The XPC-HR23B complex displays high affinity and specificity for damaged DNA in a true-equilibrium fluorescence assay

The XPC-HR23B complex is a prime candidate for the initial damage recognition step during global genome nucleotide excision repair. A specific interaction between the XPC-HR23B complex and various types of damaged DNA substrates has been demonstrated in recent work by electrophoretic mobility shift assays or immunoprecipitation. Although these studies allowed the estimation of relative binding affinities for the different types of lesions, the presence of large amounts of competitor DNA or the need for glutaraldehyde fixation prevented the quantification of equilibrium constants. We have performed a quantitative study on the binding of XPC to damaged DNA using fluorescence anisotropy measurements. The XPC-HR23B complex binds with high affinity (K(D) approximately 1-3 nM) to fluorescent 36 bp DNA fragments containing a single cisplatin 1,3-intrastrand adduct or a six-nucleotide mispaired region. From stoichiometric titration experiments, it is concluded that approximately 70% of the XPC-HR23B preparation is active in DNA binding. Binding experiments employing fluorescent probes with a single defined photoproduct reveal a 30-fold preference of XPC for 6,4-photoproducts as compared to a cyclobutane dimer. Competition experiments with undamaged and damaged plasmid DNA indicate that the XPC-HR23B complex discriminates between damaged and undamaged sites with high specificity. The specificity factor is between 100 and 3000, depending on the number of nonspecific sites considered in the calculations. Upon addition of XPA to the XPC binding reaction mixtures, it was not possible to detect cooperative ternary complex formation on the platinated 36 bp probe.     

Replication protein A interactions with DNA. III. Molecular basis of recognition of damaged DNA

Human replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein (subunits of 70, 32, and 14 kDa) that is required for cellular DNA metabolism. RPA has been reported to interact specifically with damaged double-stranded DNA and to participate in multiple steps of nucleotide excision repair (NER) including the damage recognition step. We have examined the mechanism of RPA binding to both single-stranded and double-stranded DNA (ssDNA and dsDNA, respectively) containing damage. We show that the affinity of RPA for damaged dsDNA correlated with disruption of the double helix by the damaged bases and required RPAs ssDNA-binding activity. We conclude that RPA is recognizing single-stranded character caused by the damaged nucleotides. We also show that RPA binds specifically to damaged ssDNA. The specificity of binding varies with the type of damage with RPA having up to a 60-fold preference for a pyrimidine(6-4)pyrimidone photoproduct. We show that this specific binding was absolutely dependent on the zinc-finger domain in the C-terminus of the 70-kDa subunit. The affinity of RPA for damaged ssDNA was 5 orders of magnitude higher than that of the damage recognition protein XPA (xeroderma pigmentosum group A protein). These findings suggest that RPA probably binds to both damaged and undamaged strands in the NER excision complex. RPA binding may be important for efficient excision of damaged DNA in NER.     

Recognition of cisplatin-DNA interstrand cross-links by replication protein A

Replication protein A (RPA) is a heterotrimeric protein that is required for DNA replication and most DNA repair pathways. RPA has previously been shown to play a role in recognizing and binding damaged DNA during nucleotide excision repair (NER). RPA has also been suggested to play a role in psoralen DNA interstrand cross-link (ICL) repair, but a clear biochemical activity has yet to be identified in the ICL DNA repair pathways. Using HeLa cell extracts and DNA affinity chromatography, we demonstrate that RPA is preferentially retained on a cisplatin interstrand cross-link (ICL) DNA column compared with undamaged DNA. The retention of RPA on cisplatin intrastrand and ICL containing DNA affinity columns is comparable. In vitro electrophoretic mobility shift assays (EMSAs) using synthetic DNA substrates and purified RPA demonstrate higher affinity for cisplatin ICL DNA binding compared with undamaged DNA. The enhanced binding of RPA to the cisplatin ICL is dependent on the DNA length. As the DNA flanking the cisplatin ICL is increased from 7 to 21 bases, preferential RPA binding is observed. Fluorescence anisotropy reveals greater than 200-fold higher affinity to a cisplatin ICL containing 42-mer DNA compared with an undamaged DNA and a 3-4-fold higher affinity when compared with a cisplatin intrastrand damaged DNA. As the DNA length and stringency of the binding reaction increase, greater preferential binding of RPA to cisplatin ICL DNA is observed. These data are consistent with a role for RPA in the initial recognition and initiation of cisplatin ICL DNA repair.     

Interactions of mammalian proteins with cisplatin-damaged DNA

We have undertaken the systematic isolation and characterization of mammalian proteins which display an affinity for cisplatin-damaged DNA. Fractionation of human cell extracts has led to the identification of two classes of proteins. The first includes proteins that bind duplex DNA in the absence of cisplatin damage and retain their affinity for DNA in the presence of cisplatin-DNA adducts. The DNA-dependent protein kinase (DNA-PK) falls into this class. The inhibition of DNA-PK phosphorylation activity by cisplatin-damaged DNA has led to the hypothesis that cisplatin sensitization of mammalian cells to ionizing radiation may be mediated by DNA-PK. The second class of proteins identified are those which display a high relative affinity for cisplatin-damaged DNA and a low affinity for undamaged duplex DNA. Proteins that fall into this class include high mobility group 1 protein (HMG-1), replication protein A (RPA) and xeroderma pigmentosum group A protein (XPA). Each protein has been isolated and purified in the lab. The interaction of each protein with cisplatin-damaged DNA has been assessed in electrophoretic mobility shift assays. A series of DNA binding experiments suggests that RPA binds duplex DNA via denaturation and subsequent preferential binding to the undamaged DNA strand of the partial duplex. DNA substrates prepared with photo-reactive base analogs on either the damaged or undamaged DNA strand have also been employed to investigate the mechanism and specific protein-DNA interactions that occur as each protein binds to cisplatin-damaged DNA. Results suggest both damage and strand specificity for RPA and XPA binding cisplatin-damaged DNA.     

Crosslinking of nucleotide excision repair proteins with DNA containing photoreactive damages

Photoreactive DNA duplexes mimicking substrates of nucleotide excision repair (NER) system were used to analyze the interaction of XPC-HR23B, RPA, and XPA with damaged DNA. Photoreactive groups in one strand of DNA duplex (arylazido-dCMP or 4-thio-dUMP) were combined with anthracenyl-dCMP residue at the opposite strand to analyze contacts of NER factors with damaged and undamaged strands. Crosslinking of XPC-HR23B complex with photoreactive 48-mers results in modification of XPC subunit. XPC-HR23B did not crosslink with DNA duplex bearing bulky residues in both strands while this modification does not prevent interaction of DNA with XPA. The data on crosslinking of XPA and RPA with photoreactive DNA duplexes containing bulky group in one of the strands are in favor of XPA preference to interact with the damaged strand and RPA preference for the undamaged strand. The results support the understanding and set the stage for dynamically oriented experiments of how the pre-incision complex is formed in the early stage of NER.     

Order of assembly of human DNA repair excision nuclease.

Human excision nuclease removes DNA damage by concerted dual incisions bracketing the lesion. The dual incisions are accomplished by sequential and partly overlapping actions of six repair factors, RPA, XPA, XPC, TFIIH, XPG, and XPF.ERCC1. Of these, RPA, XPA, and XPC have specific binding affinity for damaged DNA. To learn about the role of these three proteins in damage recognition and the order of assembly of the excision nuclease, we measured the binding affinities of XPA, RPA, and XPC to a DNA fragment containing a single (6-4) photoproduct and determined the rate of damage excision under a variety of reaction conditions. We found that XPC has the highest affinity to DNA and that RPA has the highest selectivity for damaged DNA. Under experimental conditions conducive to binding of either XPA + RPA or XPC to damaged DNA, the rate of damage removal was about 5-fold faster for reactions in which XPA + RPA was the first damage recognition factor presented to DNA compared with reactions in which XPC was the first protein that had the opportunity to bind to DNA. We conclude that RPA and XPA are the initial damage sensing factors of human excision nuclease.     

Strand-specific binding of RPA and XPA to damaged duplex DNA

The nucleotide excision repair (NER) pathway is a major pathway used to repair bulky adduct DNA damage. Two proteins, xeroderma pigmentosum group A protein (XPA) and replication protein A (RPA), have been implicated in the role of DNA damage recognition in the NER pathway. The particular manner in which these two damage recognition proteins align themselves with respect to a damaged DNA site was assessed using photoreactive base analogues within specific DNA substrates to allow site-specific cross-linking of the damage recognition proteins. Results of these studies demonstrate that both RPA and XPA are in close proximity to the adduct as measured by cross-linking of each protein directly to the platinum moiety. Additional studies demonstrate that XPA contacts both the damaged and undamaged strands of the duplex DNA. Direct evidence is presented demonstrating preferential binding of RPA to the undamaged strand of a duplex damaged DNA molecule.     

Double-check probing of DNA bending and unwinding by XPA-RPA: an architectural function in DNA repair.

The multiprotein factor composed of XPA and replication protein A (RPA) is an essential subunit of the mammalian nucleotide excision repair system. Although XPA-RPA has been implicated in damage recognition, its activity in the DNA repair pathway remains controversial. By replacing DNA adducts with mispaired bases or non-hybridizing analogues, we found that the weak preference of XPA and RPA for damaged substrates is entirely mediated by indirect readout of DNA helix conformations. Further screening with artificially distorted substrates revealed that XPA binds most efficiently to rigidly bent duplexes but not to single-stranded DNA. Conversely, RPA recognizes single-stranded sites but not backbone bending. Thus, the association of XPA with RPA generates a double-check sensor that detects, simultaneously, backbone and base pair distortion of DNA. The affinity of XPA for sharply bent duplexes, characteristic of architectural proteins, is not compatible with a direct function during recognition of nucleotide lesions. Instead, XPA in conjunction with RPA may constitute a regulatory factor that monitors DNA bending and unwinding to verify the damage-specific localization of repair complexes or control their correct three-dimensional assembly.     

NMR study on the interaction between RPA and DNA decamer containing cis-syn cyclobutane pyrimidine dimer in the presence of XPA: implication for damage verification and strand-specific dual incision in nucleotide excision repair

In mammalian cells, nucleotide excision repair (NER) is the major pathway for the removal of bulky DNA adducts. Many of the key NER proteins are members of the XP family (XPA, XPB, etc.), which was named on the basis of its association with the disorder xerodoma pigmentosum. Human replication protein A (RPA), the ubiquitous single-stranded DNA-binding protein, is another of the essential proteins for NER. RPA stimulates the interaction of XPA with damaged DNA by forming an RPA–XPA complex on damaged DNA sites. Binding of RPA to the undamaged DNA strand is most important during NER, because XPA, which directs the excision nucleases XPG and XPF, must bind to the damaged strand. In this study, nuclear magnetic resonance (NMR) spectroscopy was used to assess the binding of the tandem high affinity DNA-binding domains, RPA-AB, and of the isolated domain RPA-A, to normal DNA and damaged DNA containing the cyclobutane pyrimidine dimer (CPD) lesion. Both RPA-A and RPA-AB were found to bind non- specifically to both strands of normal and CPD- containing DNA duplexes. There were no differences observed when binding to normal DNA duplex was examined in the presence of the minimal DNA-binding domain of XPA (XPA-MBD). However, there is a drastic difference for CPD-damaged DNA duplex as both RPA-A and RPA-AB bind specifically to the undamaged strand. The strand-specific binding of RPA and XPA to the damaged duplex DNA shows that RPA and XPA play crucial roles in damage verification and guiding cleavage of damaged DNA during NER.     

Damaged DNA-binding protein DDB stimulates the excision of cyclobutane pyrimidine dimers in vitro in concert with XPA and replication protein A

Human cells contain a protein that binds to UV-irradiated DNA with high affinity. This protein, damaged DNA-binding protein (DDB), is a heterodimer of two polypeptides, p127 and p48. Recent in vivo studies suggested that DDB is involved in global genome repair of cyclobutane pyrimidine dimers (CPDs), but the mechanism remains unclear. Here, we show that in vitro DDB directly stimulates the excision of CPDs but not (6-4)photoproducts. The excision activity of cell-free extracts from Chinese hamster AA8 cell line that lacks DDB activity was increased 3-4-fold by recombinant DDB heterodimer but not p127 subunit alone. Moreover, the addition of XPA or XPA + replication protein A (RPA), which themselves enhanced excision, also enhanced the excision in the presence of DDB. DDB was found to elevate the binding of XPA to damaged DNA and to make a complex with damaged DNA and XPA or XPA + RPA as judged by both electrophoretic mobility shift assays and DNase I protection assays. These results suggest that DDB assists in the recognition of CPDs by core NER factors, possibly through the efficient recruitment of XPA or XPA.RPA, and thus stimulates the excision reaction of CPDs.     

RPA repair recognition of DNA containing pyrimidines bearing bulky adducts

Recognition of new DNA nucleotide excision repair (NER) substrate analogs, 48-mer ddsDNA (damaged double-stranded DNA), by human replication protein A (hRPA) has been analyzed using fluorescence spectroscopy and photoaffinity modification. The aim of the present work was to find quantitative characteristics of RPA-ddsDNA interaction and RPA subunits role in this process. The designed DNA structures bear bulky substituted pyrimidine nitrogen bases at the inner positions of duplex forming DNA chains. The photoreactive 4-azido-2,5-difluoro-3- pyridin-6-yl (FAP) and fluorescent antracenyl, pyrenyl (Antr, Pyr) groups were introduced via different linker fragments into exo-4N of deoxycytidine or 5C of deoxyuridine. J-dU-containing DNA was used as a photoactive model of undamaged DNA strands. The reporter group was a fluorescein residue, introduced into the 5'-phosphate end of one duplex-forming DNA strand. RPA-dsDNA association constants and the molar RPA/dsDNA ratio have been calculated based on fluorescence anisotropy measurements under conditions of a 1:1 RPA/dsDNA molar ratio in complexes. The evident preference for RPA binding to ddsDNA over undamaged dsDNA distinctly depends on the adduct type and varies in the following way: undamaged dsDNA < Antr-dC-ddsDNA < mmdsDNA < FAPdU-, Pyr-dU-ddsDNA < FAP-dC-ddsDNA (K(D) = 68 +/- 1; 25 +/- 6; 13 +/- 1; 8 +/- 2, and 3.5 +/- 0.5 nM correspondingly) but weakly depends on the chain integrity. Interestingly the bulkier lesions not in all cases have a greater effect on RPA affinity to ddsDNA. The experiments on photoaffinity modification demonstrated only p70 of compactly arranged RPA directly interacting with dsDNA. The formation of RPA-ddsDNA covalent adducts was drastically reduced when both strands of DNA duplex contained virtually opposite located FAP-dC and Antr-dC. Thus RPA requires undamaged DNA strand presence for the effective interaction with dsDNA bearing bulky damages and demonstrates the early NER factors characteristic features underlying strand discrimination capacity and poor activity of the NER system toward double damaged DNA.     

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

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.     

Localization of xeroderma pigmentosum group A protein and replication protein A on damaged DNA in nucleotide excision repair

The interaction of xeroderma pigmentosum group A protein (XPA) and replication protein A (RPA) with damaged DNA in nucleotide excision repair (NER) was studied using model dsDNA and bubble-DNA structure with 5-{3-[6-(carboxyamido-fluoresceinyl)amidocapromoyl]allyl}-dUMP lesions in one strand and containing photoreactive 5-iodo-dUMP residues in defined positions. Interactions of XPA and RPA with damaged and undamaged DNA strands were investigated by DNA–protein photocrosslinking and gel shift analysis. XPA showed two maximums of crosslinking intensities located on the 5′-side from a lesion. RPA mainly localized on undamaged strand of damaged DNA duplex and damaged bubble-DNA structure. These results presented for the first time the direct evidence for the localization of XPA in the 5′-side of the lesion and suggested the key role of XPA orientation in conjunction with RPA binding to undamaged strand for the positioning of the NER preincision complex. The findings supported the mechanism of loading of the heterodimer consisting of excision repair cross-complementing group 1 and xeroderma pigmentosum group F proteins by XPA on the 5′-side from the lesion before damaged strand incision. Importantly, the proper orientation of XPA and RPA in the stage of preincision was achieved in the absence of TFIIH and XPG.     

Overexpression and purification of human XPA using a baculovirus expression system

The xeroderma pigmentosum group A protein (XPA) is an essential component of the eukaryotic nucleotide excision repair (NER) process. Recombinant human XPA was expressed in baculovirus-infected insect cells as a [His](6)-tagged fusion protein. A two-column purification procedure resulted in greater than 90% purity for the recombinant protein with a final yield of 0.53 mg from 200 ml of infected cells. The recombinant protein migrated as a doublet of 44 and 42 kDa upon SDS-PAGE consistent with that observed for the native protein. XPA can interact with a number of proteins including replication protein A (RPA) which has been implicated in the initial recognition of damaged DNA. Using a modified ELISA, we demonstrate that the recombinant XPA fusion protein also forms a complex with RPA independent of DNA. The ability of XPA to bind damaged DNA was assessed in an electrophoretic mobility shift assay using globally cisplatin-damaged DNA. The results revealed a slight preference for DNA damaged with cisplatin consistent with its proposed role in the recognition of damaged DNA. The recombinant XPA fusion protein was able to complement cell-free extracts immunodepleted of XPA restoring NER-catalyzed incision of cisplatin-damaged DNA in an in vitro excision repair assay.     

Xeroderma pigmentosum complementation group A protein (XPA) modulates RPA-DNA interactions via enhanced complex stability and inhibition of strand separation activity

Replication protein A (RPA) participates in many cellular functions including DNA replication and nucleotide excision repair. A direct interaction between RPA and the xeroderma pigmentosum group A protein (XPA) facilitates the assembly of a preincision complex during the processing of DNA damage by the nucleotide excision repair pathway. We demonstrate here the formation of a ternary RPA, XPA, and duplex cisplatin-damaged DNA complex as is evident by electrophoretic supershift analysis. The RPA-XPA complex displays modest specificity for damaged versus undamaged duplex DNA, and the RPA-XPA complex displays a greater affinity for binding duplex cisplatin-damaged DNA when compared with the RPA or XPA proteins alone, consistent with previous results. Using DNA denaturation assays, we demonstrate that the role of XPA is in the stabilization of the duplex DNA structure via inhibition of the strand separation activity of RPA. Rapid kinetic analysis indicates that the bimolecular k(on) of the RPA-XPA complex is 2.5-fold faster than RPA alone for binding a duplex cisplatin-damaged DNA. The dissociation rate, k(off), of the RPA-XPA complex is slower than that of the RPA protein alone, suggesting that the XPA protein stabilizes the initial binding of RPA to duplex DNA as well as maintaining the integrity of the duplex DNA. Interestingly, XPA has no effect on the k(on) of RPA for a single-stranded 40-mer DNA.     

Thermodynamic properties of damaged DNA and its recognition by xeroderma pigmentosum group A protein and replication protein A

The effects of the lesions induced by single, site-specific 1,2-GG or 1,3-GTG intrastrand adducts of cis-diamminedichloroplatinum(II) formed in oligodeoxyribonucleotide duplexes on energetics of DNA were examined by means of differential scanning calorimetry. These effects were correlated with affinity of these duplexes for damaged-DNA binding-proteins XPA and RPA; this affinity was examined by gel electrophoresis. The results confirm that rigid DNA bending is the specific determinant responsible for high-affinity interactions of XPA with damaged DNA, but that an additional important factor, which affects affinity of XPA to damaged DNA, is a change of thermodynamic stability of DNA induced by the damage. In addition, the results also confirm that RPA preferentially binds to DNA distorted so that hydrogen bonds between complementary bases are interrupted. RPA also binds to non-denaturational distortions in double-helical DNA, but affinity of RPA to these distortions is insensitive to alterations of thermodynamic stability of damaged DNA.     

Replication protein A (RPA) binding to duplex cisplatin-damaged DNA is mediated through the generation of single-stranded DNA.

Replication protein A (RPA) is a heterotrimeric protein composed of 70-, 34-, and 14-kDa subunits that has been shown to be required for DNA replication, repair, and homologous recombination. We have previously shown preferential binding of recombinant human RPA (rhRPA) to duplex cisplatin-damaged DNA compared with the control undamaged DNA (Patrick, S. M., and Turchi, J. J. (1998) Biochemistry 37, 8808-8815). Here we assess the binding of rhRPA to DNA containing site-specific cisplatin-DNA adducts. rhRPA is shown to bind 1.5-2-fold better to a duplex 30-base pair substrate containing a single 1,3d(GpXpG) compared with a 1,2d(GpG) cisplatin-DNA intrastrand adduct, consistent with the difference in thermal stability of DNA containing each adduct. Consistent with these data, a 21-base pair DNA substrate containing a centrally located single interstrand cisplatin cross-link resulted in less binding than to the undamaged control DNA. A series of experiments measuring rhRPA binding and concurrent DNA denaturation revealed that rhRPA binds duplex cisplatin-damaged DNA via the generation of single-stranded DNA. Single-strand DNA binding experiments show that rhRPA binds 3-4-fold better to an undamaged 24-base DNA compared with the same substrate containing a single 1,2d(GpG) cisplatin-DNA adduct. These data are consistent with a low affinity interaction of rhRPA with duplex-damaged DNA followed by the generation of single-stranded DNA and then high affinity binding to the undamaged DNA strand.     

Equilibrium and stop-flow kinetic studies of fluorescently labeled DNA substrates with DNA repair proteins XPA and replication protein A.

Nucleotide excision repair (NER) is a crucial pathway in the maintenance of genome stability requiring at least two dozen proteins. XPA and RPA have essential roles in the damage recognition step of NER. To better understand the mechanism of their interactions with DNA, we utilized equilibrium and stop-flow kinetic approaches with fluorescently labeled oligonucleotides. Fluorescein is a bona fide NER lesion because a circular plasmid with a single defined fluorescein was repaired by efficient extracts from Xenopus oocyte nuclei. Single-stranded and double-stranded oligonucleotides 5'-labeled with fluorescein were used in the subsequent studies. Oligonucleotide fluorescence was quenched upon specific binding to full-length recombinant Xenopus XPA (xXPA) and/or human RPA. The binding was highly sensitive to the buffer conditions. Analysis of equilibrium binding data with ds DNA and xXPA revealed a single dissociation constant (K(d)) of 24.4 nM. Stopped-flow kinetic experiments were described by a first-order on-rate constant k(on) of 9.03 x 10(8) M(-1) s(-1) and k(off) of 26.1 s(-1). From the ratio of off-rate to on-rate, a calculated K(d) of 28.9 nM was obtained, revealing that the kinetic and equilibrium studies were consistent. The affinity of xXPA for ds undamaged DNA determined in our spectrofluorometry experiments was up to 3 orders of magnitude higher than previously reported values using different substrates, conditions, and assays [gel-shifts (EMSA), filter-binding, anisotropy, and surface plasmon resonance]. The same substrate DNA containing a 4-bp mismatch in the middle yielded a K(d) five times higher (158 nM), indicating weaker binding by xXPA. The differences in K(d) values for these two substrates were mainly attributable to the k(on), rather than k(off) rates. Fluorescence intensity changes upon interaction of xXPA with ss 50-mer were too low to calculate an accurate K(d). Although recombinant human RPA binding to the ds 50-mer was very weak (K(d) > 1 mM), stop-flow and equilibrium measurements to ss oligonucleotide yielded K(d) values of 96 and 20.3 nM, respectively, which correlated with previously reported values using gel mobility shift assays and a similarly sized poly-dT. Equilibrium and stop-flow measurements to the cognate and mismatched ds oligonucleotides using both xXPA and hRPA yielded a 2- to 3-fold increase in the K(d).     

Photocrosslinking locates a binding site for the large subunit of human replication protein A to the damaged strand of cisplatin-modified DNA.

The repair proteins XPA, XPC and replication protein A (RPA) have been implicated in the primary recognition of damaged DNA sites during nucleotide excision repair. Detailed structural information on the binding of these proteins to DNA lesions is however lacking. We have studied the binding of human RPA (hRPA) and hRPA-XPA-complexes to model oligonucleo-tides containing a single 1, 3-d(GTG)-cisplatin-modification by photocrosslinking and electrophoretic mobility shift experiments. The 70 kDa subunit of hRPA can be crosslinked with high efficiency to cisplatin-modified DNA probes carrying 5-iodo-2"-deoxyuridin (5-IdU) as crosslinking chromophore. High efficiency crosslinking is dependent on the presence of the DNA lesion and occurs preferentially at its 5"-side. Examination of the crosslinking efficiency in dependence on the position of the 5-IdU chromophore indicates a specific positioning of hRPA with respect to the platination site. When hRPA and XPA are both present mainly hRPA is crosslinked to the DNA. Our mobility shift experiments directly show the formation of a stable ternary complex of hRPA, XPA and the damaged DNA. The affinity of the XPA-hRPA complex to the damaged DNA is increased by more than one order of magnitude as compared to hRPA alone.