Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2005 Jun 14;44(23):8438-8448.
doi: 10.1021/bi048057b.

DNA damage induced hyperphosphorylation of replication protein A. 2. Characterization of DNA binding activity, protein interactions, and activity in DNA replication and repair

Steve M Patrick #  1 , Greg G Oakley #  2 , Kathleen Dixon  3 , John J Turchi  1
Affiliations

DNA damage induced hyperphosphorylation of replication protein A. 2. Characterization of DNA binding activity, protein interactions, and activity in DNA replication and repair

Steve M Patrick et al. Biochemistry. .

Abstract

Replication protein A (RPA) is a heterotrimeric protein consisting of 70-, 34-, and 14- kDa subunits that is required for many DNA metabolic processes including DNA replication and DNA repair. Using a purified hyperphosphorylated form of RPA protein prepared in vitro, we have addressed the effects of hyperphosphorylation on steady-state and pre-steady-state DNA binding activity, the ability to support DNA repair and replication reactions, and the effect on the interaction with partner proteins. Equilibrium DNA binding activity measured by fluorescence polarization reveals no difference in ssDNA binding to pyrimidine-rich DNA sequences. However, RPA hyperphosphorylation results in a decreased affinity for purine-rich ssDNA and duplex DNA substrates. Pre-steady-state kinetic analysis is consistent with the equilibrium DNA binding and demonstrates a contribution from both the k(on) and k(off) to achieve these differences. The hyperphosphorylated form of RPA retains damage-specific DNA binding, and, importantly, the affinity of hyperphosphorylated RPA for damaged duplex DNA is 3-fold greater than the affinity of unmodified RPA for undamaged duplex DNA. The ability of hyperphosphorylated RPA to support DNA repair showed minor differences in the ability to support nucleotide excision repair (NER). Interestingly, under reaction conditions in which RPA is maintained in a hyperphosphorylated form, we also observed inhibition of in vitro DNA replication. Analyses of protein-protein interactions bear out the effects of hyperphosphorylated RPA on DNA metabolic pathways. Specifically, phosphorylation of RPA disrupts the interaction with DNA polymerase alpha but has no significant effect on the interaction with XPA. These results demonstrate that the effects of DNA damage induced hyperphosphorylation of RPA on DNA replication and DNA repair are mediated through alterations in DNA binding activity and protein-protein interactions.

PubMed Disclaimer

Figures

FIGURE 1
FIGURE 1
Anisotropy and stopped-flow kinetic analysis of hyper-phosphorylated RPA binding pyrimidine ssDNA. (A) Fluorescence polarization analysis of RPA (filled circles) and hyperphosphorylated RPA (open circles) binding to dT30 DNA substrates in reactions supplemented with 1 M NaCl. (B) Kinetic traces were performed at a constant RPA concentration (6.25 nM) and increasing concentrations of DNA (62.5, 75, 87.5, 100, and 112.5 nM). The traces were fit to a single-exponential decay. The observed rate constants (kobs) were plotted versus DNA concentration and fit to a straight line. The slope of the line provides the bimolecular rate constant, kon, for hyperphosphorylated RPA binding the dT30 ssDNA. Each point on the graph represents the average of at least three individual experiments, and the error bars represent the standard deviation.
FIGURE 2
FIGURE 2
Anisotropy and stopped-flow analysis of RPA binding purine ssDNA. (A) Fluorescence polarization analysis of RPA (filled circles) and hyperphosphorylated RPA (open circles) binding to a purine-rich 30 base DNA substrate. (B) Stopped-flow kinetic analysis of RPA binding purine ssDNA. The plot of kobs versus DNA concentration for rhRPA (filled circles) and hyperphosphorylated RPA (open circles) binding to a 30 bp purine-rich DNA are presented. The points on the graph are the average of three to four separate experiments, and the error bars represent the standard deviation.
FIGURE 3
FIGURE 3
Hyperphosphorylated RPA binding duplex undamaged and cisplatin-damaged 30-mer DNA. (A) EMSAs were performed using the following indicated amounts of rhRPA (lanes 1–5 and 11–15) or hyperphosphorylated RPA (lanes 6–10 and 16–20) and 50 fmol of either 30 bp undamaged (lanes 1–10) or 1,2d(GpG) cisplatin-containing DNA (lanes 11–20). The products were separated on a 4% native polyacrylamide gel and visualized by autoradiography. Lanes 1, 6, 11, and 16 without added RPA; lanes 2, 7, 12, and 17, 50 ng (425 fmol) RPA; lanes 3, 8, 13, and 18, 100 ng (850 fmol); lanes 4, 9, 14, and 19, 150 ng (1.275 pmol); lanes 5, 10, 15, and 20, 200 ng (1.7 pmol). (B) Quantification of increasing concentrations of rhRPA (filled symbols) and hyper-phosphorylated RPA (open symbols) binding undamaged (circles) and 1,2d(GpG) cisplatin-damaged DNA (squares). The results are the average of two individual experiments, and the error bars represent the range of values.
FIGURE 4
FIGURE 4
Anisotropy and stopped-flow of RPA binding duplex DNA. DNA binding analysis of phosphorylated RPA on duplex DNA substrates. (A) Fluorescence polarization analysis of RPA (filled circles) and hyperphosphorylated RPA (open circles) binding to a duplex DNA containing a cisplatin 1,2 d(GpG) adduct. (B) Fluorescence polarization analysis of RPA (filled circles) and hyperphosphorylated RPA (open circles) binding undamaged duplex DNA. (C) Stopped-flow kinetic analysis of rhRPA and hyperphosphorylated RPA binding duplex 1,2d(GpG) and 1,3d(GpXpG) cisplatin damaged 30-mer DNA. The kinetic traces from 10 to 12 measurements were fit to a double exponential decay, and the observed rate constants for the fast phase of the reaction were plotted versus DNA concentration (62.5–112.5 nM). The plots of the 1,2d(GpG) (circles) and 1,3d(GpXpG) (squares) cisplatin-damaged DNA for rhRPA (filled symbols) and hyperphosphorylated RPA (open symbols) were fit to a straight line. Each point on the graph represents the average of three to four individual experiments, and the error bars represent the standard deviation.
FIGURE 5
FIGURE 5
In vitro DNA repair assay using rhRPA and hyperphosphorylated RPA. HeLa crude extracts were added to a 120 bp DNA containing a cisplatin 1,3d(GpXpG) DNA adduct. Incision of the DNA substrate was visualized on 8% polyacrylamide–7 M urea sequencing gels. (A) Lane 1 is the control with no added extract, and lane 2 (C) is the control with the addition of crude extract; lane 3 (ID) is the RPA immunodepleted extracts; lanes 4 and 5 are immunodepleted extracts with the addition of 300 ng of rhRPA or hyperphosphorylated RPA, respectively; lanes 6 and 7 are immunodepleted extracts with the addition of 0.1 μM okadaic acid for rhRPA and hyperphosphorylated RPA, respectively. (B) Quantification of incision using the control value (crude extract containing endogenous RPA) as 100% incised. (C) Western blot analysis of the incision reactions. Lanes 1 and 2 are input controls for rhRPA and hyperphosphorylated RPA prior to the incision reaction, respectively; lane 3 is the endogenous RPA present in the crude extract; lane 4 is the RPA immunodepleted extracts; lanes 5 and 6 are the rhRPA and hyperphosphorylated RPA following the incision reaction, respectively; lanes 7 and 8 are the rhRPA and hyperphosphorylated RPA following the incision reaction in the presence of okadaic acid, respectively.
FIGURE 6
FIGURE 6
In vitro DNA replication assay using rhRPA and hyperphosphorylated RPA. (A) Western blot analysis of RPA-p34 in the replication reactions. In vitro DNA replication reactions were prepared using RPA-immunodepleted extract as described in Materials and Methods. The extracts were supplemented with buffer (lanes 1 and 4), rhRPA (lanes 2 and 5), or hyperphosphorylated RPA (lanes 3 and 6). In lanes 2 and 3, the RPA was added prior to T-antigen, while in lanes 5 and 6 the RPA was added as the last component to the reaction mix. Reactions were incubated for 2 h, and then proteins were separated by SDS–PAGE and RPA p34 phosphorylation status was measured by Western blot. (B) Analysis of DNA replication products by agarose gel electrophoresis. Lane 1 is the control without T-antigen added, and lane 2 is the complete reaction including T-antigen; lane 3 is from a reaction performed with the extract immunodepleted of RPA; lanes 4 and 5 are immunodepleted extracts with 150 ng of rhRPA or hyperphosphorylated RPA added prior to T-antigen. Lanes 6–10 are from identical reactions except the RPA was added as the last component to the reactions. (C) Quantification of the pZ189 replication generating Form I DNA. PhosphorImager analysis of the gel was performed quantifying Form I DNA, and the activity obtained with the immunodepleted extract supplemented with rhRPA was set to represent 100% replication.
FIGURE 7
FIGURE 7
Hyperphosphorylation of RPA differentially alters protein–protein interactions. (A) Co-immunoprecipitation of RPA with either XPA or DNA pol α Purified rhRPA (lanes 1 and 2), hyperphosphorylated RPA (lanes 3 and 4), and hyperphosphorylated RPA that was treated with CIP (lanes 5 and 6) was mixed with purified XPA or DNA pol α for 30 min on ice. The mixture was incubated further with anti-XPA or anti-DNA pol α overnight at 4 °C. The proteins that were immunoprecipitated with protein G-agarose (P) or remained in the supernatant (S) were separated by SDS–PAGE and RPA-p34 was detected by Western blotting. The arrowheads indicate the position of the RPA-p34 subunit. (B) Protein–protein interaction between RPA and DNA pol α or XPA detected with an ELISA assay. A modified ELISA was performed as described under Materials and Methods. Wells were coated with 100 ng of either rhRPA or hyperphosphorylated RPA. The wells were then blocked and 0.5×, 1×, or 2× molar ratio of the secondary protein was added and incubated. Primary antibody directed against either XPA or DNA pol α was added followed by the addition of the HRP-conjugated secondary antibody. The absorbance at 652 nm was measured over a period of 15 min. The results are presented as “Relative affinity for RPA” with the value obtained for rhRPA representing 100%.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Wold MS. Replication protein A: a heterotrimeric, single-stranded DNA-binding protein required for eukaryotic DNA metabolism. Annu. Rev. Biochem. 1997;66:61–92. - PubMed
    1. Iftode C, Daniely Y, Borowiec J. Replication protein A (RPA): the eukaryotic SSB, Crit. Rev. Biochem. Mol. Biol. 1999;34:141–180. - PubMed
    1. Brosh RM, Orren DK, Nehlin JO, Ravn PH, Kenny MK, Machwe A, Bohr VA. Functional and physical interaction between WRN helicase and human replication protein A. J. Biol. Chem. 1999;274:18341–18350. - PubMed
    1. He Z, Henricksen LA, Wold MS, Ingles CJ. RPA involvement in the damage-recognition and incision steps of nucleotide excision repair. Nature. 1995;374:566–569. - PubMed
    1. Bohr VA, Cooper M, Orren D, Machwe A, Piotrowski J, Sommers J, Karmakar P, Brosh R. Werner syndrome protein: biochemical properties and functional interactions. Exp. Gerontol. 2000;35:695–702. - PubMed

Publication types

MeSH terms