Functional characterization of Rad18 domains for Rad6, ubiquitin, DNA binding and PCNA modification

Rad18 is a ubiquitin E3 ligase that monoubiquitinates PCNA on stalled replications forks. This allows recruitment of damage-tolerant polymerases for damage bypass and DNA repair. In this activity, the Rad18 protein has to interact with Rad6, the E2 ubiquitin-conjugating enzyme, ubiquitin, PCNA and DNA. Here we analyze the biochemical interactions of specific domains of the Rad18 protein. We found that the Rad6/Rad18 complex forms stable dimers in vitro. Consistent with previous findings, both the Ring domain and a C-terminal region contribute to the Rad6 interaction, while the C-terminus is not required for the interaction with PCNA. Surprisingly we find that the C2HC zinc finger is important for interaction with ubiquitin, apparently analogous to the interactions of classical zinc fingers with ubiquitin such as found in the UBZ and UBM domains in Y-family polymerases. Finally we find that the SAP domain, but not the zinc finger domain, is capable of DNA binding in vitro.     

Rad18 guides poleta to replication stalling sites through physical interaction and PCNA monoubiquitination

The DNA replication machinery stalls at damaged sites on templates, but normally restarts by switching to a specialized DNA polymerase(s) that carries out translesion DNA synthesis (TLS). In human cells, DNA polymerase eta (poleta) accumulates at stalling sites as nuclear foci, and is involved in ultraviolet (UV)-induced TLS. Here we show that poleta does not form nuclear foci in RAD18(-/-) cells after UV irradiation. Both Rad18 and Rad6 are required for poleta focus formation. In wild-type cells, UV irradiation induces relocalization of Rad18 in the nucleus, thereby stimulating colocalization with proliferating cell nuclear antigen (PCNA), and Rad18/Rad6-dependent PCNA monoubiquitination. Purified Rad18 and Rad6B monoubiquitinate PCNA in vitro. Rad18 associates with poleta constitutively through domains on their C-terminal regions, and this complex accumulates at the foci after UV irradiation. Furthermore, poleta interacts preferentially with monoubiquitinated PCNA, but poldelta does not. These results suggest that Rad18 is crucial for recruitment of poleta to the damaged site through protein-protein interaction and PCNA monoubiquitination.     

Rad18 E3 ubiquitin ligase activity mediates Fanconi anemia pathway activation and cell survival following DNA Topoisomerase 1 inhibition

Camptothecin (CPT) and related chemotherapeutic drugs induce formation of DNA Topoisomerase I (Top1) covalent or cleavage complexes (Top1ccs) that block leading-strand DNA synthesis and elicit DNA Double Stranded Breaks (DSB) during S phase. The Fanconi Anemia (FA) pathway is implicated in tolerance of CPT-induced DNA damage yet the mechanism of FA pathway activation by Top1 poisons has not been studied. We show here that the FA core complex protein FANCA and monoubiquitinated FANCD2 (an effector of the FA pathway) are rapidly mobilized to chromatin in response to CPT treatment in several human cancer cell lines and untransformed primary human dermal fibroblasts. FANCD2 depletion using siRNA leads to impaired recovery from CPT-induced inhibition or DNA synthesis, persistence of γH2AX (a DSB marker) and reduced cell survival following CPT treatment. The E3 ubiquitin ligase Rad18 is necessary for CPT-induced recruitment of FANCA and FANCD2 to chromatin. Moreover, Rad18-depletion recapitulates the DNA synthesis and survival defects of FANCD2-deficiency in CPT-treated cells. It is well-established that Rad18 promotes FA pathway activation and DNA damage tolerance in response to bulky DNA lesions via a mechanism involving PCNA monoubiquitination. In contrast, PCNA monoubiquitination is not involved in Rad18-mediated FA pathway activation or cell survival following acquisition of CPT-induced DSB. Moreover, while Rad18 is implicated in recombinational repair of DSB via an E3 ligase-independent mechanism, we demonstrate that Rad18 E3 ligase activity is essential for appropriate FA pathway activation and DNA damage tolerance after CPT treatment. Taken together, our results define a novel pathway of Rad18-dependent DSB repair that is dissociable from known Rad18-mediated DNA repair mechanisms based on its independence from PCNA ubiquitination and requirement for E3 ligase activity.     

Rad18 E3 ubiquitin ligase activity mediates Fanconi anemia pathway activation and cell survival following DNA topoisomerase 1 inhibition

Camptothecin (CPT) and related chemotherapeutic drugs induce formation of DNA topoisomerase I (Top1) covalent or cleavage complexes (Top1ccs) that block leading-strand DNA synthesis and elicit DNA Double Stranded Breaks (DSB) during S phase. The Fanconi Anemia (FA) pathway is implicated in tolerance of CPT-induced DNA damage yet the mechanism of FA pathway activation by Top1 poisons has not been studied. We show here that the FA core complex protein FANCA and monoubiquitinated FANCD2 (an effector of the FA pathway) are rapidly mobilized to chromatin in response to CPT treatment in several human cancer cell lines and untransformed primary human dermal fibroblasts. FANCD2 depletion using siRNA leads to impaired recovery from CPT-induced inhibition or DNA synthesis, persistence of γH2AX (a DSB marker) and reduced cell survival following CPT treatment. The E3 ubiquitin ligase Rad18 is necessary for CPT-induced recruitment of FANCA and FANCD2 to chromatin. Moreover, Rad18-depletion recapitulates the DNA synthesis and survival defects of FANCD2-deficiency in CPT-treated cells. It is well-established that Rad18 promotes FA pathway activation and DNA damage tolerance in response to bulky DNA lesions via a mechanism involving PCNA monoubiquitination. In contrast, PCNA monoubiquitination is not involved in Rad18-mediated FA pathway activation or cell survival following acquisition of CPT-induced DSB. Moreover, while Rad18 is implicated in recombinational repair of DSB via an E3 ligase-independent mechanism, we demonstrate that Rad18 E3 ligase activity is essential for appropriate FA pathway activation and DNA damage tolerance after CPT treatment. Taken together, our results define a novel pathway of Rad18-dependent DSB repair that is dissociable from known Rad18-mediated DNA repair mechanisms based on its independence from PCNA ubiquitination and requirement for E3 ligase activity.Key words: camptothecin, Rad18, topoisomerase I, double strand breaks, Fanconi anemia     

Mechanistic analysis of PCNA poly‐ubiquitylation by the ubiquitin protein ligases Rad18 and Rad5

Poly‐ubiquitylation is a common post‐translational modification that can impart various functions to a target protein. Several distinct mechanisms have been reported for the assembly of poly‐ubiquitin chains, involving either stepwise transfer of ubiquitin monomers or attachment of a preformed poly‐ubiquitin chain and requiring either a single pair of ubiquitin‐conjugating enzyme (E2) and ubiquitin ligase (E3), or alternatively combinations of different E2s and E3s. We have analysed the mechanism of poly‐ubiquitylation of the replication clamp PCNA by two cooperating E2–E3 pairs, Rad6–Rad18 and Ubc13–Mms2–Rad5. We find that the two complexes act sequentially and independently in chain initiation and stepwise elongation, respectively. While loading of PCNA onto DNA is essential for recognition by Rad6–Rad18, chain extension by Ubc13–Mms2–Rad5 is only slightly enhanced by loading. Moreover, in contrast to initiation, chain extension is tolerant to variations in the attachment site of the proximal ubiquitin moiety. Our results provide information about a unique conjugation mechanism that appears to be specialised for a regulatable pattern of dual modification.     

Symmetry and asymmetry of the RING-RING dimer of Rad18

The human ubiquitin-conjugating enzyme Rad6 (E2), with ubiquitin ligase enzyme Rad18 (RING E3), monoubiquitinates proliferating cell nuclear antigen at stalled replication forks in DNA translesion synthesis. Here, we determine the structure of the homodimeric Rad18 RING domains by X-ray crystallography and classify it to RING-RING dimers that dimerize through helices adjacent to the RING domains and through the canonical RING domains. Using NMR spectroscopy and site-directed mutagenesis, we demonstrate that the Rad6b binding site, for the Rad18 RING domain, strongly resembles that of other E2/E3 RING/U-box complexes. We show that the homodimeric Rad18 RING domain can recruit two Rad6b E2 enzymes, whereas the full-length Rad18 homodimer binds only to a single Rad6b molecule. Such asymmetry is a common feature of RING-RING heterodimers and has been observed for the CHIP U-box homodimer. We propose that asymmetry may be a common feature of dimeric RING E3 ligases.     

Rad18-mediated Translesion Synthesis of Bulky DNA Adducts Is Coupled to Activation of the Fanconi Anemia DNA Repair Pathway

Fanconi anemia (FA) is a cancer susceptibility syndrome characterized by sensitivity to DNA-damaging agents. The FA proteins (FANCs) are implicated in DNA repair, although the precise mechanisms by which FANCs process DNA lesions are not fully understood. An epistatic relationship between the FA pathway and translesion synthesis (TLS, a post-replication DNA repair mechanism) has been suggested, but the basis for cross-talk between the FA and TLS pathways is poorly understood. We show here that ectopic overexpression of the E3 ubiquitin ligase Rad18 (a central regulator of TLS) induces DNA damage-independent mono-ubiquitination of proliferating cell nuclear antigen (PCNA) (a known Rad18 substrate) and FANCD2. Conversely, DNA damage-induced mono-ubiquitination of both PCNA and FANCD2 is attenuated in Rad18-deficient cells, demonstrating that Rad18 contributes to activation of the FA pathway. WT Rad18 but not an E3 ubiquitin ligase-deficient Rad18 C28F mutant fully complements both PCNA ubiquitination and FANCD2 activation in Rad18-depleted cells. Rad18-induced mono-ubiquitination of FANCD2 is not observed in FA core complex-deficient cells, demonstrating that Rad18 E3 ligase activity alone is insufficient for FANCD2 ubiquitylation. Instead, Rad18 promotes FA core complex-dependent FANCD2 ubiquitination in a manner that is secondary to PCNA mono-ubiquitination. Taken together, these results demonstrate a novel Rad18-dependent mechanism that couples activation of the FA pathway with TLS.     

Domains required for dimerization of yeast Rad6 ubiquitin-conjugating enzyme and Rad18 DNA binding protein.

The RAD6 gene of Saccharomyces cerevisiae encodes a ubiquitin-conjugating enzyme required for postreplicational repair of UV-damaged DNA and for damage-induced mutagenesis. In addition, Rad6 functions in the N end rule pathway of protein degradation. Rad6 mediates its DNA repair role via its association with Rad18, whose DNA binding activity may target the Rad6-Rad18 complex to damaged sites in DNA. In its role in N end-dependent protein degradation, Rad6 interacts with the UBR1-encoded ubiquitin protein ligase (E3) enzyme. Previous studies have indicated the involvement of N-terminal and C-terminal regions of Rad6 in interactions with Ubr1. Here, we identify the regions of Rad6 and Rad18 that are involved in the dimerization of these two proteins. We show that a region of 40 amino acids towards the C terminus of Rad18 (residues 371 to 410) is sufficient for interaction with Rad6. This region of Rad18 contains a number of nonpolar residues that have been conserved in helix-loop-helix motifs of other proteins. Our studies indicate the requirement for residues 141 to 149 at the C terminus, and suggest the involvement of residues 10 to 22 at the N terminus of Rad6, in the interaction with Rad18. Each of these regions of Rad6 is indicated to form an amphipathic helix.     

PCNA modifications for regulation of post-replication repair pathways

Stalled DNA replication forks activate specific DNA repair mechanism called post-replication repair (PRR) pathways that simply bypass DNA damage. The bypassing of DNA damage by PRR prevents prolonged stalling of DNA replication that could result in double strand breaks (DSBs). Proliferating cell nuclear antigen (PCNA) functions to initiate and choose different bypassing pathways of PRR. In yeast, DNA replication forks stalled by DNA damage induces monoubiquitination of PCNA at K164, which is catalyzed by Rad6/Rad18 complex. PCNA monoubiquitination triggers the replacement of replicative polymerase with special translesion synthesis (TLS) polymerases that are able to replicate past DNA lesions. The PCNA interaction motif and/or the ubiquitin binding motif in most TLS polymerases seem to be important for the regulation of TLS. The TLS pathway is usually error-prone because TLS polymerases have low fidelity and no proofreading activity. PCNA can also be further polyubiquitinated by Ubc13/ Mms2/Rad5 complex, which adds an ubiquitin chain onto monoubiquitinated K164 of PCNA. PCNA polyubiquitination directs a different PRR pathway known as error-free damage avoidance, which uses the newly synthesized sister chromatid as a template to bypass DNA damage presumably through template switching mechanism. Mammalian homologues of all of the yeast PRR proteins have been identified, thus PRR is well conserved throughout evolution. Mutations of some PRR genes are associated with a higher risk for cancers in mice and human patients, strongly supporting the importance of PRR as a tumor suppressor pathway.     

Ubiquitin carrier protein-catalyzed ubiquitin transfer to histones. Mechanism and specificity

Ubiquitinated derivatives of histones H2A and H2B, in which the carboxyl terminus of ubiquitin is joined to epsilon-amino groups of specific lysine residues of each histone, occur in vivo. Certain ubiquitin carrier proteins (E2s) catalyze ubiquitin transfer to histones (Pickart, C. M., and Rose, I. A. (1985) J. Biol. Chem. 260, 1573-1581). The catalytic activities of these purified ubiquitin carrier proteins have been quantitatively characterized with purified histones, in order to determine if one or more of them exhibits specificity for H2A over other histones (H3,H4) which are not known to be ubiquitinated in vivo. The results show the following. 1) No E2 exhibits strong specificity for H2A over the other histones. 2) For a given histone, kinetics of formation of its monoubiquitinated adduct do not differ strongly among the E2s; sigmoid kinetics (nH = 2) are generally observed, with values of K 0.5 ranging from 2-6 microM. 3) E214K catalyzes primarily monoubiquitination. 4) E220K catalyzes multiple ubiquitination (up to three ubiquitin/histone) by a processive mechanism that involves joining of ubiquitin carboxyl termini to multiple histone lysine residues. 5) E235K also catalyzes processive ubiquitination, with formation of polyubiquitinated products exhibiting a lag phase. Many of the polyubiquitinated adducts produced at low histone concentration are larger than expected for monoubiquitination of every histone-lysine residue, and polyubiquitination is selectively inhibited by substitution of reductively methylated ubiquitin for ubiquitin. These results suggest that E235K uniquely catalyzes ubiquitin transfer to lysine residues of previously conjugated ubiquitin molecule(s). The implications of these results for biological mechanisms of histone ubiquitination are discussed.     

Stability of Rad51 recombinase and persistence of Rad51 DNA repair foci depends on post-translational modifiers,ubiquitin and SUMO

The DNA double-strand breaks are particularly deleterious, especially when an error-free repair pathway is unavailable, enforcing the error-prone recombination pathways to repair the lesion. Cells can resume the cell cycle but at the expense of decreased viability due to genome rearrangements. One of the major players involved in recombinational repair of DNA damage is Rad51 recombinase, a protein responsible for presynaptic complex formation. We previously showed that an increased level of this protein promotes the usage of illegitimate recombination. Here we show that the level of Rad51 is regulated via the ubiquitin-dependent proteolytic pathway. The ubiquitination of Rad51 depends on multiple E3 enzymes, including SUMO-targeted ubiquitin ligases. We also demonstrate that Rad51 can be modified by both ubiquitin and SUMO. Moreover, its modification with ubiquitin may lead to opposite effects: degradation dependent on Rad6, Rad18, Slx8, Dia2, and the anaphase-promoting complex, or stabilization dependent on Rsp5. We also show that post-translational modifications with SUMO and ubiquitin affect Rad51's ability to form and disassemble DNA repair foci, respectively, influencing cell cycle progression and cell viability in genotoxic stress conditions. Our data suggest the existence of a complex E3 ligases network that regulates Rad51 recombinase's turnover, its molecular activity, and access to DNA, limiting it to the proportions optimal for the actual cell cycle stage and growth conditions, e.g., stress. Dysregulation of this network would result in a drop in cell viability due to uncontrolled genome rearrangement in the yeast cells. In mammals would promote the development of genetic diseases and cancer.     

The Rad5 helicase activity is dispensable for error-free DNA post-replication repair

DNA post-replication repair (PRR) functions to bypass replication-blocking lesions and is subdivided into two parallel pathways: error-prone translesion DNA synthesis and error-free PRR. While both pathways are dependent on the ubiquitination of PCNA, error-free PRR utilizes noncanonical K63-linked polyubiquitinated PCNA to signal lesion bypass through template switch, a process thought to be dependent on Mms2-Ubc13 and a RING finger motif of the Rad5 ubiquitin ligase. Previous in vitro studies demonstrated the ability of Rad5 to promote replication fork regression, a function dependent on its helicase activity. To investigate the genetic and mechanistic relationship between fork regression in vitro and template switch in vivo, we created and characterized site-specific mutations defective in the Rad5 RING or helicase activity. Our results indicate that both the Rad5 ubiquitin ligase and the helicase activities are exclusively involved in the same error-free PRR pathway. Surprisingly, the Rad5 helicase mutation abolishes its physical interaction with Ubc13 and the K63-linked PCNA polyubiquitin chain assembly. Indeed, physical fusions of Rad5 with Ubc13 bypass the requirement for either the helicase or the RING finger domain. Since the helicase domain overlaps with the SWI/SNF chromatin-remodelling domain, our findings suggest a structural role of this domain and that the Rad5 helicase activity is dispensable for error-free lesion bypass.     

SHIP2 (SH2 Domain-containing Inositol Phosphatase 2) SH2 Domain Negatively Controls SHIP2 Monoubiquitination in Response to Epidermal Growth Factor

The SH2 domain containing inositol 5-phosphatase SHIP2 contains several interacting domains that are important for scaffolding properties. We and others have previously reported that SHIP2 interacts with the E3 ubiquitin ligase c-Cbl. Here, we identified human SHIP2 monoubiquitination on lysine 315. SHIP2 could also be polyubiquitinated but was not degraded by the 26 S proteasome. Furthermore, we identified a ubiquitin-interacting motif at the C-terminal end of SHIP2 that confers ubiquitin binding capacity. However, this ubiquitin-interacting motif is dispensable for its monoubiquitination. We showed that neither c-Cbl nor Nedd4-1 play the role of ubiquitin ligase for SHIP2. Strikingly, monoubiquitination of the ΔSH2-SHIP2 mutant (lacking the N-terminal SH2 domain) is strongly increased, suggesting an intrinsic inhibitory effect of the SHIP2 SH2 domain on its monoubiquitination. Moreover, SHIP2 monoubiquitination was increased upon 30 min of epidermal growth factor stimulation. This correlates with the loss of interaction between the SHIP2 SH2 domain and c-Cbl. In this model, c-Cbl could mask the monoubiquitination site and thereby prevent SHIP2 monoubiquitination. The present study thus reveals an unexpected and novel role of SHIP2 SH2 domain in the regulation of its newly identified monoubiquitination.     

Role of a non-canonical surface of Rad6 in ubiquitin conjugating activity

Rad6 is a yeast E2 ubiquitin conjugating enzyme that monoubiquitinates histone H2B in conjunction with the E3, Bre1, but can non-specifically modify histones on its own. We determined the crystal structure of a Rad6∼Ub thioester mimic, which revealed a network of interactions in the crystal in which the ubiquitin in one conjugate contacts Rad6 in another. The region of Rad6 contacted is located on the distal face of Rad6 opposite the active site, but differs from the canonical E2 backside that mediates free ubiquitin binding and polyubiquitination activity in other E2 enzymes. We find that free ubiquitin interacts weakly with both non-canonical and canonical backside residues of Rad6 and that mutations of non-canonical residues have deleterious effects on Rad6 activity comparable to those observed to mutations in the canonical E2 backside. The effect of non-canonical backside mutations is similar in the presence and absence of Bre1, indicating that contacts with non-canonical backside residues govern the intrinsic activity of Rad6. Our findings shed light on the determinants of intrinsic Rad6 activity and reveal new ways in which contacts with an E2 backside can regulate ubiquitin conjugating activity.     

Rad18 regulates DNA polymerase kappa and is required for recovery from S-phase checkpoint-mediated arrest

We have investigated mechanisms that recruit the translesion synthesis (TLS) DNA polymerase Polkappa to stalled replication forks. The DNA polymerase processivity factor PCNA is monoubiquitinated and interacts with Polkappa in cells treated with the bulky adduct-forming genotoxin benzo[a]pyrene dihydrodiol epoxide (BPDE). A monoubiquitination-defective mutant form of PCNA fails to interact with Polkappa. Small interfering RNA-mediated downregulation of the E3 ligase Rad18 inhibits BPDE-induced PCNA ubiquitination and association between PCNA and Polkappa. Conversely, overexpressed Rad18 induces PCNA ubiquitination and association between PCNA and Polkappa in a DNA damage-independent manner. Therefore, association of Polkappa with PCNA is regulated by Rad18-mediated PCNA ubiquitination. Cells from Rad18(-/-) transgenic mice show defective recovery from BPDE-induced S-phase checkpoints. In Rad18(-/-) cells, BPDE induces elevated and persistent activation of checkpoint kinases, indicating persistently stalled forks due to defective TLS. Rad18-deficient cells show reduced viability after BPDE challenge compared with wild-type cells (but survival after hydroxyurea or ionizing radiation treatment is unaffected by Rad18 deficiency). Inhibition of RPA/ATR/Chk1-mediated S-phase checkpoint signaling partially inhibited BPDE-induced PCNA ubiquitination and prevented interactions between PCNA and Polkappa. Taken together, our results indicate that ATR/Chk1 signaling is required for Rad18-mediated PCNA monoubiquitination. Recruitment of Polkappa to ubiquitinated PCNA enables lesion bypass and eliminates stalled forks, thereby attenuating the S-phase checkpoint.     

A conditional yeast E1 mutant blocks the ubiquitin-proteasome pathway and reveals a role for ubiquitin conjugates in targeting Rad23 to the proteasome

E1 ubiquitin activating enzyme catalyzes the initial step in all ubiquitin-dependent processes. We report the isolation of uba1-204, a temperature-sensitive allele of the essential Saccharomyces cerevisiae E1 gene, UBA1. Uba1-204 cells exhibit dramatic inhibition of the ubiquitin-proteasome system, resulting in rapid depletion of cellular ubiquitin conjugates and stabilization of multiple substrates. We have employed the tight phenotype of this mutant to investigate the role ubiquitin conjugates play in the dynamic interaction of the UbL/UBA adaptor proteins Rad23 and Dsk2 with the proteasome. Although proteasomes purified from mutant cells are intact and proteolytically active, they are depleted of ubiquitin conjugates, Rad23, and Dsk2. Binding of Rad23 to these proteasomes in vitro is enhanced by addition of either free or substrate-linked ubiquitin chains. Moreover, association of Rad23 with proteasomes in mutant and wild-type cells is improved upon stabilizing ubiquitin conjugates with proteasome inhibitor. We propose that recognition of polyubiquitin chains by Rad23 promotes its shuttling to the proteasome in vivo.     

Supramolecular complex formation between Rad6 and proteins of the p53 pathway during DNA damage-induced response

The HR6A and -B genes, homologues of the yeast Rad6 gene, encode ubiquitin-conjugating enzymes that are required for postreplication repair of DNA and damage-induced mutagenesis. Using surface plasmon resonance, we show here that HR6 protein (referred as Rad6) physically interacts with p53. Analysis of proteins coimmunoprecipitated with Rad6 antibody from metabolically labeled normal MCF10A human breast epithelial cells not only confirmed Rad6-p53 interactions in vivo but also demonstrated for the first time that exposure of MCF10A cells to cisplatin or adriamycin (ADR) induces recruitment of p14ARF into Rad6-p53 complexes. Further analysis of ADR-induced p53 response showed that stable Rad6-p53-p14ARF complex formation is associated with a parallel increase and decrease in monoubiquitinated and polyubiquitinated p53, respectively, and arrest in G(2)/M phase of the cell cycle. Interestingly, the ADR-induced suppression of p53 polyubiquitination correlated with a corresponding decline in intact Hdm2 protein levels. Treatment of MCF10A cells with MG132, a 26S proteasome inhibitor, effectively stabilized monoubiquitinated p53 and rescued ADR-induced downregulation of Hdm2. These data suggest that ADR-induced degradation of Hdm2 occurs via the ubiquitin-proteasome pathway. Rad6 is present in both the cytoplasmic and nuclear compartments of normal MCF10A cells, although in response to DNA damage it is predominantly found in the nucleus colocalizing with ubiquitinated p53, whereas Hdm2 is undetectable. Consistent with in vivo data, results from in vitro ubiquitination assays show that Rad6 mediates addition of one (mono-) to two (multimono-) ubiquitin molecules on p53 and that inclusion of Mdm2 is essential for its polyubiquitination. The data presented in the present study suggest that Rad6-p53-p14ARF complex formation and p53 ubiquitin modification are important damage-induced responses that perhaps determine the fidelity of DNA postreplication repair.