polymerase partial differential. This finding along with the previous observation that aRPA supports early steps of nucleotide excision repair and recombination indicates that aRPA can support DNA repair synthesis that requires polymerase delta, PCNA, and RFC and support a role for aRPA in DNA repair.

Figure 1

The affect of RPA and aRPA on pol α when polymerase is pre-mixed with the DNA substrate. (A) Schematic illustrating experimental setup and order of addition of proteins. Asterisk indicates the location of the ³²-P label. (B) DNA pol α extension assays where pol α (1 nM) has been pre-incubated with the DNA substrate (20 nM 3′-OH ends). Following pre-incubation, either RPA (50 nM) or aRPA (50 nM) was added and the reaction initiated by the addition of dNTP’s (10 μM). Reaction products were separated by electrophoresis on a denaturing polyacrylamide sequencing gel and visualized on a Fuji FLA-7000 phosphorimager. (C) The results from three independent experiments were quantified and are presented. For each experiment the amount of DNA in each band was determined. Percent primer extension was calculated by determining the ratio of all extended products (+1 – +42 nt) to total DNA (all products plus unextended primer (+0)). Error bars indicate standard deviation. (D) The amount of products at +17 and +25 nt in the gel shown in 1(B) were quantified by dividing the sum of the amount of DNA at +17 nt and +25 nt by total DNA.

Figure 2

The effect on pol α when either RPA or aRPA are pre-bound to the DNA substrate. (A) Schematic illustrating experimental setup and order of addition of proteins. Asterisk indicates the location of the ³²-P label. (B) DNA pol α extension assays where either RPA (50 nM) or aRPA (50 nM) was pre-incubated with the DNA substrate (20 nM 3′-OH ends). Following pre-incubation, pol α (1 nM) was added and the reaction initiated by the addition of dNTP’s (10 μM). Reaction products were separated and visualized as described in Figure 1. (C) The results from three independent experiments were quantified and are presented. Percent primer extension was determined as described in Figure 1. Error bars indicate standard deviation.

Figure 3

Titration of RPA and aRPA in pol α extension assay. (A) (B) Pol α was pre-incubated with the DNA substrate (20 nM 3′-OH ends) as described in Figure 1. Following pre-incubation, RPA (closed circles) or aRPA (closed triangles) was added and the reaction initiated by the addition of (A) dNTP’s (10 μM) or (B) only dCTP (10 μM). (C) (D) RPA (closed circles) or aRPA (closed triangles) was pre-incubated with the DNA substrate (20 nM 3′-OH ends) as described in Figure 2. Following pre-incubation, pol α (1 nM) was added and the reaction initiated by the addition of (C) dNTP’s (10 μM) or (D) dCTP (10 μM). Primer extension was quantified as described in Figure 1. Average of two independent experiments are shown with error bars to indicate the range of the data.

Figure 4

Enzyme linked immunosorbant assay in which interactions were measured between different forms of RPA and either (A) pol α, (B) PCNA, (C) RFC or (D) pol δ. Forms of RPA used: RPA (closed circles) and aRPA (closed triangles). The data from each experiment was normalized to the highest absorbance in each experiment, averaged and plotted. Error bars indicate the average of two or more independent replicates. BSA was used to determine nonspecific background (< 0.1) in each assay and subtracted. (B) PCNA (closed squares) was also placed directly on the place as a positive control.

Figure 5

Mechanism of pol α inhibition. (A) Schematic illustrating experimental setup, order of addition of proteins and pre-incubation of proteins and DNA substrate. (B) DNA pol α extension assays where either RPA (50 nM) or aRPA (50 nM) was pre-incubated with the DNA substrate (20 nM 3′-OH ends) and the other form of RPA was pre-incubated with pol α (1 nM). The pre-incubated samples were mixed and the reaction initiated by the addition of dNTP’s (10 μM). Reaction products were separated by electrophoresis on a denaturing polyacrylamide sequencing gel and visualized by phosphorimaging. (C) The results from two independent experiments were quantified and are presented. Percent primer extension was determined as described in Figure 1. Error bars indicate range of data.

Figure 6

Primer RNA-DNA synthesis in the presence of RPA or aRPA. Primer RNA-DNA synthesis was measured by incorporation of 32P-dCTP in a 1 min labeling reaction that contained pSKori DNA (400 ng), Tag (800 ng), pol α (100 ng) and Topo I (200 ng), RPA (900 ng) and aRPA (900 ng) as indicated. After deproteination, the DNA was denatured with formamide and subjected to electrophoresis on a 12% acrylamide sequencing gel followed by detection of the labeled DNA with a phosphorimager. Major band in lane on right represents a 42 nucleotide single stranded DNA marker. Arrow indicates predominant length primers.

Figure 7

pol δ synthesis on singly-primed single-stranded M13mp18 template. (A) pol δ activity was assayed on singly-primed single-stranded M13mp18 (50 fmol) in reaction initiated by the addition of dNTPs (150 μM). Addition of individual components is indicated by a plus sign: pol δ (20 nM), RPA (555 nM), aRPA (555 nM), PCNA (50 nM) and RFC (50 nM). The time of the reaction is indicated (0, 5, 15 min). Reaction products were separated by electrophoresis on a denaturing polyacrylamide sequencing gel and visualized by phosphorimaging. (B) The results from three independent experiments were quantified and are presented. Primer extension was quantified by dividing either short products (≤ 40 nt, dark gray) or long products (≥ 41 nt, light gray) by total DNA. Error bars indicate the standard deviation of the data for the long products. (The standard deviation for the short products is shown in supplemental figure 1.) (C) Expanded time course of complete reactions (pol δ (20 nM), PCNA (50 nM) and RFC (50 nM)) with the addition of either RPA (555 nM, dark gray) or aRPA (555 nM, light gray)

Figure 8

Salt dependence of pol δ activity. pol δ activity was assayed on singly-primed single-stranded M13mp18 (50 fmol) in varying concentrations of NaCl initiated by the addition of dNTPs (150 μM). Reactions components: pol δ (20 nM)-open circles; pol δ (20 nM), RPA (555 nM), PCNA (50 nM) and RFC (50 nM)-closed circles; pol δ (20 nM), aRPA (555 nM), PCNA (50 nM) and RFC (50 nM)-closed triangles and pol δ (20 nM), PCNA (50 nM) and RFC (50 nM)-open diamonds. (A) Reaction products were separated by electrophoresis on a denaturing polyacrylamide sequencing gel and visualized by phosphorimaging. (B) The results from two independent experiments were quantified and are presented. Percent long products (≥ 41 nt) were calculated as described in Figure 7. Error bars indicate the range of the data.