DNA damage-induced accumulation of Rad18 protein at stalled replication forks in mammalian cells involves upstream protein phosphorylation

BBRC Biochemical and Biophysical Research Communications 323 (2004) 831–837 www.elsevier.com/locate/ybbrc

DNA damage-induced accumulation of Rad18 protein at stalled replication forks in mammalian cells involves upstream protein phosphorylationq Andrey Nikiforova, Maria Svetlovaa, Lioudmila Solovjevaa, Lioudmila Sasinab, Joseph Siinoc, Igor Nazarovc, Morton Bradburyc,d, Nikolai Tomilina,c,* b

a Institute of Cytology, Russian Academy of Sciences, Tikchoretskii Av. 4, 194064 St. Petersburg, Russia Institute of Experimental Medicine, Russian Academy of Medical Sciences, Pavlov Str. 12, 197376 St. Petersburg, Russia c Department of Biological Chemistry, UC Davis School of Medicine, Davis, CA 95616, USA d Bioscience Division, MSM 888, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA

Received 21 August 2004

Abstract Rad18 protein is required for mono-ubiquitination of PCNA and trans-lesion synthesis during DNA lesion bypass in eukaryotic cells but it remains unknown how it is activated after DNA damage. We expressed GFP-tagged human (h)Rad18 in Chinese hamster cells and found that it can be completely extracted from undamaged nuclei by Triton X-100 and methanol. However, several hours after treatment with methyl methanesulfonate (MMS) Triton-insoluble form of GFP–hRad18 accumulates in S-phase nuclei where it colocalizes with PCNA. This accumulation is suppressed by inhibitors of protein kinases staurosporine and wortmannin but is not effected by roscovitine. We also found that methyl methanesulfonate induces phosphorylation of Ser-317 in protein kinase Chk1 and Ser-139 in histone H2AX and stimulates formation of single-stranded DNA at replication foci. Together, our results suggest that MMS-induced accumulation of hRad18 protein at stalled forks involves protein phosphorylation which may be performed by S-phase checkpoint kinases.
2004 Elsevier Inc. All rights reserved. Keywords: DNA lesion bypass; Methyl methanesulfonate; Human Rad18 protein; Insolubilization

Ultraviolet light and some chemical agents induce DNA lesions which can be eliminated by nucleotide excision repair [1,2]. This repair strongly decreases initially induced number of lesions but some of them enter replication and block it, since high fidelity DNA polymerases cannot insert normal nucleotides opposite damaged nucleotides in template strand [3,4]. To restore replication such lesions should be bypassed and in q Abbreviations: PRR, postreplication repair; MMS, methyl methanesulfonate; GFP, green fluorescent protein; TLS, trans-lesion synthesis; PCNA, proliferating cell nuclear antigen. * Corresponding author. Fax: +1 530 752 3516, +7 812 247 0341. E-mail address: [email protected] (N. Tomilin).

0006-291X/$ - see front matter
2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.08.165

eukaryotic cells two possible mechanisms of this bypass or postreplication repair (PRR) were identified: (1) through DNA polymerase switch when high-fidelity replicative DNA polymerase is transiently replaced by a special DNA polymerase which is able to perform trans-lesion synthesis, TLS [5–7], (2) through template switch when replication fork reversal and formation of a Holliday junction (HJ) allow blocked DNA polymerase to use undamaged daughter strand as template [8,9]. In yeast Saccharomyces cerevisiae lesion bypass is controlled by members of the Rad6 gene epistasis group which contains genes Rad18, Rad5, Mms2, Ubc13, Rad30, and Rev3 [10,11], and also depends on Pol30 gene encoding replication accessory protein PCNA

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[12]. Rad6p is a ubiquitin-conjugating enzyme forming stable complex with ring-finger protein Rad18p [13]. PCNA is mono-ubiquitinated by the Rad6p/Rad18p complex [14] and this modification is required for TLS [15]. Human homolog of yeast Rad18 gene is identified and it has been shown that human (h)Rad18 protein interacts with the human homologs of the Rad6 protein (HHR6A and HHR6B) and is involved in PRR [16,17]. Inactivation of Rad18 gene in mice leads to sensitivity of cells to UV and methyl methanesulfonate (MMS) and to an increased frequency of homologous recombination [18]. Rad18-dependent mono-ubiquitination of PCNA in UV-irradiated human cells stimulating its ability to bind trans-lesion DNA polymerase g is recently demonstrated [19]. However, how mammalian Rad18 protein is activated after DNA damage remains unknown. Complex genetic control of PRR indicates that the lesion bypass is not a result of simple competition between different polymerases for blocked sites but is a tightly regulated process involving many proteins and their modifications and may also involve intra-S-phase checkpoint pathways induced by stalling of replication forks [20]. For example, Chk1 kinase is working in DNA damage checkpoints as downstream effector of ATR (Ataxia telangiectasia mutated and Rad3-related) kinase [21,22] which accumulates at stalled replication foci [23]. PRR in mammalian cells is sensitive to caffeine [24] which is also known as an inhibitor of the ATR–Chk1 pathway [25]. In this study we constructed plasmid encoding GFP-tagged hRad18 and analyzed dynamic changes of its localization and mobility in stably transfected Chinese hamster cells. The results obtained suggest that accumulation of this protein at stalled replication forks after DNA damage requires upstream protein phosphorylation.

Materials and methods Cells, transfections, and treatments. Chinese hamster V79-4 lung fibroblasts and human A539 cells were obtained from ATCC. Cells were cultivated in RPMI-1640 medium supplemented with 10% of fetal calf serum. Transfections with indicated plasmids were done using TRANS-FAST reagent (Promega) and stable expressing clones were isolated by selection in growth medium containing 1 mg/ml G418. Expressing clones were identified by GFP fluorescence and then expanded. Treatments of cells with methyl methanesulfonate (MMS, Aldrich) were done by addition of this drug to cells in growth medium at indicated concentration for 1 h. Construction of plasmids encoding GFP-tagged proteins. DNA fragments containing cDNAs of human Fen1 were amplified from human poly(A)-containing RNA using single-tube Titan RT-PCR Kit (Roche). Poly(A)-containing RNA was isolated from cultivated human A539 cells using mRNA isolation Kit (Roche). DNA fragment containing full hRad18 cDNA was amplified using as template plasmid obtained from S. Tateishi. Sequences of primers used to amplify human Rad18 and human Fen1 cDNAs, respectively, hRAD18-N1: GACTCCCTGGCCGAGTCTC; hRAD18-N2: CAAAGCTGG TACCTGTGTGAAATGTC; FEN-C1: CTGTGTTGCCATGGGA

ATTC; FEN-C2: TTCCCCTTTTAAACTTCCCTGC. For construction of expression plasmids we used N-terminal (hRad18) and C-terminal (Fen1) fusion GFP-TOPO vectors from Invitrogen and chemically competent TOP10 cells. Individual recombinant clones were screened by PCR and presence of expected inserts was confirmed by sequencing. Plasmid DNAs were isolated using a Qiagen Kit. Construction of plasmids encoding GFP-tagged histone H2AX and its mutated variants was described earlier [26]. Immunofluorescence analysis of PCNA in fixed cells. Before fixation for 10 min in 4% formaldehyde in PBS at 4 C cells were washed in PBS and then extracted for 20 min at RT in cytoskeleton (CSK) buffer containing 10 mM Pipes, pH 6.8, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM dithiothreitol, 1 mM EGTA, 0.5% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 5 lg/ml aprotinin, and 5 lg/ml leupeptin, then washed in PBS, and extracted again for 20 min at
20 C in 100% methanol. PCNA was visualized as described earlier [27] using mouse monoclonal antibodies PC-10 (Santa Cruz, 1:100, 30 min), biotinylated sheep-anti-mouse IgG (Sigma, 1:100), and avidin–Texas Red (Vector, 1:200). Mouse monoclonal antibodies against 5-bromodeoxyuridine used in detection of single-stranded DNA in situ were obtained from Roche. Immunoblotting. After washing with PBS and pre-extraction with CSK buffer and methanol as described in previous section cells were collected using rubber policeman and resuspended in lysis buffer containing 50 mM Tris–HCl buffer, pH 8, 150 mM NaCl, 1% Nonidet P-40, 1% deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1 mM EDTA, 1 mM PMSF, 5 lg/ml aprotinin, and 5 lg/ml leupeptin. After 30–60 min incubation at 4 C with occasional vortexing lysates was centrifuged 10 min at 9000g at 4 C and concentration of total protein was measured in supernatants using BCA Kit (Roche) and adjusted to be the same in different probes. Then equal volumes of 2· Laemmli buffer (50 mM Tris–HCl, pH 6.8, 0.8 mM EDTA, 4% SDS, 0.25% bromphenol blue, 20% glycerol, and 200 mM dithiothreitol) were added to the probes, and incubated for 5 min at 96 C, and centrifuged for 1 min at 14,000g and proteins present in supernatants were separated in denaturing polyacrylamide gels (7.5–12%). After transfer of proteins to nitrocellulose filter (Hybond C) in the buffer containing 25 mM Tris, 192 mM glycine, and 20% methanol for 16 h at 30 V, filter was blocked by incubation for overnight in TBS buffer (20 mM Tris–HCl, pH 8, 150 mM NaCl, and 0.1% Tween 20) containing 5% of non-fat dried milk. All antibodies were diluted in TBS plus 1% milk and incubations were carried out for 1 h at room temperature and washings in TBS. Primary mouse monoclonal antibodies against GFP were from Clontech (JL-8, 1:1000) and against PCNA—from Santa Cruz (PC-10, 1:1000). Secondary antibodies (sheep-anti-mouse IgG conjugated with horseradish peroxidase) were from Amersham and signal was detected using ECL Kit. Affinity purified rabbit polyclonal antibodies against phosphorylated Ser-317 of human Chk1 were obtained from Bethyl Laboratories and mouse monoclonal antibodies against pS139 of histone H2AX (clone JBW301) were obtained from Upstate. Microscopy. Images of fixed and living cells have been obtained using a Zeiss confocal laser scanning system 510 or a Zeiss LSM5 Pascal system equipped with Plan-NEOFLUAR 63/1.3 objective, helium–neon laser of 543 nm wavelength, and/or argon laser of 458/ 488 nm wavelengths.

Results Expression of human GFP–hRad18 protein in undamaged Chinese hamster cells We established stable clones of Chinese hamster V79 cells expressing GFP–hRad18 which were selected in the

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proteins remaining after extraction changes dramatically compared to unextracted cells. It should be noted that GFP–hRad18 is also completely extracted from undamaged S-phase cells containing foci of Triton-insoluble PCNA (see next sections) which is known to be associated with replication foci [28], indicating that hRad18 is not tightly bound to the normal replication machinery. Methyl methanesulfonate treatment induces accumulation of Triton X-100-insoluble form of GFP–hRad18 protein Fig. 1. Expression of GFP–hRad18 protein in stable clone of Chinese hamster cells detected by fluorescent microscopy (A) and by immunoblotting with antibodies against GFP (B). (C) Coomassie-stained gel with samples used in Western blotting (B). Bottom image in (A), right lanes in (B,C) show results obtained with cells extracted with CSK buffer containing 0.5% Triton X-100 and 100% methanol. In (A) cells were fixed with 4% formaldehyde before (top) or after extraction (bottom). Letters U and E mean unextracted and extracted variants, respectively.

growth medium containing G418. These clones show normal proliferation rate and both diffuse and focal distribution of GFP is observed in the nuclei (Fig. 1A, top image). Analysis of cell lysates of these clones using Western blotting with antibodies against GFP showed the presence of a major protein band (Fig. 1B, left lane). Apparent molecular mass of this band was higher than expected for the fusion of GFP with hRad18 protein but we believe that the detected band is actually GFP– hRad18, since the presence of fusion transcripts of expected size was detected in indicated clones by RTPCR and sequencing (not shown), and since protein band of identical size was also detected by immunoblotting in transiently transfected cells. Increased apparent size of GFP–hRad18 can be caused by posttranslational modifications of this protein or its anomalous migration in polyacrylamide gels but it is unlikely that this increase is caused by rearrangements of GFP–hRad18 plasmid after its integration into chromosome. When an accessory factor of DNA polymerase d PCNA is recuited to the sites of DNA synthesis in undamaged S-phase mammalian cells it becomes resistant to extraction with a buffer containing Triton X100 [28]. This protein also changes its extractability in non-S cells after UV-irradiation [29] consistent with its established involvement in nucleotide excision repair [30]. Similar behavior after DNA damage shows two other NER proteins XPA [31] and CSA [32]. We examined extractability of GFP–hRad18 from undamaged Chinese hamster cells. It is seen from Figs. 1A and B that GFP–hRad18 protein is almost completely extracted from undamaged cells by 0.5% Triton X-100 and methanol, indicating that in the absence of DNA damage GFP–hRad18 is not stably bound to the nucleus. As can be seen from Fig. 1C, spectrum of cellular

Like UV-irradiation, damage of mammalian cells by alkylating agent MMS inhibits DNA synthesis and significant slowing of replication fork movement and a high rate of fork stalling is directly demonstrated [33] using DNA fiber immunofluorescence. This inhibition leads to accumulation of S-phase cells with stalled replication foci containing colocalized PCNA and DNA polymerase g and significant increase of such cells is detectable at 2–16 h after MMS treatment [34]. To study whether DNA damage effects GFP–hRad18 extractability, we analyzed whether this protein remains

Fig. 2. Analysis of GFP–hRad18 and phosphorylation of histone GFP–H2AX after treatment of cells stably expressing these proteins with methyl methanesulfonate (MMS). In all variants treatment with 0.01% MMS was for 1 h and then cells were incubated in growth medium for 5 h. In (A) and (B) cells were pre-extracted with CSK buffer and methanol. Staurosporine (ST, 200 nM) and wortmannin (WM) were added before MMS and then present during MMS treatment and incubations. Right plot in (A) show results of densitometry from two independent experiments, mean ± SD. GFP– hRad18 was detected using antibodies against GFP (Clontech), PCNA (A) was detected using specific mouse antibodies PC-10 (Santa Cruz Co.). (C) shows induction of phosphorylation of GFP–H2AX in Chinese hamster clones expressing GFP fusions of normal human H2AX (WT) or mutated H2AX variant with replacement of Ser-139 for alanine. Blot was probed with monoclonal mouse antibodies against c-H2AX obtained from Upstate Biotech Co. (D) shows induction of Chk1 phosphorylated at Ser-317, blot was probed with specific antibodies obtained from Bethyl Co. In all lanes of one and the same blot equal amount of total protein (measured using Bradford reagent) was loaded.

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soluble in Triton X-100 and methanol after MMS. Endogenous PCNA is used as an internal marker of gel loading and protein solubility. It is seen from Fig. 2A that in contrast to the control undamaged cells GFP– hRad18 from MMS-treated cells is poorly extracted with Triton X-100 and methanol, indicating that MMS induces accumulation of a form of GFP–hRad18 which is resistant to extraction by the indicated solvents. It is also found that an inhibitor of protein phosphorylation staurosporine (ST, 200 nM) suppresses MMS-induced accumulation of GFP–hRad18 (Fig. 2A). This result correlates with our earlier observations that staurosporine inhibits postreplication repair in human cells [35] and suggests that the indicated accumulation may be functionally important. Using immunoblotting we also detected an increase of total GFP–hRad18 protein after treatment of cells with MMS (data not shown). It is unlikely that this increase is caused by stimulation of expression of GFP– hRad18 fusion gene which is under control of CMV promoter in the plasmid used here but it is possible that MMS-induced changes of the overall amount of GFP–hRad18 and its extractability are caused by DNA damage-dependent phosphorylation of this protein (Nikiforov et al., unpublished observations). Triton/methanol-insoluble form of GFP–hRad18 in MMS-treated S-phase cells associates with Triton-insoluble PCNA As can be seen from Fig. 3A, in the absence of DNA damage PCNA-positive (S-phase) cells are free of extraction-resistant GFP–hRad18 but several hours after MMS treatment S-phase nuclei also contain GFP–hRad18 (Fig. 3A), indicating that MMS-induced Triton-insoluble form of GFP–hRad18 accumulates in S-phase cells. Consistent with biochemical results (Fig. 2A) this accumulation is strongly suppressed by

200 nM staurosporine (Figs. 3A and B). Colocalization of GFP and PCNA signals suggests that GFP–hRad18 actually accumulates at stalled replication foci. Quantitation of these cytological observations with normalization of GFP–hRad18 signal relative to PCNA signal (Fig. 3B) confirms results obtained using Western blotting (Fig. 2A). It should be noted that in contrast to UV-irradiation which induces Triton-insoluble PCNA in G1/G2 cells [29] in MMS-treated cells we did not observe the formation of Triton-insoluble PCNA in non-S-phase cells under our experimental conditions. Together, the results indicate that MMS treatment induces phosphorylation-dependent accumulation of Triton-insoluble GFP–hRad18 at stalled replication foci. Inhibition of MMS-induced accumulation of hRad18 by wortmannin and induction by MMS of phosphorylation of Chk1 and histone H2AX Staurosporine inhibits many different protein kinases, e.g., protein kinase C, the cyclin-dependent kinases (Cdk), and the protein kinase Chk1 [36]. MMS induces ATR- and replication block-dependent phosphorylation of Chk1 in Xenopus oocyte extracts [37] and another alkylating agent N-methyl-N 0 -nitroN-nitrosoguanidine induces ATR-dependent Chk1pS317 phosphorylation in HeLa cells [38]. Replication stress also leads to the ATR-dependent phosphorylation of Ser-139 in histone H2AX which is inhibited by 200 mM wortmannin [39] and UV-dependent induction of c-H2AX was detected at stalled replication foci in xeroderma pigmentosum variant cells [40]. We found that the addition of an inhibitor of Cdk kinases roscovitive (10 lM) does not effect the accumulation of GFP–hRad18 (not shown). In contrast, MMS-induced accumulation of GFP–hRad18 is almost completely inhibited by 200 lM wortmannin and 20 lM concen-

Fig. 3. Accumulation of GFP–hRad18 protein at stalled replication foci in Chinese hamster cells treated with methyl methanesulfonate (MMS). In all variants cells were pre-extracted with cytoskeleton buffer containing 0.5% Triton X-100 and methanol as described in Materials and methods and then fixed in 4% formaldehyde, bars in (A) is 5 lm. In (A) endogeneous PCNA was visualized using primary mouse antibodies clone PC-10, biotinylated sheep-anti-mouse IgG, and avidin–Texas red. In (B) GFP fluorescence intensity analyzed in 100–150 PCNA-positive (S-phase) cells for each variant was normalized relative to red PCNA signal. Treatment with 0.01% MMS was 1 h and then cells were incubated in growth medium without MMS for 4 h. In indicated variants (ST) 200 nM staurosporine was present during MMS treatment and 4 h incubation in growth medium.

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tration of this drug leads to partial inhibition (Fig. 2B). Since mammalian protein kinases ATM and DNA-PK are known to be almost completely inhibited by 20 lM wortmannin [41], these observations suggest that ATR kinase (which is relatively resistant to wortmannin) is likely to be involved in MMS-induced accumulation of hRad18 protein in mammalian cells but we cannot exclude the possible involvement of ATM kinase as well. Earlier we had detected weak phosphorylation of endogenous H2AX in MMS-treated Chinese hamster cells using immunocytochemistry [27] and here we studied MMS-induced phosphorylation of GFPtagged human H2AX in stable V79 clone expressing this protein using Western blotting. As a control, another clone expressing GFP-tagged mutant H2AX protein was used in which Ser-139 was replaced by alanine [26]. It can be seen from Fig. 2C that MMS induces phosphorylation of GFP–H2AX containing wild type (wt) histone sequence but there is no phosphorylation of mutated GFP–H2AX (sa). We also looked for the induction of phosphorylation of Ser-317 in Chk1 using Western blots probed with specific antibodies [38]. It is seen from Fig. 2D that Chk1-pS317 can be detected in V79 cells after treatment with MMS. These results support the view that activated Chk1 kinase may be involved in MMS-induced accumulation of GFP–hRad18 in Chinese hamster cells.

Detection at stalled replication foci in MMS-treated cells of single-stranded DNA ATR kinase is known to be activated after the binding of ATR–ATRIB complex to single-stranded (ss) DNA coated with RPA complex [42]. Using antibodies which react with 5-bromodeoxyuridine only in ssDNA discrete nuclear foci were detected earlier in mammalian cells treated with ionizing radiation [43] but these foci can arise at sites of random double-strand breaks. Here we used similar antibodies to detect ssDNA in MMS-treated Chinese hamster cells which DNA was substituted with 5-iododeoxyuridine (IdU). To identify replication foci these experiments were done with V79 cells stably expressing human GFP-tagged flap endonuclease Fen1 in which distinct nuclear foci can be detected colocalizing with PCNA foci in normally proliferating and MMS-treated cells (not shown). Results obtained with GFP-Fen1 expressing cells grown for 3 days in the medium containing IdU, then treated with MMS and further incubated in growth medium for 4 h are shown in Fig. 4. It is seen that the bright foci of ssDNA can be detected in the nuclei 4 h after treatment with 0.01% MMS and these foci colocalize with the focal GFP-Fen1. The foci of ssDNA can also be seen in some nuclei one hour after DNA damage

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Fig. 4. Visualization of single-strand DNA after MMS treatment of Chinese hamster cells stably expressing GFP-tagged replication protein Fen-1. Cells were grown in the medium containing 10 lM 5-iododeoxyuridine for 3 days, then treated 1 h with 0.01% MMS and incubated in growth medium without MMS for 4 h, then fixed, and ssDNA labeled as described in [43] using secondary antibodies linked to Texas red. Colocalized foci of GFP-Fen1 and single-stranded DNA are marked by arrows. Bar 10 lm.

but they were not found in control undamaged cells. This result indicates that ssDNA is actually induced by MMS and this DNA can activate ATR–Chk1 checkpoint pathway [42].

Discussion Methyl methanesulfonate is able to activate DNA damage checkpoints in yeast [20] and ATR-dependent accumulation of some replication and repair proteins (Rad1, RPA70, and Pola) at stalled replication forks has been observed after MMS damage in experiments with Xenopus interphase extracts [37]. However, these proteins are not directly implicated in PRR and functional significance of their accumulation for DNA repair remains unclear. ATR pathway is not required for replication fork stalling after MMS damage [33,37] which is apparently caused by direct block of replicative polymerases and fork movement by alkylated bases or apurinic sites. It is found in this study that GFP–hRad18 protein can be completely extracted by Triton X-100 and methanol from normally proliferating Chinese hamster cells but MMS treatment leads to accumulation of Triton-insoluble form of GFP–hRad18 at stalled replication foci. We have also shown here that MMS-induced accumulation of GFP–hRad18 is sensitive to inhibitors of protein kinases involved in DNA damage checkpoint control. Staurosporine which inhibited indicated accumulation (Figs. 2A and 3) is an inhibitor of Chk1 but can also inhibit other kinases including cyclin-dependent kinases [36]. However, specific inhibitor of Cdk kinases roscovitine did not effected MMS-induced accumulation of hRad18 and well-known inhibitor of the checkpoint kinases ATM and ATR wortmannin showed clear suppression of this accumulation (Fig. 2B) consistent with the view that the ATR–Chk1 pathway [21,22] may be involved. ATR accumulates at stalled replication forks [23] and this is driven by formation of ssDNA at stalled forks which binds RPA complex and then binds and

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activates ATR–ATRIP complex [42]. ATR–ATRIP phosphorylates Rad17 complex [42], Ser317 and Ser345 of Chk1 [21,22,38], and Ser139 of histone H2AX [39]. We found that ssDNA is actually formed at replication sites in MMS-treated Chinese hamster cells (Fig. 4) and that MMS induces phosphorylation of Ser317 in Chk1 (Fig. 2D) and Ser139 in histone H2AX (Fig. 2C). Together, these results suggest that DNA damage checkpoint pathways are involved in the MMS-induced accumulation of hRad18 protein at stalled replication forks and possibly in lesion bypass. Concerning events which lead after activation of ATR–Chk1 pathway to insolubilization of hRad18 and its accumulation at stalled replication foci several models can be considered. The simplest is that hRad18 is directly phosphorylated by activated ATR or Chk1 kinases. Chk1 has broad site specificity and can phosphorylate a large number of different proteins but preferred target for ATR kinase is SQ subsequence which is absent from hRad18 protein. Other model is that ATR and Chk1 phosphorylate protein which is bound to hRad18, e.g., HHR6A or HHR6B, which can effect hRad18 solubility. It is also possible that activated ATR or Chk1 phosphorylate some replication or chromatin proteins accumulated at stalled replication foci, e.g., RPA, Rad1/Rad9 complex or Rad17 [42], and this leads to the binding of hRad18 complex. It is established that mammalian Rad18 protein is required for postreplication repair [16,18] but question arises whether its accumulation at stalled replication forks detected here is functionally significant or just reflects secondary effects not related to lesion bypass. It has been shown recently that hRad18 is involved in PCNA mono-ubiquitination after UV-irradiation of human cells which is important for the tight binding of trans-lesion DNA polymerase g [19]. This polymerase is also associated with replication foci in undamaged cells [34] and interacts with non-ubiquitinated PCNA [44]. Mutational inactivation of Polg subdomains responsible for its binding to mono-ubiquitinated PCNA leads to the loss of Polg ability to complement UV-sensitivity of XP variant cell line XP30RO detected in the presence of caffeine [19]. Most of known XP variant cell lines show very low UV-sensitivity factor (1.1– 1.5) but are strongly (3–5 times) sensitized by checkpoint abrogator caffeine [25,45]. This means that the Polg-dependent trans-lesion synthesis contributes to UV resistance only when caffeine-sensitive lesion bypass subpathway [24] is inactive. However, Rad18-deficient cells show significant UV- and MMS-sensitivity in the absence of caffeine [16,18] indicating that the Rad18mediated functions in postreplication repair are not limited by its functions in recruitment of Polg [19] which is apparently insensitive to caffeine. Therefore, Rad18 may be required not only for recruitment of Polg but also for other important events during postreplication repair,

e.g., elimination of stalled Pold and/or for template switch [8,9] and these events may require checkpoint kinase-dependent accumulation of Rad18 protein at stalled forks. Further studies of DNA damage-induced accumulation of hRad18 are clearly required to establish actual significance and mechanism of this phenomenon.

Acknowledgments We thank S. Tateishi for plasmid containing hRad18 cDNA, A. Lehmann, K. Tanaka, and M.Cordeiro-Stone for helpful discussions. This research was supported by the Office of Science (BER), US Department of Energy, Grant No. DE-FG0301ER63070, and the Russian Fund for Basic Research Grant 02-04-49145. References [1] R.B. Setlow, Shedding light on proteins, nucleic acids, cells, humans and fish, Mutat. Res. 511 (2002) 1–14. [2] P.C. Hanawalt, Oncogene 21 (2002) 8949–8956. [3] R.B. Setlow, The photochemistry, photobiology, and repair of polynucleotides, Prog. Nucleic Acid Res. Mol. Biol. 8 (1968) 257– 295. [4] W.D. Rupp, P. Howard-Flanders, Discontinuities in the DNA synthesized in an excision-defective strain of Escherichia coli following ultraviolet irradiation, J. Mol. Biol. 31 (1968) 291–304. [5] J.R. Nelson, C.W. Lawrence, D.C. Hinkle, Deoxycytidyl transferase activity of yeast REV1 protein, Science 272 (1996) 1646–1649. [6] C. Masutani, R. Kusumoto, A. Yamada, N. Dohmae, M. Yokoi, M. Yuasa, M. Araki, S. Iwai, K. Takio, F. Hanaoka, The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase eta, Nature 399 (1999) 700–704. [7] G. Spivak, P.C. Hanawalt, Translesion DNA synthesis in the dihydrofolate reductase domain of UV-irradiated CHO cells, Biochemistry 31 (1992) 6794–6800. [8] N.P. Higgins, K. Kato, B. Strauss, A model for replication repair in mammalian cells, J. Mol. Biol. 101 (1976) 417–425. [9] Z. Li, W. Xiao, J.J. McCormick, V.M. Maher, Identification of a protein essential for a major pathway used by human cells to avoid UV- induced DNA damage, Proc. Natl. Acad. Sci. USA 99 (2002) 4459–4464. [10] V. Bailly, S. Lauder, S. Prakash, L. Prakash, Yeast DNA repair proteins Rad6 and Rad18 form a heterodimer that has ubiquitin conjugating, DNA binding, and ATP hydrolytic activities, J. Biol. Chem. 272 (1997) 23360–23365. [11] C.A Torres-Ramos, S. Prakash, L. Prakash, Requirement of RAD5 and MMS2 for postreplication repair of UV-damaged DNA in Saccharomyces cerevisiae, Mol. Cell. Biol. 22 (2002) 2419–2426. [12] C.A. Torres-Ramos, B.L. Yoder, P.M. Burgers, S. Prakash, L. Prakash, Requirement of proliferating cell nuclear antigen in RAD6-dependent postreplicational DNA repair, Proc. Natl. Acad. Sci. USA 93 (1996) 9676–9681. [13] H.D. Ulrich, S. Jentsch, Two RING finger proteins mediate cooperation between ubiquitin-conjugating enzymes in DNA repair, EMBO J. 19 (2000) 3388–3397. [14] C. Hoege, B. Pfander, G.L. Moldovan, G. Pyrowolakis, S. Jentsch, RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO, Nature 419 (2002) 135–141.

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