Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2016 Apr 7.
Published in final edited form as: Structure. 2015 Mar 12;23(4):724–733. doi: 10.1016/j.str.2015.02.008

STRUCTURALLY DISTINCT UBIQUITIN- AND SUMO-MODIFIED PCNA: IMPLICATIONS FOR THEIR DISTINCT ROLES IN THE DNA DAMAGE RESPONSE

Susan E Tsutakawa 1,#, Chunli Yan 2,#, Xiaojun Xu 2, Christopher P Weinacht 3, Bret D Freudenthal 4,‡, Kun Yang 3, Zhihao Zhuang 3, M Todd Washington 4, John A Tainer 1,5,6,*, Ivaylo Ivanov 2,*
PMCID: PMC4394044  NIHMSID: NIHMS667080  PMID: 25773143

SUMMARY

Proliferating cell nuclear antigen (PCNA) is a pivotal replication protein, which also controls cellular responses to DNA damage. Posttranslational modification of PCNA by SUMO and ubiquitin modulate these responses. How the modifiers alter PCNA-dependent DNA repair and damage tolerance pathways is largely unknown. We used hybrid methods to identify atomic models of PCNAK107-Ub and PCNAK164-SUMO consistent with small angle X-ray scattering (SAXS) data of these complexes in solution. We show that SUMO and ubiquitin have distinct modes of association to PCNA. Ubiquitin adopts discrete docked binding positions. By contrast, SUMO associates by simple tethering and adopts extended flexible conformations. These structural differences are the result of the opposite electrostatic potentials of SUMO and Ub. The unexpected contrast in conformational behavior of Ub-PCNA and SUMO-PCNA has implications for interactions with partner proteins, interacting surfaces accessibility, and access points for pathway regulation.

INTRODUCTION

Dynamic assembly, coordinated access to DNA, and conformational switching are critical aspects of DNA replication and repair, essential processes upon which life depends. In both processes, the sliding clamp proliferating cell nuclear antigen (PCNA) (Ivanov et al., 2006; Kelman, 1997; Krishna et al., 1994; Moldovan et al., 2007). acts as a master coordinator of multiple pathways controlling replication and DNA damage responses (DDR). PCNA's toroidal shape allows it to topologically encircle DNA while also binding to core replisomal constituents, numerous repair and cell cycle control proteins (Jonsson and Hubscher, 1997; Maga and Hubscher, 2003; Moldovan et al., 2007; Stolimenov and Helleday, 2009). In this capacity, the sliding clamp acts as a platform for the assembly of the replication machinery on DNA. Emerging evidence suggests that posttranslational modifications (PTMs) of PCNA play a critical role in coordinating DNA damage responses to suppress genome instability. This added level of regulation is realized through the interplay of PTMs and partner protein recruitment to PCNA. Specifically, reversible covalent attachment of ubiquitin or ubiquitin-like (UBL) proteins (Bienko et al., 2005; Hicke, 2001; Hoege et al., 2002; Perry et al., 2008; Ulrich, 2005, 2012; Ulrich and Walden, 2010) (e.g. SUMO) selects among alternative damage response pathways such as homologous recombination or translesion synthesis (TLS). The structural basis for such selection has so far remained elusive. Ubiquitin and SUMO both attach to their PCNA target through a glycine residue from the end of their flexible carboxy termini. This glycine forms an isopeptide linkage with a specific lysine side chain on the target. Despite sharing a common β-GRASP fold, SUMO and ubiquitin elicit distinct functional outcomes, and characteristically have been portrayed as having antagonistic roles (Perry et al., 2008). However, recent studies (Stelter and Ulrich, 2003) have significantly attenuated this picture by showing the two modifiers could act in concert to affect cellular fates.

UV irradiation or exposure to DNA damaging agents commonly results in lesions that cause replication fork stalling. The cellular response is mono-ubiquitination of PCNA at a conserved lysine K164, which in turn triggers translesion synthesis (TLS) – an essential damage tolerance pathway (Hoege et al., 2002; Stelter and Ulrich, 2003). In TLS the replicative polymerase is transiently exchanged with a specialized TLS polymerase capable of progressing past lesions in the template strand (Yang et al., 2013). Ubiquitin attachment provides an additional binding surface for TLS polymerases with an ubiquitin-binding motif (Bienko et al., 2005). By contrast, PCNA SUMOylation at positions K164 or K127 leads to suppression of homologous recombination through the recruitment of the antirecombinogenic helicase Srs2 (Pfander et al., 2005). Srs2 interacts with SUMO-PCNA through its carboxy-terminal domain, which harbors tandem receptor motifs: a SUMO interaction motif (SIM) and a non-canonical PIP-box (Armstrong et al., 2012; Freudenthal et al., 2011). Both motifs are required for specific recognition of SUMO–PCNA. One possible mechanism for Srs2 to suppress homologous recombination is through disruption of Rad51 filaments (Krejci et al., 2003; Veaute et al., 2003), thereby assisting the ubiquitin-dependent TLS pathway. A novel alternative mechanism has been proposed wherein Srs2 binding to SUMO-PCNA dissociates the replicative and TLS Pols from the repair synthesis machinery and, thus, prevents the synthesis dependent extension of recombination intermediates (Burkovics et al., 2013). This latter mechanism requires only Srs2 recruitment through the SIM motif but neither its translocase activity nor interaction with Rad51. PCNA ubiquitination and SUMOylation offer striking examples of crosstalk between pathways controlled by distinct PTMs (Stelter and Ulrich, 2003; Ulrich, 2005). While attachment of a single ubiquitin is the dominant DDR response in mammalian cells, yeast PCNA can also undergo polyubiquitination through the non-proteasomal K63 linkage (Hoege et al., 2002). Polyubiquitination of PCNA at K164 channels DDR to yet another pathway - error-free damage bypass by template switching. Additionally, in yeast PCNA mono-ubiquitination at position K107 helps cells to overcome defects in DNA ligation through an S-phase checkpoint activation (Das-Bradoo et al., 2010; Nguyen et al., 2013). Damage-related ubiquitination at other PCNA positions has been recently identified: PCNA was ubiquitinated at K248 after UV irradiation (Povlsen et al., 2012) and ubiquitinated at K168 after colchicine treatment (Xu et al., 2010). How could PTMs introduced at different positions on PCNA result in such vastly divergent functional outcomes? Furthermore, how do posttranslational modifications of PCNA facilitate recruitment of subsequent effector proteins in these pathways?

To answer these questions we modeled PCNA covalently modified by ubiquitin at residue K107 and SUMO at K164 using a multiscale computational protocol. Models consistent with solution X-ray scattering data (SAXS) were identified (Hura et al., 2009; Putnam et al., 2007; Rambo and Tainer, 2010, 2011), which allowed us to assess the structural differences of PTM-PCNA complexes (PCNAK107-Ub and PCNAK164-SUMO) and compare these with PCNAK164-Ub from a previous study. Here we show that SUMO and Ub have distinct modes of interaction with PCNA and that the position of ubiquitin attachment, 107 versus 164, alters conformation. Ubiquitin bound to PCNA can dynamically adopt multiple discrete docked conformations. By contrast, SUMO is flexibly tethered, with no substantial docking interactions with PCNA. Our hybrid structural analysis reveals the biologically relevant conformations of modified PCNA in solution that effector proteins would first encounter and interact with.

RESULTS

Distinct architectures of modified PCNA complexes uncovered by SAXS data

We conducted small angle X-ray scattering (SAXS) experiments to probe the overall architecture and flexibility of Saccharomyces cerevisiae PCNAK107-Ub and PCNAK164-SUMO complexes in solution. Covalent attachment in the PCNA-Ub complex was achieved by chemical cross-linking with a K107C PCNA mutant (Chen et al., 2010). The PCNA-SUMO complex was formed by split-fusion (Freudenthal et al., 2011; Freudenthal et al., 2010). The split-fusion and chemical ligation methods yield linkages close to the enzymatically produced isopeptide bond (Chen et al., 2010; Tsutakawa et al., 2011) SDS-PAGE analysis showed that there were three modifications per PCNA homotrimer.

SAXS data were collected for PCNAK107-Ub and PCNAK164-SUMO complexes and compared to the previously obtained scattering profile of PCNAK164-Ub and simulated profiles from available crystal structures (split-fusion PCNAK164-Ub (Freudenthal et al., 2010) and PCNAK164-SUMO (Armstrong et al., 2012); PDB ID 3L10 and 3V60, respectively). All three experimental scattering profiles (Figure 1) are distinct, revealing that the three PTM complexes adopt conformations with different levels of compactness in solution. PCNAK164-Ub is slightly less compact than PCNAK107-Ub with the corresponding P(r) distribution tailing to higher values of r (Figure 1B). SUMOylated PCNA presents an I(q) profile (Figure 1A) with similar overall shape to the SAXS profile of the 3V60 crystal structure, but considerably more extended (Figure 1B). Fitting to the SAXS data, the crystal structures 3L10 and 3V60 produced high χfree values consistent with significant discrepancies between the conformations adopted in the crystal structures and the solution phase ensembles. Guinier plots (Figure 1C) for PCNAK107-Ub and PCNAK164-SUMO were linear, indicating that the samples were not aggregated. The radius of gyration, Rg, is accurately defined by SAXS regardless of sample concentration and, therefore, provides an important constraint for possible solution structures (Rambo and Tainer, 2011). The Rg values (Table S1) based on the Guinier analyses were 35.5 Å and 35.6 Å for PCNAK107-Ub and PCNAK164-Ub, respectively. These values are comparable to the 36.05 Å Rg calculated from the 3L10 crystal structure. The PCNAK164-SUMO complex features a significantly larger Rg of 47.1 Å suggesting that SUMO may occupy more extended positions on PCNA compared to ubiquitin. To determine the compactness of the solution conformation, we compared the Porod-Debye constant, as determined from a linear regression analysis (I vs. q) of the top of the first peak in the Porod-Debye plot (q4*I(q) vs. q4). The Porod-Debye constant reflects the protein density, volume compared to the Rg (Rambo and Tainer, 2011). Well-folded proteins have a constant of 4, while globular domains connected by linkers, such as Mre11-Rad50 (Deshpande et al., 2014) can be as low as 3. PCNAK164-SUMO, PCNAK107-Ub and PCNAK164-Ub had Porod-Debye constants of 3.5, 4, and 3.7, respectively. We previously showed that PCNAK164-Ub adopted both docked and flexible conformations, in agreement with the Porod-Debye constant of 3.7. The Porod-Debye constant for PCNAK164-SUMO suggests a more flexible structure, while that for PCNAK107-Ub is consistent with a compact structure. To further examine the compactness of the different modified PCNA complexes, we used a Volume of Correlation (Vc)-based dimensionless Kratky plot (Figure 1D). (Durand et al., 2010; Reyes et al.) The three modified PCNA show a ranking in order of compactness of PCNAK107-Ub, PCNAK164-Ub, and PCNAK164-SUMO (at 1.7 on the abscissa) consistent with the Porod-Debye constants.

Figure 1. Distinct architectures of PCNA-Ub and PCNA-SUMO complexes from SAXS analysis.

Figure 1

Open in a new tab

(A) SAXS profiles of PCNAK164-SUMO (blue), PCNAK107-Ub (green) and PCNAK164-Ub (red); (B) P(r) functions for PCNAK164-SUMO (blue), PCNAK107-Ub (green) and PCNAK164-Ub (red); (C) Guinier analyses of SAXS data for PCNAK107-Ub (green) and PCNAK164-SUMO (blue) showing relative linearity of the samples in Guinier region, indicating lack of aggregation. (D) A dimensionless Kratky plot indicates PCNAK164-SUMO being significantly less compact than PCNAK164-Ub, which is slightly less compact than PCNAK107-Ub (green). All P(r) distributions were normalized dividing the values by the peak height.

Ubiquitin- and SUMO-PCNA conformations from conjugated protein docking

To further explore the substantial differences between ubiquitin and SUMO positioning in the modified PCNA complexes, we constructed computational models of ubiquitin (SUMO) ligated to PCNA. We used a recently developed protocol for conjugated protein docking in Rosetta 3.4. The method samples the conformational space available to ligated proteins (Baker et al., 2013; Leaver-Fay et al., 2011; Saha et al., 2011) using a standard Rosetta Metropolis Monte Carlo random sampling tool (Rohl et al., 2004). The protocol involved docking a single Ub (SUMO) moiety to Saccharomyces cerevisiae PCNA conjugated at the K107 or K164 position, respectively. The short linker between the modifier and the PCNA attachment point limited accessible conformations. This allowed us to locally saturate the PCNA surface with decoys and achieve extensive sampling. Scoring results from Rosetta are shown in Figure 2. For PCNAK107-Ub, the all-atom score of the docking decoys is plotted as a function of the Cα root-mean-square deviation (RMSD) from the lowest scoring PCNA-Ub structure. For PCNAK164-SUMO, the crystal structure 3V60 (Armstrong et al., 2012) was used as a reference to compute the Cα RMSD. Figure 2 revealed a clear distribution pattern for the decoys, which clustered into bands around specific RMSD values. For PCNA-Ub the lowest scoring decoys formed score “funnels” indicating several preferred position for Ub to reside on the surface of PCNA. The majority of PCNAK107-Ub decoys had Rosetta energy scores ranging from −1360 to −1100. For PCNAK164-SUMO most scores were found in a much narrower range between −1030 to −920. Collectively, the higher Rosetta scores, the narrow score range and the absence of low-scoring outliers for the PCNA-SUMO complex implied significantly weaker association between PCNA and SUMO as compared to ubiquitin. Next, we selected the lowest-scoring structurally distinct models from the Rosetta output (Figure 2). These docking positions (one chosen from each score funnel) were refined using all-atom explicit solvent molecular dynamics (MD) with the program NAMD (Kale et al., 1999; Phillips JC et al., 2005). The MD simulation and analysis allowed us to eliminate conformations where the Ub or SUMO moiety interacted primarily through polar contacts and consequently moved away from the PCNA surface during MD refinement. Nine unique docked positions for each system were selected to build triplet structures of modified PCNA. These structures after the MD refinements (sorted by Rosetta score) are presented in Figure S2.

Figure 2. Rosetta score versus RMSD plots for ubiquitinated and SUMOylated PCNA.

Figure 2

Open in a new tab

(A) Decoys from PCNAK107-Ub docking are shown in gray. Lowest-scoring structurally distinct models (selected for building triplets) are shown in red. One model (blue dot) departed from its binding position during MD and was not considered for MES analysis. (B) Decoys from PCNAK164-SUMO docking are shown in gray. Lowest-scoring models (selected for building triplets) are shown in red. Four models (blue dots) departed during MD and were not considered for MES analysis. One partially flexible position (purple) was subsequently included in MES analysis.

Optimal positions of Ubiquitin/SUMO on PCNA from Minimal Ensemble Search Against Experimental SAXS data

As the experimentally tested PCNA would have three modifications per homotrimer, we generated triplet models with three ubiquitin or three SUMO moieties linked to homotrimeric PCNA for comparison to the experimental SAXS data. Thirteen positions for PCNAK107-Ub (including the 3L10 crystal structure and 3 detached flexible Ub positions identified by averaging from the MD ensemble) and thirteen positions for PCNAK164-SUMO (including the 3V60 crystal structure and 3 detached flexible SUMO positions) were used to build triplet structures for the modified complexes. All possible combinations of positions were generated with three Ub or SUMO moieties per PCNA homotrimer.

SAXS data encodes all interatomic distance information even from low-populated flexible conformations (Hammel, 2012; Putnam et al., 2007; Schneidman-Duhovny et al., 2010; Tsutakawa et al., 2011). The experimental data can be compared with scattering predicted from atomic models or ensembles of atomic models, allowing elimination of models not consistent with the experimental data. To implement this strategy we computed theoretical SAXS profiles for all triplet models of the modified complexes using the program FoXS and determined the fit of these profiles to the experimental scattering data. The χ and χfree (Rambo and Tainer, 2013) values were used to measure goodness of fit of the computed profiles to the experimental scattering data. Computed χ values for PCNAK107-Ub and PCNAK164-SUMO are plotted in Figure 3A, B as a function of Cα RMSD for each conformation. The single best fit to the experimental data was χ=0.78 (χfree=0.82) for PCNAK107-Ub and χ=2.55 (χfree=2.97) for PCNAK164-SUMO, respectively. However, in a flexible system, multiple conformations will contribute to the SAXS profile. Therefore, a minimal ensemble search (MES) (Pelikan et al., 2009) was employed to identify a small subset of conformations that optimally represent the scattering data. In the case of PCNAK107-Ub an excellent agreement to the experimental curve was obtained (χ=0.74; χfree=0.78) (Figure 3C and Figure S3A) by a set of two distinct conformers with ensemble contributions of 59% and 41% (Figure 3G). In these conformers the Ub occupied primarily docked positions, closely associating with the PCNA surface. In the case of PCNAK164-SUMO, however, the ensemble was dominated by two flexible, extended conformations (45% and 31% occupancy) and a third flexible but more compact conformation (24% occupancy) (Figure 3H). MES notably improved the goodness-of-fit to the PCNA-SUMO SAXS data (χ=2.16, χfree=2.41, Figure 3D and Figure S3B). Conversely, large discrepancies between the crystal structure 3V60 and the solution SAXS profile were observed (χ=6.70, χfree=7.65, Figure 3B) indicating that the conformation found in the crystal is not prevalent in the solution phase ensemble.

Figure 3. Ub primarily adopts docked positions in PCNAK107-Ub while SUMO occupies extended positions in PCNAK164-SUMO.

Figure 3

Open in a new tab

A minimal ensemble search (MES) produces the best fit to the experimental SAXS data for PCNAK107-Ub and PCNAK164-SUMO. (A) χ values for the triplet PCNAK107-Ub structures plotted against RMSD. Conformations selected by MES are highlighted in blue and magenta, respectively. (B) χ values for the triplet PCNAK164-SUMO structures plotted against RMSD. Conformations selected by MES are highlighted in blue, magenta and red, respectively. (C) Overlaid experimental SAXS profile for PCNAK107-Ub (green), computed profile for 3L10 crystal structure (red dotted line) and computed profile from MES model (black). (D) Overlaid experimental SAXS profile for PCNAK164-SUMO (blue), computed profile for 3V60 crystal structure (red dotted line) and computed profile from MES model (black). (E) P(r) functions for PCNAK107-Ub (green), 3L10 crystal structure (red dotted line) and MES model (black). (F) P(r) functions, for PCNAK164-SUMO (blue), 3V60 crystal structure (red dotted line) and MES model (black). (G) The two most populated atomic structures from MES analysis of PCNAK107-Ub in surface representation. (H) The three most populated atomic structures from MES analysis of PCNAK164-SUMO in surface representation. The P-loop is shown in purple; the K107 and K164 attachment points are depicted in red. PCNA, Ub and SUMO are shown in gray, green and blue, respectively. The MES occupancies for the three conformations are labeled in blue, magenta and red, respectively. All P(r) distributions were normalized diving the values by the peak height.

In all models selected by MES we calculated the occupancy of docked versus extended flexible positions for PCNAK107-Ub and PCNAK164-SUMO. In the PCNAK107-Ub complex, the ubiquitins were all positioned on the back side of PCNA. Approximately half of the Ub population were docked along the P-loop, similar to the position observed in the crystal structure. One third was at the subunit interface. The remainder (20%) was located near the central cavity. All three positions allow dsDNA to pass through PCNA and, intriguingly, would place Ub in proximity to the dsDNA backbone passing through PCNA (Figure S4). The attachment position at K107 is on the back face of the clamp close to the subunit interface. Thus, switching the point of attachment from K164 to K107 leads an increased probability to locate the Ub moiety on the back face of PCNA. Unlike the PCNAK164-Ub study, the MES did not identify any flexible conformations. The goodness of the fit of these back positions to the experimental SAXS data was consistent with this observation.

In the case of PCNA-SUMO, extended positions were identified in the MES calculation. As shown in Figure 3D, MES analysis identifies SUMO with 84% occupancy in two extended flexible positions. Interestingly, MES did not pick any positions docked to the PCNA surface despite inclusion of the 3V60 crystallographic position in the MES optimization. This outcome indicates that SUMO is largely flexible in solution. It is notable that the docked positions observed by crystallography (Armstrong et al., 2012; Freudenthal et al., 2011) (3PGE and 3V60) have significant crystallographic contacts with few intramolecular contacts.

Detailed interactions in the MES identified models

To identify specific structural features that might drive the closer association of Ub to PCNA, we inspected the two most populated MES triplet models. The primary position near the P loop was originally observed in the crystal structure and is based on a hydrophobic contact of the major protein interacting face on Ub, Ile44 Ub and Val70Ub with Met188PCNA and Val186PCNA. The Ub position above the subunit interface was stabilized through several salt bridges Arg42Ub-Glu7PCNA, Lys48Ub-Glu59PCNA, Glu51Ub-Lys5PCNA, and Glu58Ub-Arg61PCNA. The position near the central cavity had two salt bridges Asp39Ub-K107PCNA and Asp58Ub-Lys242PCNA. A stacking of aliphatic chains between Arg72Ub-Asp109PCNA is also observed at the interface between PCNA and Ub. This Ub position could also be explained by a general electrostatic attraction between the acidic face of ubiquitin moving toward the basic central cavity. It is notable in the last two positions that Ile44Ub and Val70Ub are exposed, perhaps promoting an interface for protein-protein interaction with Ub.

In contrast to the ubiquitin-modified PCNA, our MES results with SUMOylated PCNA suggest that covalently attached SUMO actually makes few contacts to PCNA in solution and adopts extended flexible positions. The crystal structure of PCNAK164-SUMO revealed that the interface with the modifier comprises loop regions of PCNA and SUMO, which involve acidic residues from both sides. Furthermore, the outer surface of PCNA is overwhelmingly negatively charged. We verified that these interfacial contacts from the crystal structure could not be maintained in an MD simulation. The distinct behavior by Ub and SUMO becomes apparent upon inspection of the surface electrostatic potentials for Ub, SUMO and PCNA (Figure 4). For all three dominant docked conformations of PCNAK107-Ub we observe electrostatic complementarity at the Ub/PCNA interface. By contrast, there is clear repulsion for the PCNAK164-SUMO crystal structure where both surfaces are negatively charged. Thus, it is likely that the docked conformation identified in the X-ray structure is stabilized by the crystal environment and is not highly populated in solution.

Figure 4. Structural differences in PCNAK107-Ub and PCNAK164-SUMO complexes from the anticorrelated electrostatic potential of Ub and SUMO.

Figure 4

Open in a new tab

(A) Ub docked onto PCNA in the most populated MES positions (P-loop, subunit interface and central cavity positions) and SUMO position from the 3V60 crystal structure. Ub, SUMO and PCNA are colored green, blue and gray, respectively. The IDCL and P-loops on PCNA are shown in orange and purple, respectively. The attachment positions, K107 and K164 residues, are displayed as red balls. (B) Electrostatic potential surfaces corresponding to the bound positions of Ub and SUMO on PCNA (P-loop, subunit interface and central cavity, from left to right for PCNAK107-Ub) and 3V60 structure for PCNAK164-SUMO). 100 mM NaCl concentration was introduced to screen the electrostatics mimicking physiological conditions. The potential varies from −5KBT/e to +5KBT/e and is depicted from red to blue, respectively.

DISCUSSION

PCNA monoubiquitination and SUMOylation are reversible dynamic modifications that orchestrate cellular events in response to DNA damage. How Ub and SUMO associate with PCNA and how this association may facilitate subsequent partner interactions has not been fully established. Three general models can be envisioned: (i) simple tethering; (ii) structured interface formation or (iii) allosteric response. Under the simple tethering model, the only function of the modifier (ubiquitin or SUMO) is to provide an additional interaction interface to the protein partner carrying ubiquitin- or SUMO-interacting domain. Under the structured interface model, the modifier and effector protein can form a continuous binding interface with PCNA. In the allosteric model, Ub or SUMO conjugation induces conformational change in PCNA, which in turn facilitates recruitment of downstream effectors in the respective pathways. This allosteric hypothesis is exemplified by the SUMO-induced ordering of thymine DNA glycosylase (Baba et al., 2005; Steinacher and Schar, 2005). Our multiple-trajectory MD simulation results show little change in PCNA structure and argue against an allosteric model for the mechanism of PCNA ubiquitination or SUMOylation in effecting distinct functional responses to DNA damage.

In ubiquitinated PCNA, there appears to be formation of structured interfaces. Ubiquitin is stabilized against the PCNA surface and is able to adopt several discrete positions relative to the PCNA ring (Figure 5). PCNAK107-Ub and PCNAK164-Ub do have distinct distributions of conformations. The P-loop position observed in the crystal structure is found in both, but the predominant position for the PCNAK164-Ub complex, which is along the side of the PCNA ring, is not found for PCNAK107-Ub. The side position would not be easily accessible when the Ub is linked via K107. The newly identified positions found for PCNAK107-Ub, on the back side of PCNA, are interesting as they are close to where the DNA would pass though the PCNA ring and as the major protein-protein interaction interface for Ub is exposed. To describe this situation where covalently bound ubiquitin occupies docked positions against the PCNA surface, we use the concept of segmental flexibility and define it as the stabilization of “functionally-relevant” positions in otherwise flexible systems. Both PCNAK107-Ub and PCNAK164-Ub exhibit such segmental flexibility. Ubiquitin in docked positions could control the orientation of effector proteins upon initial encounter of PCNA-Ub and determine the side from which partner proteins approach PCNA. Thus, our findings highlight the importance of spatial constraints for ubiquitin, which could allow Ub to be recognized by TLS Pol in combination with binding to PCNA. Furthermore, segmental flexibility is likely a common characteristic of eukaryotic ubiquitin regulatory systems. As other lysines on the edge of the PCNA ring are also uniquely modified (Povlsen et al., 2012; Xu et al., 2010), we postulate that these distinct covalent modification sites provide a specific conformational range for the downstream effector proteins, such as we observed for K107 and K164.

Figure 5. Distinct types of interfaces are exposed dependent on the different mode of association of ubiquitin and SUMO to PCNA.

Figure 5

Open in a new tab

The PCNA trimer is shown in gray; the Ub modifier in green; SUMO in blue; the attachment positions are indicated by a red dots; curved arrows indicate flexible attachment. Approximate occupancy (%) of the identified distinct positions of the modifiers on PCNA given below each model.

By contrast, the PCNAK164-SUMO complex adopted extended flexible conformations wherein SUMO was covalently bound to K164 but was not otherwise interacting with the surface of PCNA (Figure 5). Therefore, for SUMOylated PCNA recruitment, our results support simple tethering, consistent with the model of the Srs2 interaction with SUMOylated PCNA that adopted two positions (Armstrong et al., 2012). Flexible tethering of SUMO is required for successful Srs2 recruitment and consistent with the observation that Srs2 could not interact in cis with both SUMO and PCNA in the conformation of PCNAK164-SUMO crystal structure. Additionally, the structural requirement for tethered flexibility is supported by the fact that the K127 and K164 SUMOylation sites, more than 30 Å apart from each other, were genetically equivalent in their ability to interact with Srs2 (Papouli et al., 2005; Pfander et al., 2005). This implies the crystallographic position is not required for Srs2 interaction.

The structurally distinct modes of association of Ub and SUMO to the sliding clamp (Figure 5) present radically different conformations and accessibility of the interacting surface for partner proteins. PCNA-SUMO binds two structurally distinct proteins: Srs2 and Rad18. Srs2 is an anti-recombinase that has been shown to possess two activities to prevent recombination. The first is the ability to disrupt Rad51 nucleoprotein filaments (Krejci et al., 2003; Veaute et al., 2003), which could be on the same partially duplex DNA passing through the PCNA ring, on the partially duplex sister strand, or on another partial duplex in the vicinity. The second is the ability to inhibit DNA polymerases from extending recombination intermediates (Burkovics et al., 2013). Again, these recombination intermediates could be on the same duplex of the PCNASUMO, on the sister partial, or on another duplex in the vicinity of PCNA. For both of these Srs2 activities, a greater degree of flexibility is important for delivering the catalytic domain of Srs2 to the appropriate location. Rad18 is a SUMO-directed E3 ubiquitin ligase responsible for catalyzing the ubiquitylation of PCNA. The SUMO moiety on PCNA-SUMO binds Rad18 and positions it to allow ubiquitylation of K164 on other PCNA subunits of the trimer (Parker and Ulrich, 2012). Since Rad18 is structurally unrelated to Srs2, PCNA-SUMO must bind different, structurally unrelated proteins and deliver their catalytic domains to various positions around the PCNA ring. Thus, an expanded spatial range would be required to accommodate the interactions and activities of binding partners.

PCNA-Ub, by contrast, is only known to bind one class of structurally related proteins, the Y-family translesion synthesis polymerases. The basis of the interactions between PCNAUb and the various Y-family polymerases is similar. The conserved ubiquitin-binding motifs (UBMs) and ubiquitin-binding zinc-finger domains (UBZs) of these polymerases are located in their intrinsically disordered C-terminal regions. Moreover, the catalytic domains of these polymerases only function on the partial duplex on the front face of the PCNA ring. Thus, consistent with our structural results, a more restricted spatial range and tighter binding of the Ub modifier to PCNA is required to support the function of these complexes. One indicator for the biological significance of the newly identified PCNA-Ub interfaces is slight sensitivity to DNA damage, which is consistent with a partial or complete defect in translesion synthesis. Substitutions of Ub-interacting residues in yeast PCNA all confer an increased sensitivity to DNA damaging agents. For example, the ubiquitin at the subunit interface forms a hydrogen bond with Lys5 of PCNA and is close (within 4 Å) to Arg61 of PCNA. A substitution of either Lys5 or Arg61 increases the sensitivity to ultraviolet (UV) radiation, hydroxyurea (HU), and methyl methansulfonate (MMS) (Ayyagari et al., 1995). Similarly, the ubiquitin near the PCNA cavity forms hydrogen bonds with Asp109 and is close to Glu189. A substitution of Asp109 increases the sensitivity to UV radiation and MMS, and a substitution of Glu189 increased the sensitivity to UV radiation (Ayyagari et al., 1995). Lastly, the Ub position along the PCNA ring, identified as the predominant position for PCNAK164-Ub, includes hydrogen bonds with Ser177 and Ser179 of PCNA. Although to our knowledge no substitutions of these residues have been reported, a substitution in Gly178, located in-between these two residues, increases the sensitivity to UV radiation and leads to a complete loss of error-prone translesion synthesis (Zhang et al., 2006). In none of these substitutions is the growth of the cells affected.

More generally, our work underscores the power of hybrid structural methods and our results afford insights into how PCNA post-translational modifications by Ub and SUMO provide both the necessary specificity and flexibility to regulate recruitment and coordinated actions of effector proteins to promote genomic stability.

EXPERIMENTAL PROCEDURES

SAXS analysis of PCNAK164-SUMO and PCNAK107-Ub

SUMOylated and cross-linked yeast PCNA were purified using established protocols (Chen et al., 2010; Freudenthal et al., 2011; Freudenthal et al., 2010). SAXS data were collected at the SIBYLS 12.3.1 beamline at the Advanced Light Source, LBNL (Classen et al., 2013; Classen et al., 2010; Hura et al., 2009). Scattering measurements were performed on 20 μl samples at 15 °C (PCNA K164-SUMO) or 22 °C (PCNA K107-Ub) loaded into a helium-purged sample chamber, 1.5 m from the Mar165 detector. Prior to data collection, modified PCNA were purified by size exclusion chromatography on a 24 mL Superose6 column equilibrated in 20 mM Tris pH 7.5, 150 mM NaCl, 5% glycerol. Data were collected on both the original gel filtration fractions and samples concentrated ~2x from individual fractions (Table S2 and Figure S1). Fractions prior to the void volume and concentrator eluates were used for buffer subtraction. Sequential exposures (0.5, 0.5, 5, and 0.5 s for PCNAK164-SUMO and 0.5, 0,5, 2, 5, 0.5 s for PCNAK107-Ub) were taken at 12 keV to maximize signal to noise with visual checks for radiation-induced damage to the protein. Although scattering of the split-fusion PCNAK164-SUMO showed radiation-induced aggregation, the scattering of the cross-linked PCNAK107-Ub showed a decrease in slope, indicative that the molecules in solution were becoming smaller. It is likely due to irradiation breaking the disulfide bond. The first and second 0.5-second exposures overlaid, so the first exposure was assumed to have only minimal damage. The best data, based on signal-to-noise and Guinier, were collected on split-fusion PCNAK164-SUMO (2.7 mg/ml, 0.5 s exposure) and cross-linked PCNAK107-Ub (1.1 mg/ml, 0.5 s exposure). Scattering data including χ2free (Rambo and Tainer, 2013) was analyzed using SCATTER available at beamline 12.3.1 (Classen et al., 2013; Rambo and Tainer, 2011). The Porod exponent was determined from a linear regression analysis (I vs q) of the top of the first peak in the Porod-Debye plot (q4*I(q) vs q4) of the scattering data. P(r) plots were calculated using Gnom implemented in the ATSAS package.

Computational models and protocols

To model the PCNAK107-Ub and PCNAK164-SUMO complexes, we employed a chemically conjugated docking protocol written as a part of the Rosetta 3.4 suite (Baker et al., 2013; Leaver-Fay et al., 2011; Saha et al., 2011). We used the standard Rosetta scoring function − score12 to rank and select the top-scoring models (Kuhlman and Baker, 2000; Rohl et al., 2004). The protocol was designed to search the conformational space available to ubiquitin chemically conjugated via an isopeptide bond and samples rotations about torsional angles in the vicinity of the isopeptide bond. To initiate the sampling for PCNAK107-Ub we used the 1UBQ structure for ubiquitin (Vijay-Kumar et al., 1987) and 1PLQ structure for yeast PCNA(Krishna et al., 1994). For the PCNAK164-SUMO complex, we used the structure of SUMOylated PCNA (PDB ID: 3V60) (Armstrong et al., 2012). The initial PDB structures were minimized with the Rosetta relax protocol while employing all-heavy-atom constraints prior to conjugated docking. For the isopeptide linker, protocol UBQ_Gp_LYX-cterm was used (Baker et al., 2013). Torsions allowed to change included: the χ angles of Lys107 or Lys164 of PCNA, the isopeptide bond and both Φ and Ψ angles for the Gly76, Gly75 and Arg74 of ubiquitin (Gly98, Gly97 and Ile96 of SUMO). Sampling was performed with a standard Rosetta Metropolis Monte Carlo search protocol (Rohl et al., 2004). Results were automatically filtered according to the solvent accessible surface area (SASA) buried at the protein interface (> 500 Å2) and total score of the docked complex (< 0). In total the sampling produced 4,791 decoys (i.e. Ub/SUMO docking poses) for PCNA-Ub and 4,499 decoys for PCNA-SUMO using 20,000 Monte Carlo cycles per trajectory. All Rosetta docking calculations were performed on the Stampede supercomputer at the Texas Advanced Computing Center (TACC).

Model refinement

Outliers with best scores from Rosetta docking were refined using all-atom explicit solvent molecular dynamics. In setting up for MD hydrogen atoms, counterions (Na+) and TIP3P solvent (Jorgensen et al., 1983) were introduced using the XLeap module in AMBER 12 (Case et al., 2005; Cornell et al., 1995; Duan et al., 2003). Additionally, 100 mM NaCl concentration was introduced to mimic physiological conditions. The systems were then minimized for 5000 steps with backbone atoms fixed followed by 5000 steps of minimization with harmonic restraints to remove unfavorable contacts. The systems were then gradually brought up to 300 K and run for 50 ps in the NVT ensemble while keeping the protein backbone restrained. The equilibration was continued for another 2 ns in the NPT ensemble and the harmonic restraints were gradually released. The 60-ns production simulations were performed in the NPT ensemble (1 atm and 300 K) without constraints. A cutoff of 10 Å was used for the short-range non-bonded interactions with a switching function at 8.5 Å. The long-range electrostatic interactions were treated with a smooth particle mesh Ewald method (Essmann et al., 1995). The r-RESPA multiple timestep method (Tuckerman et al., 1992) was adopted with a 2-fs time step for bonded, 2-fs for short-range non-bonded interactions and 4-fs for long-range electrostatic interactions. Bonds between hydrogen atoms and heavy atoms of the protein were constrained with the SHAKE algorithm. All simulations were performed with the NAMD 2.8 code (Kale et al., 1999; Phillips JC et al., 2005) using the AMBER Parm99SB force field. Models, which departed substantially from the initial Rosetta docking position during MD were eliminated from further consideration.

Minimal Ensemble Search

Thirteen positions for PCNAK107-Ub (including the 3L10 X-ray structure and 3 detached flexible Ub positions identified by averaging from the MD trajectories) and thirteen positions for PCNAK164-SUMO (including the 3V60 crystal structure and 3 detached flexible SUMO positions) were used to build triplet structures for the modified complexes. All possible combinations of positions were generated with three Ub or SUMO moieties per PCNA homotrimer. Thus, we produced a final set of 862 PCNA-Ub triplet models and 1728 PCNA-SUMO triplet models. Computation of scattering profiles from the models and comparison to the experimental data used the FoXS code (Schneidman-Duhovny et al., 2010; Schneidman-Duhovny et al., 2013). The coexistence of different conformations that contribute to the experimental scattering curve had to be taken into account by considering multiple Ub or SUMO positions. An algorithm developed by Pelikan et al. (Pelikan et al., 2009) was used to search for the minimal ensemble (MES) of conformations from the pool of all Rosetta generated triplet models. MES included only the minimum set of conformations as necessary to minimize χfree.

Supplementary Material

1
2

Highlights.

  • Ubiquitinated and SUMOylated PCNA complexes are structurally distinct in solution

  • Ubiquitin has segmental flexibility and occupies discrete positions on PCNA

  • SUMO associates by simple tethering and adopts extended flexible conformations

  • The distinct PTM-PCNA conformations underlie distinct roles in DNA damage response

ACKNOWLEDGEMENTS

This work was supported by a NSF CAREER Grant MCB-1149521 (to I.I.), Georgia State University start-up funds (to I.I.), P01 CA092584 (NCI to J.A.T.), R01 CA081967 (NCI to J.A.T), NSF Grant MCB-0953764 (to Z.Z.) and R01 GM108027 (to M.T.W.). Computational resources were provided in part by a National Science Foundation XSEDE allocation [CHE110042] and through an allocation at National Energy Research Scientific Computing Center (NERSC) supported by the U.S. Department of Energy Office of Science [contract DE-AC02-05CH11231]. This SAXS data was collected at BL12.3.1 at the Advanced Light Source (ALS), supported by the Integrated Diffraction Analysis Technologies (IDAT) program (DOE/BER), by DOE contract DE-AC02-05CH11231, and by NIH MINOS (R01GM105404).

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

AUTHOR CONTRIBUTIONS

S.E.T., M.T.W., Z.Z., J.A.T. and I.I. designed research; S.E.T., C.Y., X.X. and I.I. performed research; B.D.F. and C.P.W., M.T.W., K.Y. and Z.Z. contributed new reagents/analytic tools; S.E.T., C.Y., X.X. and I.I. analyzed data; and S.E.T., C.Y., J.A.T, M.T.W., Z.Z. and I.I. wrote the paper.

SUPPORTING INFORMATION Supplementary figures and table are provided.

REFERENCES

  1. Armstrong AA, Mohideen F, Lima CD. Recognition of SUMO-modified PCNA requires tandem receptor motifs in Srs2. Nature. 2012;483:59–63. doi: 10.1038/nature10883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ayyagari R, Impellizzeri KJ, Yoder BL, Gary SL, Burgers PM. A mutational analysis of the yeast proliferating cell nuclear antigen indicates distinct roles in DNA replication and DNA repair. Mol Cell Biol. 1995;15:4420–4429. doi: 10.1128/mcb.15.8.4420. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baba D, Maita N, Jee JG, Uchimura Y, Saitoh H, Sugasawa K, Hanaoka F, Tochio H, Hiroaki H, Shirakawa M. Crystal structure of thymine DNA glycosylase conjugated to SUMO-1. Nature. 2005;435:979–982. doi: 10.1038/nature03634. [DOI] [PubMed] [Google Scholar]
  4. Baker R, Lewis SM, Sasaki AT, Wilkerson EM, Locasale JW, Cantley LC, Kuhlman B, Dohlman HG, Campbell SL. Site-specific monoubiquitination activates Ras by impeding GTPase-activating protein function. Nat Struct Mol Biol. 2013;20:46–52. doi: 10.1038/nsmb.2430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bienko M, Green CM, Crosetto N, Rudolf F, Zapart G, Coull B, Kannouche P, Wider G, Peter M, Lehmann AR, et al. Ubiquitin-binding domains in Y-family polymerases regulate translesion synthesis. Science. 2005;310:1821–1824. doi: 10.1126/science.1120615. [DOI] [PubMed] [Google Scholar]
  6. Burkovics P, Sebesta M, Sisakova A, Plault N, Szukacsov V, Robert T, Pinter L, Marini V, Kolesar P, Haracska L, et al. Srs2 mediates PCNA-SUMO-dependent inhibition of DNA repair synthesis. EMBO J. 2013;32:742–755. doi: 10.1038/emboj.2013.9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Case DA, Cheatham TE, Darden T, Gohlke H, Luo R, Merz KM, Onufriev A, Simmerling C, Wang B, Woods RJ. The Amber biomolecular simulation programs. J Comput Chem. 2005;26:1668–1688. doi: 10.1002/jcc.20290. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chen JJ, Ai YX, Wang JL, Haracska L, Zhuang ZH. Chemically ubiquitylated PCNA as a probe for eukaryotic translesion DNA synthesis. Nat Chem Biol. 2010;6:270–272. doi: 10.1038/nchembio.316. [DOI] [PubMed] [Google Scholar]
  9. Classen S, Hura GL, Holton JM, Rambo RP, Rodic I, McGuire PJ, Dyer K, Hammel M, Meigs G, Frankel KA, Tainer JA. Implementation and performance of SIBYLS: a dual endstation small-angle X-ray scattering and macromolecular crystallography beamline at the Advanced Light Source. J Appl Crystallogr. 2013;46:1–13. doi: 10.1107/S0021889812048698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Classen S, Rodic I, Holton J, Hura GL, Hammel M, Tainer JA. Software for the high-throughput collection of SAXS data using an enhanced Blu-Ice/DCS control system. J Synchrotron Radiat. 2010;17:774–781. doi: 10.1107/S0909049510028566. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cornell WD, Cieplak P, Bayly CI, Gould IR, Merz KM, Ferguson DM, Spellmeyer DC, Fox T, Caldwell JW, Kollman PA. A 2nd Generation Force-Field for the Simulation of Proteins, Nucleic-Acids, and Organic-Molecules. J Am Chem Soc. 1995;117:5179–5197. [Google Scholar]
  12. Das-Bradoo S, Nguyen HD, Wood JL, Ricke RM, Haworth JC, Bielinsky AK. Defects in DNA ligase I trigger PCNA ubiquitylation at Lys 107. Nat Cell Biol. 2010;12:74–79. doi: 10.1038/ncb2007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Deshpande RA, Williams GJ, Limbo O, Williams RS, Kuhnlein J, Lee JH, Classen S, Guenther G, Russell P, Tainer JA, Paull TT. ATP-driven Rad50 conformations regulate DNA tethering, end resection, and ATM checkpoint signaling. EMBO J. 2014;33:482–500. doi: 10.1002/embj.201386100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Duan Y, Wu C, Chowdhury S, Lee MC, Xiong GM, Zhang W, Yang R, Cieplak P, Luo R, Lee T, et al. A point-charge force field for molecular mechanics simulations of proteins based on condensed-phase quantum mechanical calculations. J Comput Chem. 2003;24:1999–2012. doi: 10.1002/jcc.10349. [DOI] [PubMed] [Google Scholar]
  15. Durand D, Vives C, Cannella D, Perez J, Pebay-Peyroula E, Vachette P, Fieschi F. NADPH oxidase activator p67(phox) behaves in solution as a multidomain protein with semi-flexible linkers. J Struct Biol. 2010;169:45–53. doi: 10.1016/j.jsb.2009.08.009. [DOI] [PubMed] [Google Scholar]
  16. Essmann U, Perera L, Berkowitz ML, Darden T, Lee H, Pedersen LG. A smooth particle mesh Ewald method. J Chem Phys. 1995;103:8577–8593. [Google Scholar]
  17. Freudenthal BD, Brogie JE, Gakhar L, Kondratick CM, Washington MT. Crystal Structure of SUMO-Modified Proliferating Cell Nuclear Antigen. J Mol Biol. 2011;406:9–17. doi: 10.1016/j.jmb.2010.12.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Freudenthal BD, Gakhar L, Ramaswamy S, Washington MT. Structure of monoubiquitinated PCNA and implications for translesion synthesis and DNA polymerase exchange. Nat Struct Mol Biol. 2010;17:479–U123. doi: 10.1038/nsmb.1776. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hammel M. Validation of macromolecular flexibility in solution by small-angle X-ray scattering (SAXS). Eur Biophys J. 2012;41:789–799. doi: 10.1007/s00249-012-0820-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hicke L. Protein regulation by monoubiquitin. Nat Rev Mol Cell Biol. 2001;2:195–201. doi: 10.1038/35056583. [DOI] [PubMed] [Google Scholar]
  21. Hoege C, Pfander B, Moldovan GL, Pyrowolakis G, Jentsch S. RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature. 2002;419:135–141. doi: 10.1038/nature00991. [DOI] [PubMed] [Google Scholar]
  22. Hura GL, Menon AL, Hammel M, Rambo RP, Poole FL, 2nd, Tsutakawa SE, Jenney FE, Jr., Classen S, Frankel KA, Hopkins RC, et al. Robust, high-throughput solution structural analyses by small angle X-ray scattering (SAXS). Nat Methods. 2009;6:606–612. doi: 10.1038/nmeth.1353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ivanov I, Chapados BR, McCammon JA, Tainer JA. Proliferating cell nuclear antigen loaded onto double-stranded DNA: dynamics, minor groove interactions and functional implications. Nucleic Acids Res. 2006;34:6023–6033. doi: 10.1093/nar/gkl744. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jonsson ZO, Hubscher U. Proliferating cell nuclear antigen: more than a clamp for DNA polymerases. Bioessays. 1997;19:967–975. doi: 10.1002/bies.950191106. [DOI] [PubMed] [Google Scholar]
  25. Jorgensen WL, Chandrasekhar J, Madura JD, Impey RW, Klein ML. Comparison of simple potential functions for simulating liquid water. J Chem Phys. 1983;79:926–935. [Google Scholar]
  26. Kale L, Skeel R, Bhandarkar M, Brunner R, Gursoy A, Krawetz N, Phillips J, Shinozaki A, Varadarajan K, Schulten K. NAMD2: Greater scalability for parallel molecular dynamics. J Comput Phys. 1999;151:283–312. [Google Scholar]
  27. Kelman Z. PCNA: structure, functions and interactions. Oncogene. 1997;14:629–640. doi: 10.1038/sj.onc.1200886. [DOI] [PubMed] [Google Scholar]
  28. Krejci L, Van Komen S, Li Y, Villemain J, Reddy MS, Klein H, Ellenberger T, Sung P. DNA helicase Srs2 disrupts the Rad51 presynaptic filament. Nature. 2003;423:305–309. doi: 10.1038/nature01577. [DOI] [PubMed] [Google Scholar]
  29. Krishna TS, Kong XP, Gary S, Burgers PM, Kuriyan J. Crystal structure of the eukaryotic DNA polymerase processivity factor PCNA. Cell. 1994;79:1233–1243. doi: 10.1016/0092-8674(94)90014-0. [DOI] [PubMed] [Google Scholar]
  30. Kuhlman B, Baker D. Native protein sequences are close to optimal for their structures. Proc Natl Acad Sci USA. 2000;97:10383–10388. doi: 10.1073/pnas.97.19.10383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Leaver-Fay A, Tyka M, Lewis SM, Lange OF, Thompson J, Jacak R, Kaufman K, Renfrew PD, Smith CA, Sheffler W, et al. ROSETTA3: an object-oriented software suite for the simulation and design of macromolecules. Methods Enzymol. 2011;487:545–574. doi: 10.1016/B978-0-12-381270-4.00019-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Maga G, Hubscher U. Proliferating cell nuclear antigen (PCNA): a dancer with many partners. J Cell Sci. 2003;116:3051–3060. doi: 10.1242/jcs.00653. [DOI] [PubMed] [Google Scholar]
  33. Moldovan GL, Pfander B, Jentsch S. PCNA, the maestro of the replication fork. Cell. 2007;129:665–679. doi: 10.1016/j.cell.2007.05.003. [DOI] [PubMed] [Google Scholar]
  34. Nguyen HD, Becker J, Thu YM, Costanzo M, Koch EN, Smith S, Myung K, Myers CL, Boone C, Bielinsky AK. Unligated Okazaki Fragments Induce PCNA Ubiquitination and a Requirement for Rad59-Dependent Replication Fork Progression. PloS ONE. 2013;8:e66379. doi: 10.1371/journal.pone.0066379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Papouli E, Chen SH, Davies AA, Huttner D, Krejci L, Sung P, Ulrich HD. Crosstalk between SUMO and ubiquitin on PCNA is mediated by recruitment of the helicase Srs2p. Mol Cell. 2005;19:123–133. doi: 10.1016/j.molcel.2005.06.001. [DOI] [PubMed] [Google Scholar]
  36. Parker JL, Ulrich HD. A SUMO-interacting motif activates budding yeast ubiquitin ligase Rad18 towards SUMO-modified PCNA. Nucleic Acids Res. 2012;40:11380–11388. doi: 10.1093/nar/gks892. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Pelikan M, Hura GL, Hammel M. Structure and flexibility within proteins as identified through small angle X-ray scattering. Gen Physiol Biophys. 2009;28:174–189. doi: 10.4149/gpb_2009_02_174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Perry JJP, Tainer JA, Boddy MN. A SIM-ultaneous role for SUMO and ubiquitin. Trends Biochem Sci. 2008;33:201–208. doi: 10.1016/j.tibs.2008.02.001. [DOI] [PubMed] [Google Scholar]
  39. Pfander B, Moldovan GL, Sacher M, Hoege C, Jentsch S. SUMO-modified PCNA recruits Srs2 to prevent recombination during S phase. Nature. 2005;436:428–433. doi: 10.1038/nature03665. [DOI] [PubMed] [Google Scholar]
  40. Phillips JC, Braun R, Wang W, Gumbart J, Tajkhorshid E, Villa E, Chipot C, Skeel RD, Kale L, Schulten K. Scalable molecular dynamics with NAMD. J Comput Chem. 2005;26:1781–1802. doi: 10.1002/jcc.20289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Povlsen LK, Beli P, Wagner SA, Poulsen SL, Sylvestersen KB, Poulsen JW, Nielsen ML, Bekker-Jensen S, Mailand N, Choudhary C. Systems-wide analysis of ubiquitylation dynamics reveals a key role for PAF15 ubiquitylation in DNA-damage bypass. Nat Cell Biol. 2012;14:1089–1098. doi: 10.1038/ncb2579. [DOI] [PubMed] [Google Scholar]
  42. Putnam CD, Hammel M, Hura GL, Tainer JA. X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution. Q Rev Biophys. 2007;40:191–285. doi: 10.1017/S0033583507004635. [DOI] [PubMed] [Google Scholar]
  43. Rambo RP, Tainer JA. Bridging the solution divide: comprehensive structural analyses of dynamic RNA, DNA, and protein assemblies by small-angle X-ray scattering. Curr Opin Struc Biol. 2010;20:128–137. doi: 10.1016/j.sbi.2009.12.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Rambo RP, Tainer JA. Characterizing flexible and intrinsically unstructured biological macromolecules by SAS using the Porod-Debye law. Biopolymers. 2011;95:559–571. doi: 10.1002/bip.21638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Rambo RP, Tainer JA. Accurate assessment of mass, models and resolution by small-angle scattering. Nature. 2013;496:477–481. doi: 10.1038/nature12070. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Reyes FE, Schwartz CR, Tainer JA, Rambo RP. Methods for using new conceptual tools and parameters to assess RNA structure by small-angle X-ray scattering. Methods Enzymol. 2014;549:235–263. doi: 10.1016/B978-0-12-801122-5.00011-8. [DOI] [PubMed] [Google Scholar]
  47. Rohl CA, Strauss CE, Misura KM, Baker D. Protein structure prediction using Rosetta. Methods Enzymol. 2004;383:66–93. doi: 10.1016/S0076-6879(04)83004-0. [DOI] [PubMed] [Google Scholar]
  48. Saha A, Lewis S, Kleiger G, Kuhlman B, Deshaies RJ. Essential role for ubiquitin-ubiquitin-conjugating enzyme interaction in ubiquitin discharge from Cdc34 to substrate. Mol Cell. 2011;42:75–83. doi: 10.1016/j.molcel.2011.03.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Schneidman-Duhovny D, Hammel M, Sali A. FoXS: a web server for rapid computation and fitting of SAXS profiles. Nucleic Acids Res. 2010;38:W540–544. doi: 10.1093/nar/gkq461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Schneidman-Duhovny D, Hammel M, Tainer JA, Sali A. Accurate SAXS profile computation and its assessment by contrast variation experiments. Biophys J. 2013;105:962–974. doi: 10.1016/j.bpj.2013.07.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Steinacher R, Schar P. Functionality of human thymine DNA glycosylase requires SUMO-regulated changes in protein conformation. Curr Biol. 2005;15:616–623. doi: 10.1016/j.cub.2005.02.054. [DOI] [PubMed] [Google Scholar]
  52. Stelter P, Ulrich HD. Control of spontaneous and damage-induced mutagenesis by SUMO and ubiquitin conjugation. Nature. 2003;425:188–191. doi: 10.1038/nature01965. [DOI] [PubMed] [Google Scholar]
  53. Stolimenov I, Helleday T. PCNA on the crossroad of cancer. Biochem Soc Trans. 2009;37:605–613. doi: 10.1042/BST0370605. [DOI] [PubMed] [Google Scholar]
  54. Tsutakawa SE, Van Wynsberghe AW, Freudenthal BD, Weinacht CP, Gakhar L, Washington MT, Zhuang Z, Tainer JA, Ivanov I. Solution X-ray scattering combined with computational modeling reveals multiple conformations of covalently bound ubiquitin on PCNA. Proc Natl Acad Sci USA. 2011;108:17672–17677. doi: 10.1073/pnas.1110480108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Tuckerman M, Berne BJ, Martyna GJ. Reversible Multiple Time Scale Molecular-Dynamics. J Chem Phys. 1992;97:1990–2001. [Google Scholar]
  56. Ulrich HD. Mutual interactions between the SUMO and ubiquitin systems: a plea of no contest. Trends Cell Biol. 2005;15:525–532. doi: 10.1016/j.tcb.2005.08.002. [DOI] [PubMed] [Google Scholar]
  57. Ulrich HD. Ubiquitin and SUMO in DNA repair at a glance. J Cell Sci. 2012;125:249–254. doi: 10.1242/jcs.091801. [DOI] [PubMed] [Google Scholar]
  58. Ulrich HD, Walden H. Ubiquitin signalling in DNA replication and repair. Nature reviews. Mol Cell Biol. 2010;11:479–489. doi: 10.1038/nrm2921. [DOI] [PubMed] [Google Scholar]
  59. Veaute X, Jeusset J, Soustelle C, Kowalczykowski SC, Le Cam E, Fabre F. The Srs2 helicase prevents recombination by disrupting Rad51 nucleoprotein filaments. Nature. 2003;423:309–312. doi: 10.1038/nature01585. [DOI] [PubMed] [Google Scholar]
  60. Vijay-Kumar S, Bugg CE, Cook WJ. Structure of ubiquitin refined at 1.8 A resolution. J Mol Biol. 1987;194:531–544. doi: 10.1016/0022-2836(87)90679-6. [DOI] [PubMed] [Google Scholar]
  61. Xu G, Paige JS, Jaffrey SR. Global analysis of lysine ubiquitination by ubiquitin remnant immunoaffinity profiling. Nat Biotechnol. 2010;28:868–873. doi: 10.1038/nbt.1654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Yang K, Weinacht CP, Zhuang Z. Regulatory Role of Ubiquitin in Eukaryotic DNA Translesion Synthesis. Biochemistry. 2013;52:3217–3228. doi: 10.1021/bi400194r. [DOI] [PubMed] [Google Scholar]
  63. Zhang H, Gibbs PE, Lawrence CW. The Saccharomyces cerevisiae rev6-1 mutation, which inhibits both the lesion bypass and the recombination mode of DNA damage tolerance, is an allele of POL30, encoding proliferating cell nuclear antigen. Genetics. 2006;173:1983–1989. doi: 10.1534/genetics.106.058545. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS667080-supplement-1.pdf (2.3MB, pdf)
2
NIHMS667080-supplement-2.docx (207.7KB, docx)

RESOURCES