Chemical Protein Polyubiquitination Reveals the Role of a

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Chemical Protein Polyubiquitination Reveals the Role of a Noncanonical Polyubiquitin Chain in DNA Damage Tolerance Kun Yang,† Ping Gong,† Parikshit Gokhale, and Zhihao Zhuang* Department of Chemistry and Biochemistry, 214A Drake Hall, University of Delaware, Newark, Delaware 19716, United States S Supporting Information *

ABSTRACT: Polyubiquitination of proteins regulates a variety of cellular processes, including protein degradation, NF-κB pathway activation, apoptosis, and DNA damage tolerance. Methods for generating polyubiquitinated protein with defined ubiquitin chain linkage and length are needed for an in-depth molecular understanding of protein polyubiquitination. However, enzymatic protein polyubiquitination usually generates polyubiquitinated proteins with mixed chain lengths in a low yield. We report herein a new chemical approach for protein polyubiquitination with a defined ubiquitin chain length and linkage under a mild condition that preserves the native fold of the target protein. In DNA damage tolerance, K63polyubiquitinated proliferating cell nuclear antigen (PCNA) plays an important yet unclear role in regulating the selection of the error-free over error-prone lesion bypass pathways. Using the chemically polyubiquitinated PCNA, we revealed a mechanism of the K63 polyubiquitin chain on PCNA in promoting the error-free lesion bypass by suppressing the DNA translesion synthesis (TLS).

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PCNA polyubiquitination channels the DNA damage tolerance from the error-prone to the error-free lesion bypass pathway remains unclear. To obtain a molecular understanding of the roles of PCNA polyubiquitination in DNA damage tolerance, sufficient homogeneously polyubiquitinated PCNA with a defined ubiquitin chain length and linkage is required. However, enzymatic polyubiquitination usually results in protein products with a wide range of ubiquitin chain lengths. Although polyubiquitin chains with a defined length have been prepared semienzymatically10 or through solid-phase synthesis and chemical ligation,11−15 reports of chemical polyubiquitination of target proteins with defined chain length and linkage have been lacking. Very recently, elegant nonenzymatic polyubiquitination methods were reported to conjugate a K48-linked tetraubiquitin chain to a specific site in α-Synuclein and α-globin, respectively.16,17 These represent the first reports to chemically polyubiquitinate a target protein using solid-phase peptide synthesis (SPPS) under denaturing conditions. PCNA is a homotrimeric protein with a combined molecular weight of 87 kDa. To maintain the higher-order protein structure of the target protein, we have developed a strategy to polyubiquitinate PCNA with a defined ubiquitin chain length and a K63 linkage under a mild condition by photocaging and intein-mediated chemical ligation. With the well-defined polyUb-PCNA species, we obtained new insights into the role of K63-polyubiquitin chain in DNA damage tolerance.

biquitination is an important post-translational protein modification that regulates a variety of cellular processes.1 Protein ubiquitination is catalyzed by a E1-E2-E3 enzyme cascade,2 in which a single ubiquitin or a polyubiquitin chain linked through any of the seven lysine residues on ubiquitin is conjugated to a target protein. Different from K48-linked polyubiquitination that mediates protein degradation, K63linked polyubiquitination functions in many nonproteolytic pathways, including protein sorting, NF-κB pathway activation, and DNA damage tolerance (DDT).1 Although K63-linked polyubiquitination is widespread in cells, our understanding of its function remains limited, particularly in DNA damage tolerance. The difficulty of generating K63-linked polyubiquitinated proteins with defined ubiquitin chain length and in a sufficient quantity has hampered an in-depth molecular understanding of protein polyubiquitination in many fundamentally important cellular pathways. Ubiquitination of PCNA has been identified as an important regulatory event in DNA damage response.3 Following DNA damage, PCNA is monoubiquitinated by Rad6-Rad18 at Lys164 and further polyubiquitinated by Ubc13-Mms2-Rad5 through a K63-linked polyubiquitin chain.4 Monoubiquitination of PCNA acts to recruit specialized DNA polymerases to activate the DNA translesion synthesis (TLS).5 Compared to monoubiquitination of PCNA, the function of K63 polyubiquitinated PCNA in DNA damage response is less well understood. K63-linked polyubiquitination of PCNA has been shown to reduce the TLS polymerase-mediated mutagenesis in human cells.6 More recently, K63-linked polyubiquitination of PCNA was suggested to recruit human ZRANB3/AH2 to restart the stalled replication fork and facilitate the error-free lesion bypass.7−9 At present the molecular details of how © 2014 American Chemical Society

Received: February 20, 2014 Accepted: June 5, 2014 Published: June 11, 2014 1685

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Figure 1. Chemical polyubiquitination of PCNA. (a) Generation of the DiUb building block. (b) Generation of K63-linked polyUb-PCNA species using the DiUb building block by photodeprotection and disulfide exchange reaction. (c) 12% denaturing SDS-PAGE gel showing PCNA, MonoUb-, DiUb-, TriUb-, and TetraUb-PCNA species.

We first generated a ubiquitin moiety modified with a 2nitrobenzyl group at residue 63 where a cysteine was introduced to replace the original lysine (Figure 1a). The reaction product 2-nitrobenzyl-Ub (NB-Ub) was confirmed by mass spectrometry (MW of 8667 Da) (Supplementary Figure 1a). In parallel, an ubiquitin-cysteamine (Ub-SH) species was generated following the published procedure (Supplementary Figure 1b).18 From NB-Ub and Ub-SH, we generated a diubiquitin (DiUb) building block using the mouse E1 and human Ubc13-Mms210 (Figure 1a and Supplementary Figure 1c). To prepare polyubiquitinated PCNA, we first ligated the diubiquitin building block to a mutant PCNA with a single cysteine introduced at position 164 (Figure 1b) following a

previously reported chemical ubiquitination method.18 The product DiUb-PCNA was analyzed on a 12% SDS-PAGE gel (Figure 1c) and confirmed by mass spectrometry analysis. The determined molecular weight (MW) of 46,093 Da agreed well with the calculated MW of 46,090 Da (Supplementary Figure 2a). The position of modification on PCNA was confirmed by tryptic digestion of DiUb-PCNA and MS/MS analysis of the diglycine (GG)-bearing PCNA peptide (Supplementary Figure 2b and Supplementary Figure 3a). Furthermore, we confirmed the K63 DiUb linkage between the proximal and distal ubiquitin moieties by identifying a characteristic ubiquitin peptide flanking K63 conjugated with a diglycine tag (Supplementary Figure 2c and Supplementary Figure 3b). 1686

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Figure 2. Polyubiquitinated PCNA inhibits TLS opposite an abasic site by Polη. (a) DNA substrate used for the TLS assay. The two circles indicate the biotin−streptavidin blocks. X indicates the abasic site. (b) A representative 17% denaturing PAGE gel showing the DNA synthesis opposite the abasic site by Polη in the presence of 50 nM concentration of each of the PCNA species as indicated using the above abasic site-containing DNA. (c) Quantification of the incorporation efficiency opposite the abasic site by Polη with increasing concentration of the various PCNA species as indicated. (d) Pull-down of the different polyUb-PCNA species by the N-terminally His-tagged full-length Polη. A mixture of equal amounts of the various PCNA species was used as input. Control pull-down was carried out using the Ni2+ resin with no Polη bound. One-tenth of the input and the entire pulled down sample were separated on a 12% SDS-PAGE gel followed by Western blotting using anti-PCNA antibody. One-tenth of the unbound PCNA species was also loaded. (e) Quantified pull-down efficiency for the different PCNA species by Polη. The pull-down efficiency was calculated based on the band intensity in panel d as an average of three independent experiments.

PCNA, respectively, from 1.0 mg of PCNA as a starting material. We found that the polyubiquitinated PCNA species were stable in the presence of a moderate concentration of DTT or glutathione (0.1−0.5 mM) or after incubation with the yeast cell lysate (Supplementary Figure 8). With the polyubiquitinated PCNA species, we set out to investigate the potential roles of K63-linked polyubiquitination of PCNA in DNA damage tolerance. We first assayed the translesion synthesis past an abasic site by Polη in the presence of the chemically polyubiquitinated PCNA species. Agreeing with previous studies, monoubiquitinated PCNA promoted TLS opposite the abasic site by Polη19,20 (Figure 2a−c). However, the incorporation efficiency decreased as the ubiquitin chain length growing from mono- to tetra-Ub on PCNA at all three PCNA concentrations tested (Figure 2a−c). We ruled out the altered electrostatic interaction between PCNA and DNA21 and the reduced loading efficiency of PCNA by RFC in the presence of polyubiquitin chain given the similar RFC ATPase activities observed in loading polyUb-PCNAs of different chain lengths, all comparable to the unmodified PCNA (Supplementary Figure 9). Next, we investigated the interaction between Polη and the polyubiquitinated PCNA in a pull-down assay and found that the longer ubiquitin chain on PCNA increased the affinity of Polη for polyUb-PCNA species (Figure 2d,e). Polη is known to contain one ubiquitin-binding

Next, we prepared TriUb-PCNA and TetraUb-PCNA by growing the ubiquitin chain on DiUb-PCNA (Figure 1b). The 2-nitrobenzyl-modified cysteine residue (Cys63) on the distal ubiquitin of the DiUb-PCNA was first deprotected by UV irradiation at 365 nm in the presence of ascorbic acid. Following the photodeprotection, a K63-linked DiUb building block was ligated to the deprotected DiUb-PCNA to generate TetraUb-PCNA. Similarly, TriUb-PCNA was generated by ligating a single ubiquitin moiety to the deprotected DiUbPCNA. The K63 TriUb-PCNA and TetraUb-PCNA species were analyzed on a 12% SDS-PAGE gel (Figure 1c) and confirmed by mass spectrometry analysis. The determined MW of 54,573 Da and 63,222 Da agreed well with the calculated MW of 54,569 Da and 63,218 Da, respectively (Supplementary Figure 4a and Supplementary Figure 5a). MS/MS analysis of TriUb-PCNA species confirmed the correct PCNA-Ub and UbUb linkages (Supplementary Figure 4b−d and Supplementary Figure 6). The same was shown for TetraUb-PCNA (Supplementary Figure 5b−d and Supplementary Figure 7). Based on the gel, the DiUb-, TriUb-, and TetraUb-PCNA species were of high purity (>90%) and all three subunits of the PCNA trimer were homogeneously polyubiquitinated with a defined ubiquitin chain length. In a typical chemical polyubiquitination reaction, we obtained approximately 0.8, 0.5, and 0.5 mg of DiUb-PCNA, TriUb-PCNA, and TetraUb1687

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Figure 3. Polyubiquitinated PCNA suppresses the DNA synthesis of Polδ-PCNA complex. (a) DNA synthesis by the Polδ holoenzyme formed with the various PCNA species. A representative 1% alkaline agarose gel electrophoresis of the DNA product by the Polδ holoenzyme in the presence of 50 nM concentration of different PCNA species as indicated using a singly primed ssM13 DNA. (b) Quantification of the DNA synthesis product by the Polδ-(Ub)PCNA complexes with increasing concentration of the various PCNA species as indicated. (c) Pull-down of the different Ub-PCNA and PCNA species by the N-terminal MAT (metal affinity tag)-Polδ. The pull-down experiment was similar to that described in Figure 2d. (d) Quantified pull-down efficiency for the different PCNA species by Polδ. The pull-down efficiency was calculated based on the band intensity in panel c as an average of three independent experiments.

domain (UBZ) that binds a single ubiquitin moiety through its canonical Ile44 hydrophobic patch. Therefore, the binding affinity of Polη to each ubiquitin moiety in the K63 polyubiquitin chain should remain the same as Polη’s binding to the monoubiquitin moiety. Thus, we attributed the increased pull-down of polyUb-PCNA species by Polη to an avidity effect of polyubiquitin chain that promoted the association of Polη with polyUb-PCNA, in accord with an earlier observation that Polη prefers to bind longer free polyubiquitin chains.22 Given the trimeric structure of ubiquitinated PCNA, it is possible that the same Polη can bind one PCNA subunit through the PCNA interacting protein motif (PIP) and bind ubiquitin on another PCNA subunit. Such an interaction can be envisioned given a flexible conformation of Ub on PCNA as revealed by previous studies of MonoUb-PCNA.18,23 For K63-polyUb-PCNA, because of the extended conformation of the K63 ubiquitin chain, such a binding mode is less likely when Polη binds the distal ubiquitin moiety. In view of the reduced TLS efficiency across an abasic site by Polη, the K63-linked polyubiquitin chain likely traps Polη in a nonproductive mode. Alternatively, more than one Polη may bind to the same PCNA homotrimer due to the higher affinity of Polη to the polyubiquitin chain on PCNA. The resulted steric effect may lead to inhibition of the lesion bypass synthesis by Polη. To differentiate between the two possibilities, we assayed the lesion-bypass synthesis past an abasic site at increasing concentration of Polη in the presence of MonoUb- or TetraUb-PCNA (Supplementary Figure 10). The titration curve showed clear difference at the lower Polη concentrations with MonoUb-PCNA being most efficient in

promoting lesion bypass synthesis across the abasic site. However, with higher Polη concentrations (up to 100 nM) the lesion-bypass efficiency approached the same plateaued value for both MonoUb-PCNA and TetraUb-PCNA. This observation argued against the potential steric effect of multiple Polη binding to the same TetraUb-PCNA trimer and thus decreasing the lesion-bypass synthesis by Polη. The fact that higher Polη concentrations led to a similar level of DNA synthesis in the presence of the Mono- and TetraUb-PCNA species supports the notion that the inhibited lesion-bypass synthesis for polyUb-PCNA is due to the nonproductive trapping of Polη and can be overcome by higher concentration of Polη. Different from monoubiquitination, which does not affect the DNA synthesis of Polδ, we observed an overall decrease in DNA synthesis by the Polδ-(Ub)PCNA holoenzyme complex with longer polyubiquitin chain on PCNA at all three concentrations of the PCNA species tested (Figure 3a,b). We also found that the pull-down efficiency of the polyUb-PCNA species by Polδ was reduced with increased ubiquitin chain length (Figure 3c,d). One possible explanation is that the polyubiquitin chain may change the conformation of PCNA, which in turn lowers the affinity between Polδ and PCNA. However, the DNA product from the processive DNA synthesis by Polδ-PCNA is of similar size for all four UbPCNA species (Figure 3a), suggesting that the stability of the Polδ holoenzyme, as reflected by the processivity of the Polδ(Ub)PCNA holoenzyme, was not affected by the polyubiquitin chain length once the holoenzyme is formed. A more plausible 1688

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Figure 4. PolyUb-PCNA inhibits polymerase switching and the roles of K63-linked polyubiquitination of PCNA in DNA damage tolerance. (a) DNA synthesis product of Polδ assembled with different PCNA species in the absence or presence of Polη as described in Methods in Supporting Information. (b) The extent of polymerase switching reported by the decrease in DNA synthesis for the different Ub-PCNA species as quantified from the DNA synthesis product in panel a. (c) The possible roles of K63-linked polyubiquitin chain on PCNA in DNA damage tolerance. In response to DNA damage (▲) and the stalling of the Polδ-PCNA holoenzyme, polyubiquitination of PCNA can trap Polη in an unproductive mode to suppress TLS by preventing the polymerase switching between Polδ and Polη and inhibiting the activity of Polη in synthesis across the lesion. If Polδ dissociates from the stalled replication fork, the polyubiquitin chain on PCNA prevents the rebinding of Polδ to PCNA. As a result, the polyubiquitin chain on PCNA facilitates the recruitment of a protein factor (such as ZRANB3/AH2 in humans) to channel the DNA damage tolerance to the error-free branch.

explanation for the inhibition of the Polδ-PCNA complex formation is the steric clash incurred by the extended and flexible K63-polyubiquitin chain on PCNA. We envision that the flexible K63-polyubiquitin chain may prevent the rebinding of Polδ dissociated from the stalled replication fork and make the stalled replication fork accessible for other proteins required for the error-free lesion bypass pathway. TLS requires the polymerase switching between the replicative and the TLS polymerases, which is promoted by monoubiquitination of PCNA.5 To assess the effect of PCNA polyubiquitination on polymerase switching, we employed a polymerase switching assay that reports on the efficiency of Polη recruitment and the disruption of Polδ DNA synthesis.18 Remarkably, as the PCNA polyubiquitin chain length increases, we observed an overall decrease in the efficiency of polymerase switching (Figure 4a,b), supporting the role of K63-linked polyubiquitin chain in trapping Polη in a nonproductive mode, thus suppressing TLS. In recent years, progress has been made in preparing chemically monoubiquitinated protein species including histone H2B,24−26 PCNA,18,27 and α-synuclein,28,29 and important biological insights were obtained using the monoubiquitinated proteins. Compared to monoubiquitination, a higher level of complexity is expected for protein polyubiquitination given that ubiquitin chain of different lengths and linkages can be conjugated to a target protein. Despite the recent realization that ubiquitin chains with linkages different from the K48linked polyubiquitin exist in cells, our knowledge of the function of the noncanonical polyubiquitin chains remains

limited. This was in a large part due to the difficulty in generating high quality polyubiquitinated proteins with defined chain length and linkage. To date the generation of K63-linked polyubiquitinated proteins have not bee reported despite that K63-linked polyubiquitin chain is abundant in cells. In this study we demonstrated that efficient K63-polyubiquitination of PCNA can be achieved under a mild nondenaturing condition with a controlled ubiquitin chain length. The conditions reported herein for the chemical polyubiquitination are amenable for many target proteins with a higher order structure that cannot sustain the denaturing conditions. We expect that this approach can be adapted for polyubiquitination of other proteins known to be modified by ubiquitin chains. The mechanism of how K63-polyubiquitination of PCNA channels the DNA damage response to the error-free branch remains largely unknown. Using the K63-polyUb-PCNA generated, we interrogated the individual steps in the lesionbypass synthesis. Our results support a model of DDT involving the K63 polyubiquitination of PCNA (Figure 4c). Upon the stalling of the Polδ-PCNA holoenzyme, polyubiquitination of PCNA can suppress TLS by trapping Polη in a nonproductive mode to prevent the polymerase switching between Polδ and Polη and abate the Polη’s activity in synthesis opposite a lesion. Together these molecular events reduce the TLS-mediated mutagenesis. In addition polyubiquitination of PCNA prevents the Polδ-PCNA complex formation. Upon the dissociation of Polδ from the stalled replication fork, the polyubiquitin chain on PCNA prevents the rebinding of Polδ to PCNA, which vacates the protein binding sites on PCNA in the 1689

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(6) Chiu, R. K., Brun, J., Ramaekers, C., Theys, J., Weng, L., Lambin, P., Gray, D. A., and Wouters, B. G. (2006) Lysine 63polyubiquitination guards against translesion synthesis-induced mutations. PLoS Genet. 2, e116. (7) Ciccia, A., Nimonkar, A. V., Hu, Y., Hajdu, I., Achar, Y. J., Izhar, L., Petit, S. A., Adamson, B., Yoon, J. C., Kowalczykowski, S. C., Livingston, D. M., Haracska, L., and Elledge, S. J. (2012) Polyubiquitinated PCNA recruits the ZRANB3 translocase to maintain genomic integrity after replication stress. Mol. Cell 47, 396−409. (8) Yuan, J., Ghosal, G., and Chen, J. (2012) The HARP-like domaincontaining protein AH2/ZRANB3 binds to PCNA and participates in cellular response to replication stress. Mol. Cell 47, 410−421. (9) Weston, R., Peeters, H., and Ahel, D. (2012) ZRANB3 is a structure-specific ATP-dependent endonuclease involved in replication stress response. Genes Dev. 26, 1558−1572. (10) Pickart, C. M., and Raasi, S. (2005) Controlled synthesis of polyubiquitin chains. Methods Enzymol. 399, 21−36. (11) El Oualid, F., Merkx, R., Ekkebus, R., Hameed, D. S., Smit, J. J., de Jong, A., Hilkmann, H., Sixma, T. K., and Ovaa, H. (2010) Chemical synthesis of ubiquitin, ubiquitin-based probes, and diubiquitin. Angew. Chem., Int. Ed. Engl. 49, 10149−10153. (12) Virdee, S., Ye, Y., Nguyen, D. P., Komander, D., and Chin, J. W. (2010) Engineered diubiquitin synthesis reveals Lys29-isopeptide specificity of an OTU deubiquitinase. Nat. Chem. Biol. 6, 750−757. (13) Castaneda, C., Liu, J., Chaturvedi, A., Nowicka, U., Cropp, T. A., and Fushman, D. (2011) Nonenzymatic assembly of natural polyubiquitin chains of any linkage composition and isotopic labeling scheme. J. Am. Chem. Soc. 133, 17855−17868. (14) Kumar, K. S., Bavikar, S. N., Spasser, L., Moyal, T., Ohayon, S., and Brik, A. (2011) Total chemical synthesis of a 304 amino acid K48linked tetraubiquitin protein. Angew. Chem., Int. Ed. 50, 6137−6141. (15) Trang, V. H., Valkevich, E. M., Minami, S., Chen, Y. C., Ge, Y., and Strieter, E. R. (2012) Nonenzymatic polymerization of ubiquitin: single-step synthesis and isolation of discrete ubiquitin oligomers. Angew. Chem., Int. Ed. 51, 13085−13088. (16) Haj-Yahya, M., Fauvet, B., Herman-Bachinsky, Y., Hejjaoui, M., Bavikar, S. N., Karthikeyan, S. V., Ciechanover, A., Lashuel, H. A., and Brik, A. (2013) Synthetic polyubiquitinated alpha-Synuclein reveals important insights into the roles of the ubiquitin chain in regulating its pathophysiology. Proc. Natl. Acad. Sci. U.S.A. 110, 17726−17731. (17) Hemantha, H. P., Bavikar, S. N., Herman-Bachinsky, Y., HajYahya, N., Bondalapati, S., Ciechanover, A., and Brik, A. (2014) Nonenzymatic polyubiquitination of expressed proteins. J. Am. Chem. Soc. 136, 2665−2673. (18) Chen, J., Ai, Y., Wang, J., Haracska, L., and Zhuang, Z. (2010) Chemically ubiquitylated PCNA as a probe for eukaryotic translesion DNA synthesis. Nat. Chem. Biol. 6, 270−272. (19) Garg, P., and Burgers, P. M. (2005) Ubiquitinated proliferating cell nuclear antigen activates translesion DNA polymerases eta and REV1. Proc. Natl. Acad. Sci. U.S.A. 102, 18361−18366. (20) Freudenthal, B. D., Gakhar, L., Ramaswamy, S., and Washington, M. T. (2010) Structure of monoubiquitinated PCNA and implications for translesion synthesis and DNA polymerase exchange. Nat. Struct. Mol. Biol. 17, 479−484. (21) Ivanov, I., Chapados, B. R., McCammon, J. A., and Tainer, J. A. (2006) Proliferating cell nuclear antigen loaded onto double-stranded DNA: dynamics, minor groove interactions and functional implications. Nucleic Acids Res. 34, 6023−6033. (22) Plosky, B. S., Vidal, A. E., Fernandez de Henestrosa, A. R., McLenigan, M. P., McDonald, J. P., Mead, S., and Woodgate, R. (2006) Controlling the subcellular localization of DNA polymerases iota and eta via interactions with ubiquitin. EMBO J. 25, 2847−2855. (23) Tsutakawa, S. E., Van Wynsberghe, A. W., Freudenthal, B. D., Weinacht, C. P., Gakhar, L., Washington, M. T., Zhuang, Z., Tainer, J. A., and Ivanov, I. (2011) Solution X-ray scattering combined with computational modeling reveals multiple conformations of covalently bound ubiquitin on PCNA. Proc. Natl. Acad. Sci. U.S.A. 108, 17672− 17677.

stalled replication fork for recruiting protein(s) to channel the DNA damage tolerance to the error-free branch. The proteins recruited by K63-polyubiquitinated PCNA may preferentially bind both PCNA and the K63-linked polyubiquitin chain simultaneously. To date ZRANB3/AH2 in humans was suggested to be recruited by polyUb-PCNA to restart the stalled replication fork.7−9 In yeast the identity of the protein binding to polyUb-PCNA and functioning in error-free lesion bypass remains to be determined, albeit Mgs1 was suggested as a potential candidate.30 It is possible that other protein effectors recruited by K63-linked polyubiquitinated PCNA also exist in humans and yeast. The chemically polyubiquitinated PCNA provides a way of identifying the proteins by an affinity pull-down/mass spectrometry approach. Moreover, with sufficient amounts of homogeneously polyubiquitinated PCNA, in-depth biochemical and biophysical studies now become possible and will lead to a better understanding of the role of polyubiquitinated PCNA in the error-free branch of DNA damage tolerance.

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METHODS

The details of the methods are described in the Supporting Information.

ASSOCIATED CONTENT

S Supporting Information *

Methods and Supplementary Figures 1−10. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions †

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank S. Prakash and R. E. Johnson (University of Texas Medical Branch, Galveston) for the generous gift of yeast Polδ. We thank L. Haracska (Institute of Genetics, Hungarian Academy of Sciences) for the generous gift of anti-yeast PCNA antibody. We thank J. E. Azevedo for the mE1-pET28 plasmid and C. M. Pickart for the Mms2-pET16b and Ubc13-pGEX plasmids. We thank J. Wang for help with chemical polyubiquitination. This work was supported by a grant from the U.S. National Science Foundation (MCB-0953764) to Z.Z.

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REFERENCES

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(24) McGinty, R. K., Kim, J., Chatterjee, C., Roeder, R. G., and Muir, T. W. (2008) Chemically ubiquitylated histone H2B stimulates hDot1L-mediated intranucleosomal methylation. Nature 453, 812− 816. (25) Chatterjee, C., McGinty, R. K., Fierz, B., and Muir, T. W. (2010) Disulfide-directed histone ubiquitylation reveals plasticity in hDot1L activation. Nat. Chem. Biol. 6, 267−269. (26) Haj-Yahya, M., Eltarteer, N., Ohayon, S., Shema, E., Kotler, E., Oren, M., and Brik, A. (2012) N-Methylation of isopeptide bond as a strategy to resist deubiquitinases. Angew. Chem., Int. Ed. 51, 11535− 11539. (27) Eger, S., Castrec, B., Hubscher, U., Scheffner, M., Rubini, M., and Marx, A. (2011) Generation of a mono-ubiquitinated PCNA mimic by click chemistry. ChemBioChem 12, 2807−2812. (28) Hejjaoui, M., Haj-Yahya, M., Kumar, K. S. A., Brik, A., and Lashuel, H. A. (2011) Towards elucidation of the role of ubiquitination in the pathogenesis of Parkinson’s Disease with semisynthetic ubiquitinated alpha-synuclein. Angew. Chem., Int. Ed. 50, 405−409. (29) Meier, F., Abeywardana, T., Dhall, A., Marotta, N. P., Varkey, J., Langen, R., Chatterjee, C., and Pratt, M. R. (2012) Semisynthetic, sitespecific ubiquitin modification of alpha-synuclein reveals differential effects on aggregation. J. Am. Chem. Soc. 134, 5468−5471. (30) Saugar, I., Parker, J. L., Zhao, S., and Ulrich, H. D. (2012) The genome maintenance factor Mgs1 is targeted to sites of replication stress by ubiquitylated PCNA. Nucleic Acids Res. 40, 245−257.

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