Sites of UV-induced phosphorylation of the p34 subunit of replication protein A from HeLa cells.

Exposure of mammalian cells to UV radiation alters gene expression and cell cycle progression; some of these responses may ensure survival or serve as mutation-avoidance mechanisms, lessening the consequences of UV-induced DNA damage. We showed previously that UV irradiation increases phosphorylation of the p34 subunit of human replication protein A (RPA) and that this hyperphosphorylation correlated with loss of activity of the DNA replication complex. To characterize further the role of RPA hyperphosphorylation in the cellular response to UV irradiation and to determine which protein kinases might be involved, we identified by phosphopeptide analysis the sites phosphorylated in the p34 subunit of RPA (RPA-p34) from HeLa cells before and after exposure to 30 J/m2 UV light. In unirradiated HeLa cells, RPA-p34 is phosphorylated primarily at Ser-23 and Ser-29. At least four of the eight serines and one threonine in the N-terminal 33 residues of RPA-p34 can become phosphorylated after UV irradiation. Two of these sites (Ser-23 and Ser-29) are known to be sites phosphorylated by Cdc2 kinase; two others (Thr-21 and Ser-33) are consensus sites for the DNA-dependent protein kinase (DNA-PK); the fifth site (Ser-11, -12, or -13) does not correspond to the (Ser/Thr)-Gln DNA-PK consensus. All five can be phosphorylated in vitro by incubating purified RPA with purified DNA-PK. Two additional sites, probably Ser-4 and Ser-8, are phosphorylated in vivo after UV irradiation and in vitro by purified DNA-PK. The capacity of purified DNA-PK to phosphorylate many of these same sites on RPA-p34 in vitro implicates DNA-PK or a kinase with similar specificity in the UV-induced hyperphosphorylation of RPA in vivo.

RPA binds to DNA through its 70-kDa subunit (7). In vitro, the DNA binding activity of RPA is essential for DNA unwinding at the origin of replication. The p70 subunit alone, or SSB proteins from other species (8 -12), also can cause unwinding, but specific protein-protein interactions between RPA and polymerase ␣-primase and SV40 large tumor antigen are necessary for assembly of the initiation complex at the SV40 origin of replication and for DNA replication. Neither RPA-p70 alone nor heterologous SSBs substitute for RPA in these interactions (13). Thus, the RPA-p34 and RPA-p13 subunits are primarily responsible for the specificity of protein-protein interactions in DNA replication and possibly also in DNA repair. The RPA protein was shown to interact with XPA (XP group A) (14 -16), XPF-ERCC1 (XPF-excision repair cross-complementing rodent repair deficiency 1 (XP group F)) (17) and XPG (XP group G) (15,17) proteins in excision repair. RPA-p34 is phosphorylated in vitro by DNA-PK, a DNA-activated protein kinase that participates in double-strand break repair (18 -24). RPA also interacts with several transcription factors, including VP16, Gal4, and p53, and may serve as a link between transcription and DNA replication (25)(26)(27). The p53 tumor suppressor protein inhibits RPA's ability to bind to single-stranded DNA (25).
Posttranslational modification is a well recognized mechanism for modulating protein activity and protein-protein interactions. Several posttranslational modifications of the RPA subunits have been reported, including phosphorylation and poly(ADP)ribosylation (28,29), and differentially phosphorylated forms of RPA-p34 have been observed on the basis of retarded mobility on SDS-polyacrylamide gels. RPA-p34 is phosphorylated in a cell cycle-dependent manner in both human and yeast cells (28). Phosphorylation begins at the onset of S phase suggesting a possible role in regulating DNA replication. A mitosis-specific phosphorylated form of RPA-p34 also was observed. 2 We and others (30,31) previously showed that RPA-p34 is hyperphosphorylated upon exposure of cultured human cells to UV or ionizing radiation. This DNA damageinduced hyperphosphorylation is coincident with cell cycle arrest and loss of the ability of cell extracts to support DNA replication (30). These observations suggest that phosphorylation of RPA-34 serves as an essential mechanism for modulating RPA activity and its interactions with other proteins.
To understand the role of RPA phosphorylation in DNA replication, repair, and recombination, it is necessary to define characteristic phosphorylation sites in specific forms of RPA and to identify the enzyme activities responsible for their creation. This is particularly important because forms having similar migration on SDS gels may differ with respect to sites of phosphorylation and, therefore, in biological function.
Cyclin-dependent kinases and the DNA-dependent protein kinase (DNA-PK) were postulated to catalyze phosphorylation of RPA-p34 in the S phase of the cell cycle. It was shown that RPA-p34 can be phosphorylated in vitro by cyclin A/cyclin B-Cdc2 kinase complex (on Ser-23 and Ser-29) (32,33) and by DNA-PK (18,19,24,34). Interestingly, in mouse scid cells, which are mutated in the gene for the catalytic subunit of DNA-PK (DNA-PK CS ), diminished levels of hyperphosphorylation of RPA were observed after DNA damage, pointing to a role for DNA-PK CS in DNA damage-induced hyperphosphorylation of RPA-p34 (20).
Here, we describe an analysis of the sites phosphorylated in the hyperphosphorylated form of RPA-34, induced by UV irradiation. Several phosphopeptides generated from the UV-induced, hyperphosphorylated form of RPA were shown to be identical to phosphopeptides from HeLa or recombinant RPA phosphorylated in vitro by DNA-PK. Co-migrational phosphopeptide analysis identified Thr-21, Ser-23, Ser-29, Ser-33, Ser-11, -12, or -13, and probably Ser-8 and Ser-4 as sites of phosphorylation in the hyperphosphorylated form of RPA-p34 produced by exposure to UV light. The capacity of purified DNA-PK to phosphorylate many of these same sites on RPA-p34 in vitro implicates DNA-PK or a kinase with similar specificity in the UV-induced hyperphosphorylation of RPA in vivo.
Two-dimensional Phosphopeptide Mapping-32 P-Labeled proteins were extracted from dried SDS-PAGE gels and processed as described (42). For proteolytic digestion we used sequencing grade chymotrypsin purchased from Boehringer Mannheim. This source of chymotrypsin appears to be contaminated with trypsin activity; thus, our phosphopeptide maps are those expected from digestion with chymotrypsin and trypsin (chymotrypsin/trypsin). The same lot of chymotrypsin was used for all the phosphopeptide maps shown in this paper; consequently, comparisons are valid. After proteolytic digestion, peptides were separated on thin layer cellulose plates by electrophoresis at pH 1.9 in the first dimension and by ascending chromatography in isobutyric acid buffer in the second dimension (42). The radiolabeled phosphopeptides were visualized by autoradiography. Synthetic phosphopeptides were visualized after staining with ninhydrin. To improve the resolution of phosphopeptides, in some experiments the (second) chromatography dimension was performed in parallel in two different buffers (phosphochromatography buffer and isobutyric acid buffer (42)), which allowed us to resolve spots that comigrated or nearly comigrated when analyzed in only one chromatography buffer (data not shown).
Generation of Two-dimensional Peptide Mobility Prediction Maps-The two-dimensional peptide mobility prediction program Sortpeptide (provided generously by Dr. Tony Hunter, The Salk Institute) was used in conjunction with the University of Wisconsin's GCG software to generate predicted mobilities of phosphopeptides. The methods used in this program are described (42).
In Vitro Phosphorylation of RPA by DNA-PK-Purified RPA from HeLa cells was incubated with DNA-PK for 10 min at 30°C in "replication kinase buffer" containing 5 Ci of [␥-32 P]ATP and, where indicated, with sheared calf thymus DNA (5 g/ml). Replication kinase buffer has all the ingredients of in vitro replication buffer but has a lower concentration of ATP (50 M instead of 1 mM) and does not have an ATP-regeneration system (30). These changes were necessary to obtain adequate labeling of RPA. In vitro phosphorylated RPA-p34 was immunoprecipitated and fractionated by SDS-PAGE and processed for two-dimensional phosphopeptide mapping as described above.
Analysis of Phosphorylation of Synthetic Peptides-Phosphorylation of synthetic peptides I, II, and III was carried out in 20-l reaction mixtures containing 200 M peptide, 1 Ci of [␥-32 P]ATP, 50 mM Hepes, pH 7.5, 100 mM KCl, 10 mM MgCl 2 , 0.2 mM EGTA, 5 g/ml sonicated calf thymus DNA (where indicated), and 10 units of DNA-PK or replication extracts (final protein concentration 6 mg/ml). Reactions were incubated for 10 min at 30°C and stopped by adding an equal volume of 30% acetic acid. The incorporation of radioactivity into the peptides was determined using an anion exchange resin, as described by Pearson et al. (43). For two-dimensional phosphopeptide analysis and phosphoamino acid analysis, peptides were recovered by vacuum-assisted evaporation of acetic acid (Speed Vac, Savant Instruments Inc.) and subjected to two-dimensional and phosphoamino acid analysis as described above.

RESULTS
Phosphopeptide Analysis of UV-induced Hyperphosphorylated RPA-p34 from HeLa Cells-Irradiation of cultured cells with UV light induces hyperphosphorylation of RPA-p34 (30). Western immunoblot analysis of RPA-p34 from HeLa cells 8 h after mock exposure (Fig. 1, lane 1) reveals that the rapidly migrating forms 1 and 2 are prevalent. In contrast, in a lysate from UV-irradiated HeLa cells (30 J/m 2 ), additional phosphorylated forms that migrate more slowly (forms 3-5) are observed (Fig. 1, lane 2). Form 1 is the unphosphorylated form of RPA-p34 since it is absent on autoradiograms of RPA-p34 labeled in vivo with [ 32 P]orthophosphate (30).
To determine the sites of phosphorylation on the slower migrating forms, we conducted two-dimensional analysis of chymotryptic/tryptic phosphopeptides of the in vivo radiolabeled p34 subunit of RPA. gel containing the individual forms were excised, and the proteins were extracted, digested with chymotrypsin/trypsin, and resulting phosphopeptides subjected to two-dimensional analysis (Fig. 3). Comparison of the maps in Fig. 3, A-C, shows that the main phosphopeptides of the faster migrating forms 2 and 3 of RPA-p34 from unirradiated cells (Fig. 3A) are essentially the same as the sum of those from form 2 (Fig. 3B) and form 3 ( Fig. 3C) from UV-irradiated cells. The fastest migrating form (form 2) of UV-irradiated RPA-p34 ( Fig. 3B) has two main phosphopeptides, marked A and B, and two minor phosphopeptides, marked X and Y. The map of form 3 (Fig. 3C) has phosphopeptides A and B but not phosphopeptide X and Y. Three other phosphopeptides, designated C, X*, and Y*, are also present; phosphopeptide C is not present in form 2 (Fig. 2B). Fig. 3D shows the phosphopeptide map of the slowest migrating hyperphosphorylated form of RPA-p34 (form 5) from UV-irradiated cells. Many additional phosphopeptides are observed in form 5 in comparison with the faster migrating forms of RPA-p34. In the form 5 map, we observed the highly labeled phosphopeptide marked p4 migrating slightly below phosphopeptide A. At the left side of the map, the most highly labeled phosphopeptide is marked as pp6, and there are about 9 other minor phosphopeptides to the left of the origin. In the lower right side of the map four additional phosphopeptides (marked p7, p7a, pp7, and pp7a) are evident.
Identification of Phosphopeptides-To assign specific spots to specific peptides of the RPA-p34 amino acid sequence (37), we generated predicted chymotryptic/tryptic phosphopeptide maps of RPA-p34 using computer software described in Boyle et al. (42) and confirmed these assignments by using synthetic phosphopeptide markers. For this analysis, we focused on amino acid residues 1-38 because Henricksen et al. (19) and Lee and Kim (44) showed that RPA-p34 phosphorylation occurs primarily in this N-terminal region. The sequences of the peptides expected to be generated by chymotrypsin/trypsin digestion of these first 38 amino acids are shown in Fig. 4; Fig. 5A shows the predicted map of the phosphopeptides. We included in the map the partial trypsin digestion products, where peptide 7 with the additional basic residue of Lys-38, marked as 7a, has an additional positive charge, resulting in a characteristic shift in the migration to the direction of cathode (compare peptides p7 and p7a). Adding phosphate groups to a peptide makes it more negatively charged and less hydrophobic so the peptide migrates further toward the anode and less far in the chromatography dimension (for example, compare phosphopeptides p7 and pp7). Since chymotrypsin, cleavage may be inhibited by phosphorylation of serine or threonine two amino acids C-terminal to the cleavage site (45,46), phosphorylation would be expected to inhibit cleavage between peptides 1 and 2, 2 and 3, 3 and 4, and 6 and 7. Therefore, we also generated predicted map positions for these incomplete digestion products of peptides 6 and 7 (Fig. 5B).
From a comparison of predicted maps and the authentic map of fast migrating forms 2 and 3 of RPA-p34, we concluded that peptides X, Y, X*, and Y* are singly and doubly phosphorylated peptides resulting from partial digestion of amino acid residues 21-37/38 (peptides p(6 ϩ 7), p(6 ϩ 7a), pp(6 ϩ 7), and pp(6 ϩ 7a), respectively). In the faster migrating form 2, singly phosphorylated peptides X and Y are present; in form 3, additional phosphorylation on the second site retards migration in both dimensions. From these results we also conclude that in form 2, phosphorylation of Ser-29 inhibits cleavage by chymotrypsin after Phe-27. In form 3, a second phosphorylation site remains to be assigned. Since Ser-29 is a characteristic Cdc2 kinase phosphorylation site, it seems probable that the second phosphorylation will be on Ser-23 which has the same sequence characteristics as Ser-29. Recently Niu et al. (24) have shown that in vitro Ser-29 is the major site phosphorylated in RPA-p34 by Cdc2-cyclin B kinase. The phosphorylated serine and threonine residues within each of the predicted peptides are indicated by the letter P. The numerals above the sequence indicate the number assigned to the chymotryptic/tryptic peptide. Each phosphorylation event on the peptide is marked with a letter p (e.g. p6 means peptide 6 phosphorylated once, pp6 means peptide 6 phosphorylated twice). Italics indicate phosphopeptides that were used in two-dimensional analysis as cold markers (chemically synthesized unlabeled phosphopeptides). Addition of phosphate to a peptide makes it more negatively charged and less hydrophobic; thus, it migrates further toward the anode and less far in the chromatography dimension (for example, compare phosphopeptides p7 and pp7). Arrows and italics indicate phosphopeptides used as internal cold markers. therefore, it may be phosphorylated on three sites or it may represent a mixture of two or more species that are doubly or singly phosphorylated on different sites. Additional (second) phosphorylations on peptide X/Y (marked as X*/Y* in Fig. 3) and phosphopeptide C (most probably a doubly phosphorylated phosphopeptide from region B) are characteristic of the more highly phosphorylated form 3, since they are not present in form 2. The peptide map presented in Fig. 3A represents the mixture of form 2 and form 3 of RPA-p34 from unirradiated cells. All phosphopeptides present in form 2 and form 3 of RPA-p34 from UV-irradiated cells are present in this map, indicating that both forms taken together are phosphorylated on the same set of sites.
Phosphorylation of Synthetic Peptides Derived from the RPA-p34 Sequence-We showed above that the UV-induced, hyperphosphorylated form of RPA-p34 is phosphorylated on at least five sites. Two of these sites, Ser-23 and Ser-29, are putative Cdc2 kinase phosphorylation sites (32,33) and two others, Thr-21 and Ser-33, have the characteristic (Ser/Thr)-Gln site for DNA-PK (47,48). The fifth site lies in peptide 4, which contains three serines, and shows some resemblance to the rat Fos amino acid sequence (peptide Arg-359 to Leu-379) (47). To further investigate the specificity of phosphorylation of peptide 4, we examined the phosphorylation of synthetic peptides derived from the RPA-p34 sequence in cell extracts and by purified DNA-PK. We have shown that in vitro replication extracts of HeLa cells can hyperphosphorylate RPA-p34 (30) in a DNAdependent manner (Fig. 6).
Among these synthetic peptides, peptide I which contains peptide 4 was most highly phosphorylated in extracts from both untreated and UV-irradiated cells and showed DNA dependence of phosphorylation (Fig. 6). Peptide II which contains a DNA-PK consensus site showed much lower phosphorylation (7-fold lower) but phosphorylation also was activated by DNA. Peptide III was not a substrate for phosphorylation under these conditions (Fig. 6). The higher incorporation of radioactivity into peptide I may result from phosphorylation at several sites. We also used these peptides as substrates for purified DNA-PK (Fig. 6C). Peptides I and II were good substrates for DNA-PK, whereas peptide III was not phosphorylated by the enzyme. Relative to peptide I, peptide II was a better substrate for DNA-PK than for DNA-dependent kinase activities present in the cell extracts.
Next we compared by two-dimensional analysis phosphopeptides generated by chymotrypsin digestion of peptide I phos-phorylated by purified DNA-PK and in in vitro replication extracts by endogenous kinases. Chymotrypsin digestion of peptide I was expected to generate four smaller peptides, three of which contain possible phosphorylation sites (Figs. 4 and 7H). Two-dimensional analysis of products obtained after digestion of peptide I phosphorylated by DNA-PK demonstrates that this enzyme can efficiently phosphorylate serines on at least two of the smaller peptides (Fig. 7A), one of which is p4 since it co-migrates with "cold marker" phosphopeptide p4. Only one serine residue is phosphorylated on peptide 4 since there is no spot on the map that could correspond to a multiply phosphorylated peptide 4 (Fig. 7, B and H). The second phosphopeptide is probably p3 (spot p3). The spot above p3 is probably p2 or perhaps peptide p(2 ϩ 3) (due to inhibited cleavage after Phe-6). We confirmed by phosphoamino acid analysis that serine but not tyrosine was phosphorylated on peptide I (data not shown). Two-dimensional analysis of phosphopeptides obtained in extracts prepared from untreated and UV-irradiated cells demonstrated that the same sites are phosphorylated on peptide I by the protein kinase activity(ies) in the extracts that are stimulated by DNA. However, it appears that p2 is more efficiently phosphorylated in cell extracts than by purified DNA-PK (Fig. 7, B-F).
In Vitro Phosphorylation of RPA Complex or RPA-p34 by DNA-PK-Since two of the phosphorylation sites identified in the UV-induced hyperphosphorylated form of RPA-p34 (i.e. Thr-21 and Ser-33) are consensus sequences ((Ser/Thr)-Gln) for phosphorylation by DNA-PK, it was of interest to investigate whether the same sites are phosphorylated by DNA-PK on the RPA-protein complex purified from HeLa cells. Kinase-free RPA complex was incubated in replication kinase buffer (to simulate in vitro replication conditions in which RPA is hyperphosphorylated). After incubation, RPA-p34 was immunoprecipitated and subjected to SDS-PAGE; the hyperphosphorylated form (Fig. 8A, band 5) was extracted from the gel and subjected to phosphopeptide analysis. The pattern of phosphopeptides seen in the resulting map (Fig. 8A) is very similar to the pattern of phosphopeptides from the UV-induced form 5 of RPA-p34 (Fig. 3D); the major spots are p4, B, X*,Y*, pp7, and pp7a. Singly phosphorylated p7 and p7a can also be seen, as can phosphopeptide pp6. From the above results, we conclude that DNA-PK can phosphorylate in vitro the same subset of sites on RPA-p34 that are phosphorylated in vivo in the UVinduced hyperphosphorylated form of RPA-p34. Among these sites are Thr-21 and Ser-23 (on pp6), Ser-29 and Ser-33 (on FIG. 6. Comparison of the ability of in vitro replication extracts and DNA-PK to phosphorylate synthetic peptides generated from the RPA-p34 sequence. Phosphorylation of synthetic peptides by in vitro replication extracts from untreated (A), UV-irradiated cells (B), and by purified DNA-PK (C) was carried out as described under "Materials and Methods." Sheared calf thymus DNA was included as indicated in the legend. Radiolabeled peptides were purified by passing them through a column with AG 1-X8 anion exchange resin. Incorporated radioactivity was quantified by Cerenkov counting.
Since we detected phosphorylation of the RPA complex by DNA-PK on RPA-p34 sites Ser-23, Ser-29, and Ser-11, -12, or -13 that are not consensus sites for DNA-PK, we wanted to verify that the observed pattern of phosphorylation of RPA-p34 depended on the DNA-PK enzyme. Therefore we used the p34 subunit of RPA expressed in bacteria as a substrate for DNA-PK to rule out the possibility that our RPA complex was contaminated by kinases or kinase activators. Recombinant RPA-p34 was purified and incubated in replication kinase buffer with DNA-PK, and phosphopeptides from the hyperphosphorylated form (Fig. 8B, band 5) were analyzed. Fig. 8B shows the resulting phosphopeptide map, which is very similar to phosphopeptide maps of the hyperphosphorylated form generated in vitro by the action of DNA-PK on RPA complex (Fig.  8A), and the hyperphosphorylated form 5 of RPA-p34 induced by UV-irradiation of HeLa cells (Fig. 3D). From these results we conclude that UV-induced hyperphosphorylation of RPA-p34 in HeLa cells may result from phosphorylation by DNA-PK or another DNA-activated enzyme with very similar phosphorylation specificity. DISCUSSION Previous studies established that RPA-p34 is phosphorylated in a cell cycle-dependent manner in both yeast and mammalian cells (28,49) beginning at the transition of G 1 to S phase and continuing until mitosis. Consistent with these in vivo studies, it was demonstrated that RPA-p34 is phosphorylated in vitro during SV40 T antigen-dependent DNA replication in a cell-free system (30,50). RPA-p34 also becomes hyperphosphorylated in mammalian cells in response to DNA damage-inducing agents, such as UV or ionizing radiation (30,31) in parallel with the arrest of DNA synthesis (30). Similar hyperphosphorylation of RPA-p34 was observed in vitro during DNA replication in extracts from HeLa cells (30). Extracts from UV-irradiated cells that exhibited reduced replication competency hyperphosphorylated RPA-p34 to higher levels than did extracts from non-irradiated cells (30).
Our studies indicate that at least three forms of RPA-34 (forms 1-3) from normally cycling HeLa cells are distinguishable by SDS-PAGE; form 1 is unphosphorylated, and forms 2 and 3 have increasing levels of phosphorylation. Hyperphosphorylated forms 4 and 5 have not been detected during the normal cell cycle and are detected only after exposing cells to DNA damaging agents (30,31) or inhibitors of DNA replication (e.g. aphidicolin, the replicative polymerase inhibitor, or mimosine, which causes depletion of dGTP and dATP (data not shown)). RPA-p34 also was hyperphosphorylated when cells were labeled for extended periods with 32 PO 4 , presumably due to induction of DNA damage by 32 P decay (data not shown and Ref. 20). We have not ruled out the possibility that in a normal cell cycle hyperphosphorylated forms 4 and 5 are present but are very short lived and, therefore, not easily detectable. Alternatively, the hyperphosphorylated forms of RPA-p34 may be specific for interrupted DNA replication and have no function in the normal cell cycle. Testing these possibilities will require additional investigation.
Since different phosphorylated forms of RPA-p34 may have different functions, we considered it crucial to characterize their states of phosphorylation. The primary aim of the present study was to identify the sites of RPA-p34 phosphorylation in RPA from HeLa cells exposed to UV radiation or not. By using two-dimensional analysis of phosphopeptides, we found that form 2 and form 3 from both control and UV-irradiated cells had the same pattern of phosphopeptides.
The phosphopeptide map of the hyperphosphorylated UVinduced form 5 of RPA-p34 revealed additional phosphorylation sites compared with forms 2 and 3: Thr-21 and Ser-23 on phosphopeptide pp6, Ser-29 and Ser-33 on phosphopeptide pp7a, and one of the three serines (Ser-11, Ser-12, or Ser-13) on phosphopeptide p4. Also, the possible presence of additional Our observation that DNA-PK was able to hyperphosphoryl- FIG. 8. Two-dimensional analysis of phosphopeptides of RPA-p34 phosphorylated in vitro by DNA-PK. Purified RPA complex from HeLa cells (A) or RPA-p34 expressed in bacteria (B) was incubated with DNA-PK as described under "Materials and Methods." Radiolabeled RPA-p34 was immunoprecipitated with RPA-p34 specific monoclonal antibody and electrophoresed on 12% SDS-PAGE. The hyperphosphorylated form 5 was extracted from the gel slice, digested with chymotrypsin/trypsin, and the resulting phosphopeptides were resolved in two dimensions by electrophoresis at pH 1.9 in the horizontal direction with the cathode on the right, followed by ascending chromatography in isobutyric acid buffer. The radiolabeled phosphopeptides were detected by autoradiography, and synthetic cold markers were visualized by staining with ninhydrin. Phosphopeptides are marked by numbers according to Fig. 4. Each phosphorylation event on the peptide is marked with the letter p.
Previous studies identified Ser-Gln and Thr-Gln sequences as potential sites of DNA-PK-mediated phosphorylation (47). In RPA-p34, four serines (Ser-33, Ser-52, Ser-72, and Ser-174) and one threonine (Thr-21) are in this context. However, all phosphorylation of RPA-p34 by DNA-PK was shown to occur on N-terminal 33 amino acids of RPA-p34 (19). Of the nine phosphorylatable amino acids (eight serines and one threonine) present in the first 33 amino acids of RPA-p34, only two (Thr-21 and Ser-33) are in the (Ser/Thr)-Gln context that appears to be favored by DNA-PK. Our results demonstrate that in vitro DNA-PK phosphorylates RPA-p34 on both context sites and on at least three additional sites: Ser-23, Ser-29, and Ser-11, -12, or -13 that are not in a (Ser/Thr)-Gln context. The context of two of these additional sites, Ser-23 and Ser-29, is similar to that of the sites phosphorylated by DNA-PK in the C-terminal domain of RNA polymerase II (47). The amino acid sequence surrounding the phosphorylation site on p4 (Ser-11, Ser-12, or Ser-13) resembles the sequence in the rat Fos protein (peptide Arg-359 to Leu-379) that was shown in vitro to be phosphorylated by DNA-PK (47). From the above discussion, we conclude that DNA-PK phosphorylates RPA-p34 on both consensus and non-consensus sites in vitro. Studies with synthetic peptides modeled on the amino acid sequence of the first 21 amino acids of RPA-p34 will be required to identify sequence elements responsible for the ability of DNA-PK to phosphorylate RPA-p34 at Ser-23, Ser-29, or Ser-11, -12, or -13.
The biological function of hyperphosphorylation of RPA-p34 is obscure. Hyperphosphorylation of RPA-p34 in vivo has been detected only when DNA replication was interrupted by damage to the DNA template or after inhibition with certain drugs. Hyperphosphorylation of RPA-p34 in vivo after UV irradiation correlates with an arrest of DNA synthesis (30). Cell extracts prepared from UV-irradiated cells have low replication competence and hyperphosphorylated RPA; addition of unphosphorylated RPA to these extracts restores replication competence, which correlates with an increase in phosphorylated form 2 and form 3 (30). These results suggest that hyperphosphorylated form 5 of RPA-p34 may be incompetent for DNA replication but not inhibitory for the competency of other forms of RPA. Also, phosphorylated forms 2 and 3 may be required for DNA replication. Two other investigations addressed the significance of RPA-p34 hyperphosphorylation in replication and repair (19,51). However, since they were using mixtures of phosphorylated forms, no firm conclusions about the replication competency of RPA complex containing form 5 of RPA-p34 can be drawn. A clear answer to the functional significance of hyperphosphorylation of RPA will require in vitro testing of the individual phosphorylated forms (with identified sites of phosphorylation) under conditions where phosphorylation/dephosphorylation can be controlled and monitored.
Recently, a homolog of RPA-p34, called RPA4, was described (46). Interestingly, it was found to be expressed in quiescent cells. Comparison of the amino acid sequences of RPA-p34 and RPA4 reveals that the least homology is found in the N-terminal portion of the proteins. The N terminus of RPA4 lacks three sites that we found to be phosphorylated in RPA-p34 after UV irradiation of cells and in vitro by DNA-PK. This observation suggests that hyperphosphorylation of the N-terminal portion of this subunit of RPA may not be needed in quiescent cells, since they do not go through the cell cycle and do not replicate their DNA. In the event of DNA damage, they would not need a means of signaling for a delay in cell cycle progression. This finding once more highlights the importance of hyperphosphorylation of RPA-p34 in cells undergoing replication.