| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | ADVANCED SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Cell Genetics, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan
2 Developmental Genetics, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan
3 Department of Oral and Maxillofacial Surgery, Sensory and Motor Organs Sciences, Faculty of Medicine and Pharmaceutical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-0811, Japan
4 Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto 862-0973, Japan
5 Department of Biology, School of Science, Kyushu University, Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
6 Graduate School of Frontier Biosciences, Osaka University, and SORST, Japan Science and Technology Agency, 1-3 Yamada-Oka, Suita, Osaka 565-0871, Japan
 |
Abstract |
|---|
 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
|---|
(Pol) for post-replication repair (PRR), the molecular mechanisms by which these factors are recruited to stalled replication forks are not well understood. We present evidence that human RAD18 complexed with RAD6B protein preferentially binds to forked and single-stranded DNA (ssDNA) structures, which are known to be localized at stalled replication forks. The SAP domain of RAD18 (residues 248–282) is crucial for binding of RAD18 complexed with RAD6B to DNA substrates. RAD18 mutated in the SAP domain fails to accumulate at DNA damage sites in vivo and does not guide DNA Pol to stalled replication forks. The SAP domain is also required for the efficient mono-ubiquitination of PCNA. The SAP domain mutant fails to suppress the ultraviolet (UV)-sensitivity of Rad18-knockout cells. These results suggest that RAD18 complexed with RAD6B is recruited to stalled replication forks via interactions with forked DNA or long ssDNA structures, a process that is required for initiating PRR. |
Introduction |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
In the budding yeast Saccharomyces cerevisiae, genes belonging to the RAD6 epistasis group are involved in the PRR pathway; RAD6 and RAD18, which encode a ubiquitin-conjugating enzyme E2 and a ubiquitin ligase E3, respectively, play a pivotal role in PRR (Bailly et al. 1994, 1997b). Saccharomyces cerevisiae Rad18 binds to single-stranded DNA (ssDNA) and forms a tight complex with ubiquitin-conjugating enzyme E2 (Bailly et al. 1997a). Mono-ubiquitination of PCNA, which is necessary for tolerance of DNA damage, was recently shown to be RAD6–RAD18-dependent (Hoege et al. 2002; Stelter & Ulrich 2003). In vertebrate cells, a single RAD18 homologue (Tateishi et al. 2000) and two RAD6 homologues (designated RAD6A and RAD6B) have been identified (Koken et al. 1991).
In mammalian cells, TLS DNA polymerases operate to circumvent replication blocks. Among TLS polymerases, which primarily belong to the Y-family, polymerase (Pol) is of great interest because the gene encoding Pol is mutated in a cancer-causing hereditary disorder, xeroderma pigmentosum variant (XPV) (Johnson et al. 1999; Masutani et al. 1999). Pol physically interacts with RAD18 (Watanabe et al. 2004; Yuasa et al. 2006). In UV-irradiated human cells, Pol and RAD18 are recruited to nuclear foci in a UV-dose-, and time-dependent manner (Kannouche et al. 2001; Watanabe et al. 2004; Masuyama et al. 2005). Pol–RAD18 foci co-localize with PCNA, suggesting that these foci are sites of stalled replication. Despite the importance of Pol and RAD18, the molecular mechanisms by which they are recruited to these sites are not well understood. Upon recruitment of Pol–RAD18 to stalled replication forks, PCNA is ubiquitinated by the RAD6–RAD18 complex. Pol and other Y-family members such as Pol
are considered to replace replicative polymerases through interaction with mono-ubiquitinated PCNA (Kannouche et al. 2004; Watanabe et al. 2004; Bienko et al. 2005; Plosky et al. 2006). A mutant Pol devoid of focus-forming activity cannot complement the XPV defect (Kannouche et al. 2001), and a mutant RAD18 lacking the Pol interaction site fails to complement the UV-sensitivity of Rad18-knockout cells (Watanabe et al. 2004). These results demonstrate the importance of RAD18–Pol recruitment to stalled replication forks in the initiation of TLS.
Diverse DNA-damaging agents can elicit mono-ubiquitination of PCNA, thus triggering TLS. A central question in signaling for PRR is whether each type of damage is detected by a different sensor or whether all types of damage are converted to a common DNA structure that is detected by a sensor protein. A possible candidate for a common DNA structure is the stalled replication fork along with ssDNA. Upon encountering a bulky lesion, the replicative polymerase stalls, whereas MCM helicase activity continues to unwind DNA (Zou & Elledge 2003; Byun et al. 2005). The uncoupling of MCM helicase and DNA polymerase activities leads to the accumulation of ssDNA, which is stabilized by the ssDNA-binding protein, replication protein A (RPA).
In this study, we have investigated the basis for the recruitment of RAD18 to sites of replication fork stalling. We show that RAD18 complexed with RAD6B protein preferentially binds to forked DNA structures and long ssDNA, which are known to be located at stalled replication forks. Our experiments identify RAD18 domains required for binding to DNA replication intermediate-like structures. RAD18 mutants incapable of binding to these DNA structures lacked various biological activities normally associated with wild-type RAD18 (RAD18WT). Our results suggest a possible molecular mechanism for the recruitment of RAD18 and Pol to stalled replication forks and for the initiation of PRR.
|
Results |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
RAD18 has multiple functional domains, including RING, zinc finger, RAD6-binding and Pol-binding domains, and a central SAP (SAF-A/B, Acinus and PIAS) (residues 248–282) motif (Fig. 1A) that has been proposed to function as a DNA-binding motif (Aravind & Koonin 2000; Ahn & Whitby 2003). The SAP motif is a helix–extended-loop–helix structure comprising two parallel helices separated by a long loop (Fig. 1B) (Zhang et al. 2001; Okubo et al. 2004). To test a possible role for this motif in DNA binding, we generated a RAD18 mutant lacking the SAP motif (RAD18
SAP) and assayed its binding to RAD6 (Fig. 1C) and Pol (Fig. 1D). RAD18SAP retained both binding activities, implying that deletion of the SAP domain does not cause nonspecific changes in RAD18 conformation. In contrast, RAD186BD (RAD18 lacking the RAD6-binding domain) and RAD18C2 (RAD18 lacking the Pol-binding domain) lost ability to bind to RAD6A/B and Pol, respectively.
|
SAP complex were purified to about 90% (Fig. 1E). Different concentrations of the purified of RAD6B–RAD18 complex (hereafter, RAD6–RAD18) were incubated with a radiolabeled 49-mer dsDNA, a 49-mer ssDNA, a 49-mer forked DNA with single-stranded region (Y-1 form) or a 49-mer forked DNA without a single-stranded region (Y-2 form) (Fig. 2A–D). The resulting protein–ssDNA complexes were detected using the electrophoretic gel mobility-shift assay (EMSA). RAD6–RAD18–dsDNA complex was almost undetectable when RAD6–RAD18 was incubated with the 49-mer dsDNA (Fig. 2A). Upon incubation of the 49-mer ssDNA probe with RAD6–RAD18, only a small amount of slowly migrating RAD6–RAD18–ssDNA complex was detected (Fig. 2B). In contrast, about 80% of the 49-mer forked DNA probe (Y-1 form) bound to RAD6–RAD18 (Fig. 2C). RAD6–RAD18 bound to the 49-mer forked DNA lacking a single-stranded region (Y-2 form) as well as to the 49-mer Y-1 DNA (Fig. 2D). Compared to the Y-1 DNA structure, the Y-2 DNA structure seems to be more stable and resembles a stalled replication fork. These results indicate that RAD6–RAD18 preferentially binds to forked DNA substrates (Fig. 2E).
|
To confirm that RAD18 is recruited to stalled replication forks, we examined the localization of RAD18 and RPA in hydroxyurea-treated human cells (Fig. 3). Treatment of human cells with hydroxyurea is known to invoke stalling of replication forks associated with RPA-coated ssDNA (Zou & Elledge 2003). RPA foci that predominantly co-localized with endogenous RAD18 were observed, suggesting that RAD18 was recruited to stalled replication forks. These results suggest that RAD18 preferentially binds to forked DNA with long branches, structures resembling stalled replication forks.
|
The SAP domain (Fig. 1A and B) has been proposed to function as a DNA-binding motif (Aravind & Koonin 2000; Ahn & Whitby 2003). To examine the role of this domain in binding of RAD6–RAD18 to DNA, we assessed the binding of a RAD18 mutant lacking the SAP motif (RAD18SAP) complexed with RAD6B to a 49-mer forked DNA substrate (Y-1 form) using the EMSA technique. RAD6–RAD18SAP failed to bind to the 49-mer forked DNA (Y-1 form) (Fig. 4A) and to a 49-mer forked DNA without a single-stranded region (Y-2 form) (Fig. 4B). These results indicate that the SAP domain is required for the efficient binding of RAD18 to forked DNA structures. To further investigate the function of the SAP domain, we made the site-directed mutants L256P (which is affected in the helix-1 region of the SAP domain), L274P (helix-2), and G269D and K271A (inter-helix loop) (Fig. 4C). To examine the effects of these mutations on binding to a 120-mer ssDNA or a 120-mer forked DNA (Y-1 form), the mutant RAD18 proteins were purified (in a complex with RAD6B) and subjected to EMSA using these DNA substrates as probes (Fig. 4D and E). All of the mutated RAD18 proteins showed reduced binding to these DNA substrates as compared to wild-type RAD18 (RAD18WT). The two RAD18 helix mutants (L256P and L274P) exhibited reduced binding to the 120-mer forked DNA probes as compared with the loop mutant (K271A and G269D) (Fig. 4E). These results indicate important roles for the SAP domain helices in mediating interactions between RAD18 and DNA replication intermediates.
|
accumulate at stalled replication sites in genotoxin-treated cells (Kannouche et al. 2002; Watanabe et al. 2004; Nakajima et al. 2006). Based on our in vitro analyses of interactions between RAD18 and DNA replication structures we hypothesized that RAD18 binds directly to stalled replication forks in vivo and subsequently helps to recruit Pol. Thus, we investigated the requirement for the SAP domain in the formation of RAD18 and Pol nuclear foci. To this end, wild-type and mutant forms of RAD18 were expressed in Rad18-null cells, and basal and genotoxin-induced GFP–RAD18 and GFP–Pol nuclear foci were visualized in the resulting cultures. GFP–RAD18WT formed nuclear foci upon UV irradiation (Fig. 5A and B). In contrast, GFP–RAD18SAP did not form nuclear foci, indicating that the SAP domain is necessary for RAD18 focus formation. The SAP-loop mutant K271A also formed nuclear foci, as did other loop mutant (G269D) (data not shown), whereas the SAP-helix mutants L256P and L274P did not. These data suggest that the efficient DNA-binding activity of RAD18 is required for its accumulation in nuclear foci in response to DNA damage.
|
focus formation. Pol is recruited to stalled replication sites in a RAD18-dependent manner upon DNA damage (Watanabe et al. 2004). We visualized GFP–Pol foci in Rad18-null cells ectopically expressing wild-type or mutant forms of RAD18 (Fig. 5C and D). RAD18WT and the SAP-loop mutant K271A restored the ability of Rad18-null cells to form GFP–Pol foci in response to UV irradiation, as did another loop mutant (G269D) (data not shown). In contrast, the SAP-helix mutants L256P and L274P failed to restore the ability of Rad18-null cells to form GFP–Pol focus. These results demonstrate a key role for the SAP domain in RAD18-dependent Pol focus formation and suggest that the efficient DNA-binding activity of RAD18 is required for the recruitment of Pol foci to stalled replication forks. SAP domain of RAD18 is required for mono-ubiquitination of PCNA and for recovery of PRR
The ubiquitin-ligase activity of RAD18 for the mono-ubiquitination of PCNA is considered to be required for the exchange of stalled replicative polymerases with TLS enzymes (Kannouche et al. 2004; Watanabe et al. 2004; Bienko et al. 2005; Plosky et al. 2006). To examine the effects of various RAD18 mutations on PCNA mono-ubiquitination, complexes of mutant RAD18 proteins with RAD6B were tested for in vitro PCNA-directed ubiquitin ligase activity (Fig. 6A). In this assay, PCNA was loaded on primer-annealed phage circular ssDNA. When the RAD18SAP or RAD18 SAP-helix mutants (L256P and L274P) was reacted with PCNA loaded on DNA, mono-ubiquitination activity for PCNA was not observed, whereas RAD18WT showed robust activity. The SAP-loop mutant K271A showed slight activity. Thus, the intact SAP domain of RAD18 was required for the mono-ubiquitination of PCNA loaded on primer-annealed ssDNA. There are two possible explanations for this result. One is that RAD18SAP failed to access the DNA on which PCNA was loaded, leading to inefficient mono-ubiquitination. The other is that the SAP domain recognizes not only DNA structures but also PCNA. To evaluate these possibilities, complexes of mutant RAD18 proteins with RAD6B were tested for in vitro ubiquitin ligase activity for PCNA in the absence of DNA and RFC (Fig. 6B). Although the efficiency of the reaction using PCNA without DNA and RFC was low compared to that of the reaction using PCNA loaded on DNA (data not shown), mono-ubiquitinated PCNA was detected after lengthy incubation of the reaction mixture. RAD18SAP or the RAD18 SAP-helix mutants L256P and L274P were reacted with PCNA without DNA and did not exhibited mono-ubiquitination activity, whereas RAD18WT showed robust activity, and the SAP-loop mutant K271A also showed mono-ubiquitination activity. These results indicate that the SAP domain of RAD18 is required not only for binding to DNA but also for PCNA mono-ubiquitination in vitro.
|
SAP or either of the RAD18 SAP-helix mutants (L256P and L274P). These results indicate that the helical structure in the SAP domain must be intact not only for binding of RAD18 to DNA, but also for PCNA mono-ubiquitination.
To further test the significance of the forked-DNA-binding and PCNA mono-ubiquitination activities of RAD18 in PRR, we examined the survival of Rad18-null cells ectopically expressing wild-type and mutant RAD18 proteins after UV irradiation. As shown in Fig. 7, the UV-sensitivity of Rad18-null mouse cells was rescued by reconstitution with RAD18WT. The UV-sensitivity of Rad18-null cells expressing one of the SAP-loop mutants (K271A) was modestly rescued, and the same was observed for other loop mutants (G269D). In contrast, reconstitution with RAD18SAP or the SAP-helix mutants L256P and L274P failed to complement the UV sensitivity of Rad18-null cells. These results suggest that the efficient binding of RAD18 to DNA structure, and the mono-ubiquitination of PCNA are critical for cell survival upon UV irradiation.
|
|
Discussion |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
We have shown that the human RAD6–RAD18 complex binds efficiently to long ssDNA structures. These findings are consistent with a previous report that the yeast Rad6–Rad18 complex binds ssDNA in vitro (Bailly et al. 1994). Furthermore, our results demonstrate that human RAD18 preferentially binds to forked DNA structures (Fig. 2). These results suggest that human RAD18 recognizes forked and long ssDNA structures, similar to the DNA intermediates formed as a consequence of replication fork stalling.
In EMSA assay, several retarded bands were shown when RAD6–RAD18 was incubated with 49-mer forked DNA substrates (Fig. 2C and D). These ladder-like bands may consist of the DNA substrates associated with different number of RAD6–RAD18. Binding of RAD6–RAD18 to various structures of DNA substrates was enhanced by increasing the length of the ssDNA (compare 49-mer DNA substrates to 120-mer DNA substrates in Fig. 2). These manner and sigmoidal curve of the binding of RAD6–RAD18 to 49-mer forked DNA or to 120-mer ssDNA suggest stoichiometric binding of RAD6–RAD18 to the DNAs (Fig. 2E and K). Lopes et al. detected long ssDNA regions on one of the replicated strands immediately behind the replication fork (a "gapped fork") in nucleotide excision repair deficient strains of S. cerevisiae irradiated with UV light (Lopes et al. 2006). They also observed ssDNA regions of up to 400 bp well behind replication forks, up to 20 kb away. These ssDNA regions ("internal ssDNA gaps") were found on both strands of the replicated DNA molecules. The existence of internal ssDNA gaps is consistent with the Rupp and Howard-Flanders model (Rupp & Howard-Flanders 1968; Lehmann & Fuchs 2006). From our results, we infer that RAD18 preferentially binds to gapped forks and to internal ssDNA gaps.
A central question in signaling for PRR is whether each type of DNA damage is detected by a different sensor or whether all types of damage involve replication fork stalling and generation of a common DNA structure that is detected by a sensor protein. To investigate the former possibility, we examined whether RAD18 preferentially binds to DNA containing a cyclobutane pyrimidine dimer (CPD). We tested binding of RAD6–RAD18 to three different sequences of 49-mer ssDNA or dsDNA with or without CPD and found no preferential binding to CPD-containing DNA (data not shown). We investigated whether the RAD18 SAP motif is a DNA-binding domain (Fig. 4). Our results demonstrate that the SAP domain is critical for binding of RAD18 to forked and long ssDNA structures. In the predicted tertiary structure of the SAP motif, the L256 and L274 residues associate with each other via hydrophobic interactions (Fig. 1B); this association probably contributes to the stability of the helix–extended-loop–helix structure. The L256P and L274P mutations in the two helices indeed abolished interactions with DNA, while the G269D and K271A mutations in the inter-helix loop region only modestly affected DNA-binding activity (Fig. 4E). Thus, the structure of the two helices appears to be important for DNA recognition. Notenboom et al. also reported that the SAP domain is required for the binding of RAD18 to ssDNA (Notenboom et al. 2007). They measured the binding affinity of the GST–SAP protein with ssDNA by surface plasmon resonance. The GST–SAP G269K271AA and H263G264L265AAA proteins (mutated in the inter-helix loop region) showed an about fourfold reduction or modestly reduced affinity compared to that of GST-fused wild-type SAP protein, while the D255L256K257AAA and I275K276Q279AAA proteins (mutated in the two helix regions) showed no significant change in affinity compared to the GST-fused wild-type SAP protein. These results are not consistent with our results. Although these disparate findings could reflect different experimental conditions because we used a full-length RAD18 protein mutated in the SAP domain, while Notenboom et al. used a GST-fused mutated SAP protein, further studies will be needed to understand structure–function relationships in the SAP domain. RAD18 has a zinc-finger domain of the "Rad18-like CCHC zinc-finger" subtype that is likely to be involved in binding to DNA. We found that RAD18C207F, a zinc-finger domain mutant of RAD18 (Miyase et al. 2005), binds DNA with comparable efficiency to wild-type RAD18, indicating that the zinc-finger domain is not required for DNA binding (data not shown).
Recognition of forked and long ssDNA structures by RAD18 triggers recruitment of RAD18 associated with Pol to stalled replication forks
Upon treatment with DNA-damaging agents, RAD18 and Pol form nuclear foci that most probably represent stalled replication sites (Watanabe et al. 2004). The specific interaction of RAD6–RAD18 with forked DNA and long ssDNA substrates suggests an attractive model for the mechanism of recruitment of RAD6–RAD18–Pol to stalled replication forks. RAD6–RAD18 is recruited to these sites via interactions of RAD18 with forked DNA and long ssDNA. SAP-mutated RAD18 failed to support PCNA-directed E3 ligase activity in vitro or in vivo. Thus, the SAP domain of RAD18 is required not only for DNA binding, but also for the mono-ubiquitination of PCNA (Fig. 6). The SAP domain in PIAS1 is also known to interact with both DNA and protein (Okubo et al. 2004). Failure of SAP-mutated RAD18 to rescue the UV-sensitivity of Rad18–/– cells is likely attributable to both failed recruitment of RAD18 associated with Pol, and defective PCNA mono-ubiquitination. Pol preferentially interacts with mono-ubiquitinated PCNA, which is generated at stalled replication forks. In contrast, RAD18 showed no interaction with mono-ubiquitinated PCNA, suggesting that recruitment of RAD18 to stalled replication forks is not dependent on mono-ubiquitination of PCNA but on recognition of forked and long ssDNA structures. We infer that RAD18 perform three major roles: recognizing stalled replication forks as a sensor protein, targeting RAD6 and Pol to stalled replication sites as a guide, and directing mono-ubiquitination of PCNA as a ubiquitin ligase.
Discrimination between stalled and advancing replication fork by RAD18
Our finding that RAD6–RAD18 preferentially binds forked and long ssDNA structures suggests that it recognizes stalled replication forks. We assume that RAD18 does not bind normal DNA replication fork structures because PCNA mono-ubiquitination was almost undetectable in cells unexposed to genotoxin treatment. It is therefore of interest to determine the mechanisms by which RAD18 may distinguish between normal and stalled replication forks. Upon encountering bulky lesions, replicative DNA polymerases are stalled, whereas the MCM helicase continues to unwind dsDNA (Byun et al. 2005). Uncoupling of the MCM helicase and DNA polymerase activities leads to an accumulation of ssDNA stabilized by RPA at the fork. In Xenopus cell-free extracts, PCNA ubiquitination induced by replication blockage requires Cdc45-mediated uncoupling of DNA polymerase and MCM helicase activities (Chang et al. 2006). Fork stabilization resulting from coating of ssDNA with RPA is likely to facilitate access of RAD6–RAD18 to the stalled replication fork and might be required for RAD18 recruitment. Another possible pathway for stalled fork stabilization might be initiated by activation of the S-phase checkpoint (S-PCP). Replication stress and DNA damage activate S-PCP signaling pathways, a major function of which is stabilizing stalled replication forks to prevent fork collapse (Tercero et al. 2003). Efficient RAD18-mediated PCNA mono-ubiquitination requires S-PCP signaling via ATR/Chk1 (Bi et al. 2006), suggesting that S-PCP signaling pathways contribute to the recruitment of RAD6–RAD18–Pol and to PCNA ubiquitination. However, in a recent study, the loss of the checkpoint mediators ATRIP and Rad1 did not prevent PCNA ubiquitination in Xenopus egg extracts (Chang et al. 2006). Similarly, PCNA ubiquitination was not dependent on the ATR homologue Rad3 in S. pombe (Frampton et al. 2006).
Although these disparate findings could reflect a difference among yeast, Xenopus, and mammalian cells, further studies will be needed to understand the potential involvement of checkpoint signaling in PRR initiation. In conclusion, the work presented here has uncovered DNA binding mode of RAD18 by which it may recognize stalled replication forks. Our results provide new insight into the molecular basis of initiation of PRR.
|
Experimental procedures |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
Wild-type RAD18 complexed with His-tagged RAD6B was expressed in insect cells (Sf9) transfected with hRAD18pVL, hRAD6BpAcH and BD BaculoGold DNA (BD Biosciences Pharmingen, San Diego, CA). The protein complex was purified using a Ni2+-loaded HiTRap Chelating HP column (Amersham, Uppsala, Sweden) and subsequently with a Resource Q column (Amersham). RAD18 lacking the SAP domain (residues 248–282) (RAD18SAP) was prepared by same method described above except that the RAD18SAPpVL plasmid instead of RAD18pVL was used. RAD18 derivatives mutated in the SAP domain, yielding the K271A, G269D, L256P or L274P protein, were prepared by same method described above except that the RAD18K271ApVL, RAD18G269DpVL, RAD18L256PpVL or RAD18L274PpVL plasmid, was used, respectively.
Synthetic DNA substrates and oligonucleotides for binding RAD18 to ssDNA
Oligonucleotides numbers 1 (49N2) and 2 (120N2) are 5'-AGCTA CCATG CCTGC ACGAA TTCGT ATCAG CGTAA TCATG GTCAT AGCT-3' and 5'-GCATC CTCAC CATCA ACTCA CAGCC CAACA TCAAC GGGAA GCCGT CCTCC GACCC CATCG TGGGC TGGGG CAGCT ACCAT GCCTG CACGA ATTCG TATCA GCGTA ATCAT GGTCA TAGCT-3', respectively. Oligonucleotides numbers 3 (49N2R) and 4 (120N2R) are complementary to the sequences of no. 1 (49N2) and no. 2 (120N2), respectively. The blunt dsDNA substrates used in binding experiments are 1 + 3 (a 49-mer dsDNA) and 2 + 4 (a 120-mer dsDNA). Oligonucleotides numbers 5 (120N2R-40 ss) and 6 (120N2R-80ss) are 5'-ATGGT AGCTG CCCCA GCCCA CGATG GGGTC GGAGG ACGGC TTCCC GTTGA TGTTG GGCTG TGAGT TGATG GTGAG GATGC-3' and 5'-TTCCC GTTGA TGTTG GGCTG TGAGT TGATG GTGAG GATGC-3', respectively. The ssDNA-tailed dsDNA substrates used in binding experiments are 2 + 5 (an 80-mer dsDNA that has a 40-mer ssDNA tail) and 2 + 6 (a 40-mer dsDNA that has 80-mer ssDNA tail). Oligonucleotides numbers 7 (F3d47), 8 (d20) and 9 (d30–2) were previously described (Komori et al. 2002; Hishida et al. 2004). Oligonucleotide number 10 (F3d120) is 5'-AGCTA TGACC ATGAT TACGA ATTGC TTGGA ATCCT GACGA ACTGT AGCAG CCCTG GCGTC GTGAT TAGTG ATGAT GAACC AGGTT ATGAC CTTGA TTTAT TTTGC ATACC TAATC ATTAT-3'. The forked DNA substrates used in binding experiments are 1 + 7 (49-mer Y-1), 1 + 7 + 8 + 9 (49-mer Y-2) and 2 + 10 (120-mer Y-1). Annealing was performed by heating the DNAs at 95 °C for 5 min followed by incubation at 37 °C for 30 min.
EMSA for DNA binding
Binding reaction mixtures contained 20 mM Tris–acetate (pH 8.0), 1 mM dithiothreitol (DTT), 10 nM 32P-labeled DNA substrate, and the indicated amount of RAD18 protein. Reactions were incubated at 37 °C for 10 min. Products were analyzed by 6% PAGE in TAE buffer and visualized and quantified using a BAS2000 image analyzer (Fujifilm, Tokyo, Japan).
Detection of PCNA mono-ubiquitination
For the in vitro assay, ubiquitination of PCNA was carried out coupling with loading of PCNA on primed circular ssDNA by replication factor C (RFC), as reported by Garg and Burgers (Garg & Burgers 2005). Briefly, PCNA (230 ng) was incubated with RAD6–RAD18 (200 ng), ubiquitin activating enzyme (E1, 125 ng), (MBL Nagoya, Japan), RFC (50 ng), RPA (300 ng), M13 phage circular ssDNA annealed with three complementary 100-mer primers (150 ng), and 5.6 µg ubiquitin in 5 µL of 10 mM HEPES buffer (pH 7.5) containing 150 mM NaCl, 0.05% Tween-20, 0.4 mM DTT, 2 mM ATP, 10 mM MgCl2, 50 mM creatine phosphate and 100 ng creatine kinase for the indicated times at 37 °C. Aliquots of the samples were subjected to SDS-PAGE, and PCNA was detected by Western blotting using an anti-PCNA (5A10) antibody (MBL). For the in vivo assay, Rad18–/– mouse cells were immortalized with the SV40 vector. The cells were transfected with the expression vector RAD18-pCAGGS using FuGENE 6 transfection reagent (Roche Applied Science, Basel, Switzerland). Forty-eight hours after transfection, the cells were irradiated with UV light (0, 20 or 40 J/m2) and cultured for an additional 4 h. Cell lysates were subjected to SDS-PAGE, and PCNA was detected by Western blotting using an anti-PCNA (PC10) antibody (Santa Cruz, Santa Cruz, CA).
Detection of RAD18 and Pol nuclear foci
To examine the subcellular distribution of human RAD18 and Pol upon UV irradiation, eGFP–RAD18 was transiently expressed in Rad18–/– mouse cells. The cells were irradiated with UV light (15 J/m2) and incubated for 6 h. The distribution of eGFP–RAD18 was examined by microscopy. To monitor the localization of Pol upon UV irradiation, eGFP–Pol and Flag-RAD18 were transiently expressed in Rad18–/– mouse cells. The cells were incubated for 48 h and then irradiated with UV (15 J/m2). UV-irradiated cells and non-irradiated controls were incubated for 6 h. The distribution of eGFP–Pol was examined using fluorescence microscopy.
UV survival assay
Rad18–/– cells were transfected with pcDNA3.1/hygro harboring a gene encoding wild-type or SAP mutant RAD18. Colonies were isolated and analyzed for RAD18 expression by Western blotting with an anti-RAD18 antibody. Clones expressing RAD18 were seeded onto new plates. Subsequently, cells were exposed to UV light (254 nm), and cultured for 7 days. The number of surviving colonies was counted.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
bDeceased in May 2006.
Communicated by: Hiroyuki Araki
* Correspondence: Email: tate{at}gpo.kumamoto-u.ac.jp
|
References |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
Aravind, L. & Koonin, E.V. (2000) SAP—a putative DNA-binding motif involved in chromosomal organization. Trends Biochem. Sci. 25, 112–114.[CrossRef][Medline]
Bailly, V., Lamb, J., Sung, P., Prakash, S. & Prakash, L. (1994) Specific complex formation between yeast RAD6 and RAD18 proteins: a potential mechanism for targeting RAD6 ubiquitin-conjugating activity to DNA damage sites. Genes Dev. 8, 811–820.
Bailly, V., Lauder, S., Prakash, S. & Prakash, L. (1997a) Yeast DNA repair proteins Rad6 and Rad18 form a heterodimer that has ubiquitin conjugating, DNA binding, and ATP hydrolytic activities. J. Biol. Chem. 272, 23360–23365.
Bailly, V., Prakash, S. & Prakash, L. (1997b) Domains required for dimerization of yeast Rad6 ubiquitin-conjugating enzyme and Rad18 DNA binding protein. Mol. Cell. Biol. 17, 4536–4543.
Bi, X., Barkley, L.R., Slater, D.M., Tateishi, S., Yamaizumi, M., Ohmori, H. & Vaziri, C. (2006) Rad18 regulates DNA polymerase 
is required for recovery from S-phase checkpoint-mediated arrest. Mol. Cell. Biol. 26, 3527–3540.
Bienko, M., Green, C.M., Crosetto, N., Rudolf, F., Zapart, G., Coull, B., Kannouche, P., Wider, G., Peter, M., Lehmann, A.R., Hofmann, K. & Dikic, I. (2005) Ubiquitin-binding domains in Y-family polymerases regulate translesion synthesis. Science 310, 1821–1824.
Broomfield, S., Hryciw, T. & Xiao, W. (2001) DNA postreplication repair and mutagenesis in Saccharomyces cerevisiae. Mutat. Res. 486, 167–184.[Medline]
Byun, T.S., Pacek, M., Yee, M.C., Walter, J.C. & Cimprich, K.A. (2005) Functional uncoupling of MCM helicase and DNA polymerase activities activates the ATR-dependent checkpoint. Genes Dev. 19, 1040–1052.
Chang, D.J., Lupardus, P.J. & Cimprich, K.A. (2006) Monoubiquitination of proliferating cell nuclear antigen induced by stalled replication requires uncoupling of DNA polymerase and mini-chromosome maintenance helicase activities. J. Biol. Chem. 281, 32081–32088.
Frampton, J., Irmisch, A., Green, C.M., Neiss, A., Trickey, M., Ulrich, H.D., Furuya, K., Watts, F.Z., Carr, A.M. & Lehmann, A.R. (2006) Postreplication repair and PCNA modification in Schizosaccharomyces pombe. Mol. Biol. Cell. 17, 2976–2985.
Garg, P. & Burgers, P.M. (2005) Ubiquitinated proliferating cell nuclear antigen activates translesion DNA polymerases and REV1. Proc. Natl. Acad. Sci. USA 102, 18361–18366.
Hishida, T., Han, Y.W., Shibata, T., Kubota, Y., Ishino, Y., Iwasaki, H. & Shinagawa, H. (2004) Role of the Escherichia coli RecQ DNA helicase in SOS signaling and genome stabilization at stalled replication forks. Genes Dev. 18, 1886–1897.
Hoege, C., Pfer, B., Moldovan, G.L., Pyrowolakis, G. & Jentsch, S. (2002) RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature 419, 135–141.[CrossRef][Medline]
Johnson, R.E., Kondratick, C.M., Prakash, S. & Prakash, L. (1999) hRAD30 mutations in the variant form of xeroderma pigmentosum. Science 285, 263–265.
Kannouche, P., Broughton, B.C., Volker, M., Hanaoka, F., Mullenders, L.H. & Lehmann, A.R. (2001) Domain structure, localization, and function of DNA polymerase , defective in xeroderma pigmentosum variant cells. Genes Dev. 15, 158–172.
Kannouche, P., Fern&ez de Henestrosa, A.R., Coull, B., Vidal, A.E., Gray, C., Zicha, D., Woodgate, R. & Lehmann, A.R. (2002) Localization of DNA polymerases and to the replication machinery is tightly co-ordinated in human cells. EMBO J. 21, 6246–6256.[Medline]
Kannouche, P.L., Wing, J. & Lehmann, A.R. (2004) Interaction of human DNA polymerase with monoubiquitinated PCNA: a possible mechanism for the polymerase switch in response to DNA damage. Mol. Cell 14, 491–500.[CrossRef][Medline]
Koken, M.H., Reynolds, P., Jaspers-Dekker, I., Prakash, L., Prakash, S., Bootsma, D. & Hoeijmakers, J.H. (1991) Structural and functional conservation of two human homologs of the yeast DNA repair gene RAD6. Proc. Natl. Acad. Sci. USA 88, 8865–8869.
Komori, K., Fujikane, R., Shinagawa, H. & Ishino, Y. (2002) Novel endonuclease in Archaea cleaving DNA with various branched structure. Genes Genet. Syst. 77, 227–241.[CrossRef][Medline]
Lehmann, A.R. & Fuchs, R.P. (2006) Gaps and forks in DNA replication: rediscovering old models. DNA Repair (Amst.) 5, 1495–1498.[CrossRef][Medline]
Lopes, M., Foiani, M. & Sogo, J.M. (2006) Multiple mechanisms control chromosome integrity after replication fork uncoupling and restart at irreparable UV lesions. Mol. Cell 21, 15–27.[CrossRef][Medline]
Masutani, C., Kusumoto, R., Yamada, A., Dohmae, N., Yokoi, M., Yuasa, M., Araki, M., Iwai, S., Takio, K. & Hanaoka, F. (1999) The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase . Nature 399, 700–704.[CrossRef][Medline]
Masuyama, S., Tateishi, S., Yomogida, K., Nishimune, Y., Suzuki, K., Sakuraba, Y., Inoue, H., Ogawa, M. & Yamaizumi, M. (2005) Regulated expression and dynamic changes in subnuclear localization of mammalian Rad18 under normal and genotoxic conditions. Genes Cells 10, 753–762.
Miyase, S., Tateishi, S., Watanabe, K., Tomita, K., Suzuki, K., Inoue, H. & Yamaizumi, M. (2005) Differential regulation of Rad18 through Rad6-dependent mono- and polyubiquitination. J. Biol. Chem. 280, 515–524.
Nakajima, S., Lan, L., Kanno, S., Usami, N., Kobayashi, K., Mori, M., Shiomi, T. & Yasui, A. (2006) Replication-dependent and-independent responses of RAD18 to DNA damage in human cells. J. Biol. Chem. 281, 34687–34695.
Notenboom, V., Hibbert, R.G., van Rossum-Fikkert, S.E., Olsen, J.V., Mann, M. & Sixma, T.K. (2007) Functional characterization of Rad18 domains for Rad6, ubiquitin, DNA binding and PCNA modification. Nucleic Acids Res. 35, 5819–5830.
Okubo, S., Hara, F., Tsuchida, Y., Shimotakahara, S., Suzuki, S., Hatanaka, H., Yokoyama, S., Tanaka, H., Yasuda, H. & Shindo, H. (2004) NMR structure of the N-terminal domain of SUMO ligase PIAS1 and its interaction with tumor suppressor p53 and A/T-rich DNA oligomers. J. Biol. Chem. 279, 31455–31461.
Plosky, B.S., Vidal, A.E., de Henestrosa, A.R., McLenigan, M.P., McDonald, J.P., Mead, S. & Woodgate, R. (2006) Controlling the subcellular localization of DNA polymerases and via interactions with ubiquitin. EMBO J. 25, 2847–2855.[CrossRef][Medline]
Rupp, W.D. & Howard-Flanders, P. (1968) Discontinuities in the DNA synthesized in an excision-defective strain of Escherichia coli following ultraviolet irradiation. DNA Repair (Amst) 31, 291–304.
Stelter, P. & Ulrich, H.D. (2003) Control of spontaneous and damage-induced mutagenesis by SUMO and ubiquitin conjugation. Nature 425, 188–191.[CrossRef][Medline]
Tateishi, S., Sakuraba, Y., Masuyama, S., Inoue, H. & Yamaizumi, M. (2000) Dysfunction of human Rad18 results in defective postreplication repair and hypersensitivity to multiple mutagens. Proc. Natl. Acad. Sci. USA 97, 7927–7932.
Tercero, J.A., Longhese, M.P. & Diffley, J.F. (2003) A central role for DNA replication forks in checkpoint activation and response. Mol. Cell 11, 1323–1336.[CrossRef][Medline]
Watanabe, K., Tateishi, S., Kawasuji, M., Tsurimoto, T., Inoue, H. & Yamaizumi, M. (2004) Rad18 guides pol to replication stalling sites through physical interaction and PCNA monoubiquitination. EMBO J. 23, 3886–3896.[CrossRef][Medline]
Yuasa, M.S., Masutani, C., Hirano, A., Cohn, M.A., Yamaizumi, M., Nakatani, Y. & Hanaoka, F. (2006) A human DNA polymerase complex containing Rad18, Rad6 and Rev1; proteomic analysis and targeting of the complex to the chromatin-bound fraction of cells undergoing replication fork arrest. Genes Cells 11, 731–744.
Zhang, Z., Zhu, L., Lin, D., Chen, F., Chen, D.J. & Chen, Y. (2001) The three-dimensional structure of the C-terminal DNA-binding domain of human Ku70. J. Biol. Chem. 276, 38231–38236.
Zou, L. & Elledge, S.J. (2003) Sensing DNA damage through ATRIP recognition of RPA–ssDNA complexes. Science 300, 1542–1548.
Received: 7 December 2007
Accepted: 1 January 2008
This article has been cited by other articles:
|
|
 |
|
  M. Suzuki, A. Niimi, S. Limsirichaikul, S. Tomida, Q. Miao Huang, S. Izuta, J. Usukura, Y. Itoh, T. Hishida, T. Akashi, et al. PCNA Mono-Ubiquitination and Activation of Translesion DNA Polymerases by DNA Polymerase {alpha} J. Biochem., July 1, 2009; 146(1): 13 - 21. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Watanabe, K. Iwabuchi, J. Sun, Y. Tsuji, T. Tani, K. Tokunaga, T. Date, M. Hashimoto, M. Yamaizumi, and S. Tateishi RAD18 promotes DNA double-strand break repair during G1 phase through chromatin retention of 53BP1 Nucleic Acids Res., April 1, 2009; 37(7): 2176 - 2193. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Niimi, S. Brown, S. Sabbioneda, P. L. Kannouche, A. Scott, A. Yasui, C. M. Green, and A. R. Lehmann Regulation of proliferating cell nuclear antigen ubiquitination in mammalian cells PNAS, October 21, 2008; 105(42): 16125 - 16130. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | ADVANCED SEARCH | TABLE OF CONTENTS |
), ssDNA (
), Y-1 DNA (
) was quantified. The following 120-mer DNA substrates were used in binding reactions: (F) 120-mer dsDNA, (G) 80-mer dsDNA with a 40-mer ssDNA tail, (H) 40-mer dsDNA with an 80-mer ssDNA tail, (I) 120-mer ssDNA, (J) 120-mer forked DNA with single-stranded region as Y-1. RAD6–RAD18 was added at various concentrations (0, 0.1, 0.2, 0.3, 0.4 and 0.6 µM). Protein–DNA complexes were analyzed by EMSA. An asterisk represents the position of 32P-labeling. (K) Binding of RAD6–RAD18 to 120-mer dsDNA (
), 40-mer dsDNA with an 80-mer ssDNA tail (
), 120-mer ssDNA (
) was quantified. (L) Binding of RAD6–RAD18 to 120-mer ssDNA was confirmed by the addition of an anti-RAD18 antibody. The 120-mer ssDNA alone (lane 1), DNA with 0.2 µM RAD6–RAD18 (lane 2), DNA with RAD6–RAD18 and the anti-RAD18 antibody (lane 3), DNA with RAD6–RAD18 and an anti-c-Myc antibody (lane 4), DNA with the anti-RAD18 antibody (lane 5) or DNA with the anti-c-Myc antibody (lane 6) was reacted in the presence of 150 mM NaCl, and the protein–DNA complexes were analyzed by EMSA.
