Translesion Polymerase Research Articles

Checkpoint Regulation of Replication Forks at Sites of DNA Damage

Statement of methods:We have used single‐molecule DNA combing to study how the checkpoint regulates individual origins and forks in response to DNA damage.imageSummary of resultsThe S‐phase DNA damage checkpoint slows replication and allows cells to replicate damaged DNA, but how slowing relates to damage tolerance remains unclear. The standard explanation — that checkpoint delays cell‐cycle progression until the damage can be repaired — does not seem to apply in the case of DNA replication. Instead, replication proceeds in the presence of the damage, but at a slower rate. Replication slowing can be achieved by inhibition of origin firing, slowing of fork progression or a combination of both. In bulk assays, 1uM 4NQO and 0.03% MMS slow S phase to a similar extent. However the combing data shows that 4NQO slows replication by reducing origin firing rate, while MMS slows S phase by stalling forks and reducing the fork rate. While reduction in origin firing rate is checkpoint dependent, reduction in fork rate has checkpoint dependent and independent components. We are currently investigating the mechanistic difference in response to different damaging agents and how the checkpoint regulates fork components to achieve replication slowingAnother key question is how forks replicate quickly through damaged DNA in the absence of the checkpoint. The MMS‐induced lesions we use are believed to stop replicative polymerases. Nonetheless, in the absence of the checkpoint, replication forks pass them without any apparent delay. This bypass seems to be mainly dependent on recombination and translesion polymerase based synthesis. We are working on how forks bypass polymerase‐stalling lesions and what effect this bypass has on genomic stability.Funding Source:NIHGMS

Loss of Pol32 in Drosophila melanogaster causes chromosome instability and suppresses variegation.

Pol32 is an accessory subunit of the replicative DNA Polymerase δ and of the translesion Polymerase ζ. Pol32 is involved in DNA replication, recombination and repair. Pol32’s participation in high- and low-fidelity processes, together with the phenotypes arising from its disruption, imply multiple roles for this subunit within eukaryotic cells, not all of which have been fully elucidated. Using pol32 null mutants and two partial loss-of-function alleles pol32rd1 and pol32rds in Drosophila melanogaster, we show that Pol32 plays an essential role in promoting genome stability. Pol32 is essential to ensure DNA replication in early embryogenesis and it participates in the repair of mitotic chromosome breakage. In addition we found that pol32 mutantssuppress position effect variegation, suggesting a role for Pol32 in chromatin architecture.

How a homolog of high-fidelity replicases conducts mutagenic DNA synthesis.

All DNA replicases achieve high fidelity by a conserved mechanism, but each translesion polymerase carries out mutagenic DNA synthesis in its own way. Here we report crystal structures of human DNA polymerase ν (Pol ν), which is homologous to high-fidelity replicases and yet error-prone. Instead of a simple open-to-closed movement of the O helix upon binding of a correct incoming nucleotide, Pol ν has a different open state and requires the finger domain to swing sideways and undergo both opening and closing motions to accommodate the nascent base pair. A single amino acid substitution in the O-helix of the finger domain improves the fidelity of Pol ν nearly ten-fold. A unique cavity and the flexibility of the thumb domain allow Pol ν to generate and accommodate a looped-out primer strand. Primer loopout may be a mechanism for DNA trinucloetide-repeat expansion.

Translesion polymerase genes polymorphisms and haplotypes influence survival of osteosarcoma patients.

Cytotoxic activity of most chemotherapeutic agents is based on their ability to induce DNA damage. Interstrand crosslinks are among the most detrimental forms of DNA damage as both DNA strands are affected. As translesion polymerases participate in their repair, they may be important for response to chemotherapeutic agents that induce such lesions, including commonly used cisplatin. Altered expression of translesion polymerase genes REV1 and REV3L may modify sensitivity to cisplatin. As osteosarcoma patients are commonly treated with cisplatin-based chemotherapy, our aim was to investigate if REV1 and REV3L polymorphisms influence survival of osteosarcoma patients treated with cisplatin-based chemotherapy. We determined the genotypes of common functional tag REV1 and REV3L polymorphisms in 66 osteosarcoma patients. Cox regression was used for survival analysis. Carriers of at least one polymorphic REV1 rs3087403 allele had significantly shorter EFS and overall survival (OS) (p = 0.004; HR = 3.79; 95%CI = 1.53-9.35 and p < 0.001; HR = 4.44; 95%CI = 1.92-10.27, respectively). Combination of REV1 rs3087403 and REV3L rs462779 polymorphisms was also significantly associated with shorter OS (ptrend<0.001) and shorter EFS (ptrend = 0.003). The results of this first study on polymorphisms in translesion polymerase genes in osteosarcoma suggest they could help predict outcome of cisplatin-based chemotherapy in osteosarcoma patients.

The steady-state level and stability of TLS polymerase eta are cell cycle dependent in the yeast S. cerevisiae.

Polymerase eta (Pol eta) is a ubiquitous translesion DNA polymerase that is capable of bypassing UV-induced pyrimidine dimers in an error-free manner. However, this specialized polymerase is error prone when synthesizing through an undamaged DNA template. In Saccharomyces cerevisiae, both depletion and overproduction of Pol eta result in mutator phenotypes. Therefore, regulation of the cellular abundance of this enzyme is of particular interest. However, based on the investigation of variously tagged forms of Pol eta, mutually contradictory conclusions have been reached regarding the stability of this polymerase in yeast. Here, we optimized a protocol for the detection of untagged yeast Pol eta and established that the half-life of the native enzyme is 80 ± 14 min in asynchronously growing cultures. Experiments with synchronized cells indicated that the cellular abundance of this translesion polymerase changes throughout the cell cycle. Accordingly, we show that the stability of Pol eta, but not its mRNA level, is cell cycle stage dependent. The half-life of the polymerase is more than fourfold shorter in G1-arrested cells than in those at G2/M. Our results, in concert with previous data for Rev1, indicate that cell cycle regulation is a general property of Y family TLS polymerases in S. cerevisiae.

Raman Spectroscopy: A Promising Technique for Analyzing Nucleic Acid Refractory to PCR Amplification

Ancient DNA (aDNA) refers to the genetic material found in dead paleontological and archeological samples. Being subject to various types of stressors, it undergoes different hydrolytic and oxidative post mortem modifications that result in the formation of DNA lesions. These lesions are found to either block the DNA polymerase during replication or induce nucleotide misincorporations. Besides, aDNA samples occur in minimal quantities; which represents an additional obstacle that researchers have to overcome in order to study aDNA. The earliest major efforts included the use of molecular cloning and polymerase chain reaction (PCR) to amplify aDNA sequences. These techniques were later found to be associated with a number of false results and unauthentic findings. There have been numerous attempts to eliminate the shortcomings of PCR and improve the quality of aDNA through avoiding contamination, repairing lesions, using translesion polymerases, etc. However, the majority of these have failed to yield accurate and specific results. Surface- enhanced resonance Raman scattering (SERRS), on the other hand, starts in the right foot by introducing a sensitive non- enzymatic approach for the specific detection of single- and double-stranded DNA. The ability of this method to evade DNA degradation is particularly important for not only studying aDNA, but also analyzing DNA refractory to PCR amplification in processed products.

Replisome-mediated Translesion Synthesis and Leading Strand Template Lesion Skipping Are Competing Bypass Mechanisms

A number of different enzymatic pathways have evolved to ensure that DNA replication can proceed past template base damage. These pathways include lesion skipping by the replisome, replication fork regression followed by either correction of the damage and origin-independent replication restart or homologous recombination-mediated restart of replication downstream of the lesion, and bypass of the damage by a translesion synthesis DNA polymerase. We report here that of two translesion synthesis polymerases tested, only DNA polymerase IV, not DNA polymerase II, could engage productively with the Escherichia coli replisome to bypass leading strand template damage, despite the fact that both enzymes are shown to be interacting with the replicase. Inactivation of the 3' → 5' proofreading exonuclease of DNA polymerase II did not enable bypass. Bypass by DNA polymerase IV required its ability to interact with the β clamp and act as a translesion polymerase but did not require its "little finger" domain, a secondary region of interaction with the β clamp. Bypass by DNA polymerase IV came at the expense of the inherent leading strand lesion skipping activity of the replisome, indicating that they are competing reactions.

Polymerase η suppresses telomere defects induced by DNA damaging agents.

Telomeres at chromosome ends are normally masked from proteins that signal and repair DNA double strand breaks (DSBs). Bulky DNA lesions can cause DSBs if they block DNA replication, unless they are bypassed by translesion (TLS) DNA polymerases. Here, we investigated roles for TLS polymerase η, (polη) in preserving telomeres following acute physical UVC exposure and chronic chemical Cr(VI) exposure, which both induce blocking lesions. We report that polη protects against cytotoxicity and replication stress caused by Cr(VI), similar to results with ultraviolet C light (UVC). Both exposures induce ataxia telangiectasia and Rad3-related (ATR) kinase and polη accumulation into nuclear foci and localization to individual telomeres, consistent with replication fork stalling at DNA lesions. Polη-deficient cells exhibited greater numbers of telomeres that co-localized with DSB response proteins after exposures. Furthermore, the genotoxic exposures induced telomere aberrations associated with failures in telomere replication that were suppressed by polη. We propose that polη's ability to bypass bulky DNA lesions at telomeres is critical for proper telomere replication following genotoxic exposures.

Analysis of SHPRH functions in DNA repair and immunoglobulin diversification.

During replication, bypass of DNA lesions is orchestrated by the Rad6 pathway. Monoubiquitination of proliferating cell nuclear antigen (PCNA) by Rad6/Rad18 leads to recruitment of translesion polymerases for direct and potentially mutagenic damage bypass. An error-free bypass pathway may be initiated via K63-linked PCNA polyubiquitination by Ubc13/Mms2 and the E3 ligase Rad5 in yeast, or HLTF/SHPRH in vertebrates. For the latter two enzymes, redundancy with a third E3 ligase and alternative functions have been reported. We have previously shown that the Rad6 pathway is involved in somatic hypermutation of immunoglobulin genes in B lymphocytes. Here, we have used knockout strategies targeting expression of the entire SHPRH protein or functionally significant domains in chicken DT40 cells that do not harbor a HLTF ortholog. We show that SHPRH is apparently redundant with another E3 ligase during DNA damage-induced PCNA modification. SHPRH plays no substantial role in cellular resistance to drugs initiating excision repair and the Rad6 pathway, but is important in survival of topoisomerase II inhibitor treatment. Removal of only the C-terminal RING domain does not interfere with this SHPRH function. SHPRH inactivation does not substantially impact on the overall efficacy of Ig diversification. Redundancy of E3 ligases in the Rad6 pathway may be linked to its different functions in genome maintenance and genetic plasticity.

Translesion polymerase η is upregulated by cancer therapeutics and confers anticancer drug resistance.

DNA repair processes are a key determinant of the sensitivity of cancer cells to DNA-damaging chemotherapeutics, which may induce certain repair genes as a mechanism to promote resistance. Here, we report the results of a screen for repair genes induced in cancer cells treated with DNA crosslinking agents, which identified the translesion polymerase η (PolH) as a p53-regulated target acting as one defense against interstrand crosslink (ICL)-inducing agents. PolH was induced by fotemustine, mafosfamide, and lomustine in breast cancer, glioma, and melanoma cells in vitro and in vivo, with similar inductions observed in normal cells such as lymphocytes and diploid fibroblasts. PolH contributions to the protection against ICL-inducing agents were evaluated by its siRNA-mediated attenuation in cells, which elevated sensitivity to these drugs in all tumor cell models. Conversely, PolH overexpression protected cancer cells against these drugs. PolH attenuation reduced repair of ICL lesions as measured by host cell reactivation assays and enhanced persistence of γH2AX foci. Moreover, we observed a strong accumulation of PolH in the nucleus of drug-treated cells along with direct binding to damaged DNA. Taken together, our findings implicated PolH in ICL repair as a mechanism of cancer drug resistance and normal tissue protection.

Hybridization of G-Quadruplex-Forming Peptide Nucleic Acids to Guanine-Rich DNA Templates Inhibits DNA Polymerase η Extension

The guanine quadruplex (G-quadruplex) is a highly stable secondary structure that forms in G-rich repeats of DNA, which can interfere with DNA processes, including DNA replication and transcription. We showed previously that short guanine-rich peptide nucleic acids (PNAs) can form highly stable hybrid quadruplexes with DNA. We hypothesized that such structures would provide a stronger block to polymerase extension on G-rich templates than a native DNA homoquadruplex because of the greater thermodynamic stability of the PNA-DNA hybrid structures. To test this, we analyzed the DNA primer extension activity of polymerase η, a translesion polymerase implicated in synthesis past G-quadruplex blocks, on DNA templates containing guanine repeats. We observed a PNA concentration-dependent decrease in the level of polymerase η extension to the end of the template and an increase in the level of polymerase η inhibition at the sequence prior to the G-rich repeats. In contrast, the addition of a complementary C-rich PNA that hybridizes to the G-rich repeats by Watson-Crick base pairing led to a decrease in the level of polymerase inhibition and an increase in the level of full-length extension products. The G-quadruplex-forming PNA exhibited inhibition (IC50=16.2±3.3 nM) of polymerase η DNA synthesis on the G-rich templates stronger than that of the established G-quadruplex-stabilizing ligand BRACO-19 (IC50=42.5±4.8 nM). Our results indicate that homologous PNA targeting of G-rich sequences creates stable PNA-DNA heteroquadruplexes that inhibit polymerase η extension more effectively than a DNA homoquadruplex. The implications of these results for the potential development of homologous PNAs as therapeutics for halting proliferating cancer cells are discussed.

Whole genome RNAi screens reveal a critical role of REV3 in coping with replication stress

REV3, the catalytic subunit of translesion polymerase zeta (polζ), is commonly associated with DNA damage bypass and repair. Despite sharing accessory subunits with replicative polymerase δ, very little is known about the role of polζ in DNA replication. We previously demonstrated that inhibition of REV3 expression induces persistent DNA damage and growth arrest in cancer cells. To reveal determinants of this sensitivity and obtain insights into the cellular function of REV3, we performed whole human genome RNAi library screens aimed at identification of synthetic lethal interactions with REV3 in A549 lung cancer cells. The top confirmed hit was RRM1, the large subunit of ribonucleotide reductase (RNR), a critical enzyme of de novo nucleotide synthesis. Treatment with the RNR-inhibitor hydroxyurea (HU) synergistically increased the fraction of REV3-deficient cells containing single stranded DNA (ssDNA) as indicated by an increase in replication protein A (RPA). However, this increase was not accompanied by accumulation of the DNA damage marker γH2AX suggesting a role of REV3 in counteracting HU-induced replication stress (RS). Consistent with a role of REV3 in DNA replication, increased RPA staining was confined to HU-treated S-phase cells. Additionally, we found genes related to RS to be significantly enriched among the top hits of the synthetic sickness/lethality (SSL) screen further corroborating the importance of REV3 for DNA replication under conditions of RS.

Swi2/Snf2-like protein Uls1 functions in the Sgs1-dependent pathway of maintenance of rDNA stability and alleviation of replication stress.

The Saccharomyces cerevisiae Uls1 belongs to the Swi2/Snf2 family of DNA-dependent ATPases and a new protein family of SUMO-targeted ubiquitin ligases. Here we show that Uls1 is implicated in DNA repair independently of the replication stress response pathways mediated by the endonucleases Mus81 and Yen1 and the helicases Mph1 and Srs2. Uls1 works together with Sgs1 and we demonstrate that the attenuation of replication stress-related defects in sgs1Δ by deletion of ULS1 depends on a functional of Rad51 recombinase and post-replication repair pathway mediated by Rad18 and Rad5, but not on the translesion polymerase, Rev3. The higher resistance of sgs1Δ uls1Δ mutants to genotoxic stress compared to single sgs1Δ cells is not the result of decreased formation or accelerated resolution of recombination-dependent DNA structures. Instead, deletion of ULS1 restores stability of the rDNA region in sgs1Δ cells. Our data suggest that Uls1 may contribute to genomic stability during DNA synthesis and channel the repair of replication lesions into the Sgs1-dependent pathway, with DNA translocase and SUMO binding activities of Uls1 as well as a RING domain being essential for its functions in replication stress response.

Polymorphisms in translesion polymerase genes influence treatment outcome in malignant mesothelioma.

We evaluated the influence of genetic variability in translesion polymerases REV1 and REV3L on the outcome of cisplatin treatment in malignant mesothelioma patients. In total, 139 malignant mesothelioma patients were genotyped for seven tag SNPs in REV1 and REV3L. Logistic regression and Cox regression were used to assess the influence of SNPs on treatment outcome. Polymorphic REV1 rs3087403 allele and REV1 TGT haplotype were associated with increased risk for leukopenia (p = 0.013 and p = 0.047, respectively) and neutropenia (p = 0.048 and p = 0.024, respectively). REV3L rs465646, rs462779 and REV3L CCGG haplotype were significantly associated with longer overall survival (p = 0.007, p = 0.022 and p = 0.013, respectively). Our results suggest for the first time that REV1 and REV3L SNPs might serve as potential predictive markers of outcome of cisplatin-based chemotherapy. Original submitted 7 October 2013; Revision submitted 15 January 2014.

Error-free versus mutagenic processing of genomic uracil--relevance to cancer.

Genomic uracil is normally processed essentially error-free by base excision repair (BER), with mismatch repair (MMR) as an apparent backup for U:G mismatches. Nuclear uracil-DNA glycosylase UNG2 is the major enzyme initiating BER of uracil of U:A pairs as well as U:G mismatches. Deficiency in UNG2 results in several-fold increases in genomic uracil in mammalian cells. Thus, the alternative uracil-removing glycosylases, SMUG1, TDG and MBD4 cannot efficiently complement UNG2-deficiency. A major function of SMUG1 is probably to remove 5-hydroxymethyluracil from DNA with general back-up for UNG2 as a minor function. TDG and MBD4 remove deamination products U or T mismatched to G in CpG/mCpG contexts, but may have equally or more important functions in development, epigenetics and gene regulation. Genomic uracil was previously thought to arise only from spontaneous cytosine deamination and incorporation of dUMP, generating U:G mismatches and U:A pairs, respectively. However, the identification of activation-induced cytidine deaminase (AID) and other APOBEC family members as DNA-cytosine deaminases has spurred renewed interest in the processing of genomic uracil. Importantly, AID triggers the adaptive immune response involving error-prone processing of U:G mismatches, but also contributes to B-cell lymphomagenesis. Furthermore, mutational signatures in a substantial fraction of other human cancers are consistent with APOBEC-induced mutagenesis, with U:G mismatches as prime suspects. Mutations can be caused by replicative polymerases copying uracil in U:G mismatches, or by translesion polymerases that insert incorrect bases opposite abasic sites after uracil-removal. In addition, kataegis, localized hypermutations in one strand in the vicinity of genomic rearrangements, requires APOBEC protein, UNG2 and translesion polymerase REV1. What mechanisms govern error-free versus error prone processing of uracil in DNA remains unclear. In conclusion, genomic uracil is an essential intermediate in adaptive immunity and innate antiviral responses, but may also be a fundamental cause of a wide range of malignancies.

Characterization of putative mutagenesis cassettes in Sinorhizobium meliloti (927.1)

The goal of this research is to biochemically characterize chromosomal and plasmid‐encoded members of Y‐family DNA polymerases and putative mutagenesis cassettes from the nitrogen‐fixing plant symbiotic bacterium Sinorhizobium meliloti. Y‐family translesion polymerases are recruited to sites of DNA damage to replicate damaged DNA that may cause disruption to the replication fork. ImuC is a C family DNA polymerase with a putative function in mutagenic DNA replication; while ImuA and ImuB appear to be important for ImuC function, their roles have yet to be elucidated. S. meliloti harbors the Y family DNA polymerase dinB and the imuABC mutagenesis cassette encoded on both the chromosome and one of the symbiotic plasmids. All of the genes of the putative mutagenesis cassettes have been subcloned into two different plasmid vectors for in vivo and in vitro experiments. We are currently optimizing conditions for protein expression in E. coli. We are also carrying out assays to determine the ability of these gene products to confer on E. coli survival to a variety of DNA damaging agents and damage‐induced mutagenesis. Supported by NSF and ACS

Mcm10 deficiency causes defective-replisome-induced mutagenesis and a dependency on error-free postreplicative repair

Mcm10 is a multifunctional replication factor with reported roles in origin activation, polymerase loading, and replication fork progression. The literature supporting these variable roles is controversial, and it has been debated whether Mcm10 has an active role in elongation. Here, we provide evidence that the mcm10–1 allele confers alterations in DNA synthesis that lead to defective-replisome-induced mutagenesis (DRIM). Specifically, we observed that mcm10–1 cells exhibited elevated levels of PCNA ubiquitination and activation of the translesion polymerase, pol-ζ. Whereas translesion synthesis had no measurable impact on viability, mcm10–1 mutants also engaged in error-free postreplicative repair (PRR), and this pathway promoted survival at semi-permissive conditions. Replication gaps in mcm10–1 were likely caused by elongation defects, as dbf4–1 mutants, which are compromised for origin activation did not display any hallmarks of replication stress. Furthermore, we demonstrate that deficiencies in priming, induced by a pol1–1 mutation, also resulted in DRIM, but not in error-free PRR. Similar to mcm10–1 mutants, DRIM did not rescue the replication defect in pol1–1 cells. Thus, it appears that DRIM is not proficient to fill replication gaps in pol1–1 and mcm10–1 mutants. Moreover, the ability to correctly prime nascent DNA may be a crucial prerequisite to initiate error-free PRR.

Three Residues of the Interdomain Linker Determine the Conformation and Single-base Deletion Fidelity of Y-family Translesion Polymerases

Dpo4 and Dbh are from two closely related Sulfolobus species and are well studied archaeal homologues of pol IV, an error prone Y-family polymerase from Escherichia coli. Despite sharing 54% amino acid identity, these polymerases display distinct mutagenic and translesion specificities. Structurally, Dpo4 and Dbh adopt different conformations because of the difference in relative orientation of their N-terminal catalytic and C-terminal DNA binding domains. Using chimeric constructs of these two polymerases, we have previously demonstrated that the interdomain linker is a major determinant of polymerase conformation, base-substitution fidelity, and abasic-site translesion synthesis. Here we find that the interdomain linker also affects the single-base deletion frequency and the mispair extension efficiency of these polymerases. Exchanging just three amino acids in the linkers of Dbh and Dpo4 is sufficient to change the fidelity by up to 30-fold, predominantly by altering the rate of correct (but not incorrect) nucleotide incorporation. Additionally, from a 2.4 Å resolution crystal structure, we have found that the three linker amino acids from Dpo4 are sufficient to allow Dbh to adopt the standard conformation of Dpo4. Thus, a small region of the interdomain linker, located more than 11 Å away from the catalytic residues, determines the fidelity of these Y-family polymerases, by controlling the alignment of substrates at the active site.

Intramolecular Telomeric G-Quadruplexes Dramatically Inhibit DNA Synthesis by Replicative and Translesion Polymerases, Revealing their Potential to Lead to Genetic Change

Recent research indicates that hundreds of thousands of G-rich sequences within the human genome have the potential to form secondary structures known as G-quadruplexes. Telomeric regions, consisting of long arrays of TTAGGG/AATCCC repeats, are among the most likely areas in which these structures might form. Since G-quadruplexes assemble from certain G-rich single-stranded sequences, they might arise when duplex DNA is unwound such as during replication. Coincidentally, these bulky structures when present in the DNA template might also hinder the action of DNA polymerases. In this study, single-stranded telomeric templates with the potential to form G-quadruplexes were examined for their effects on a variety of replicative and translesion DNA polymerases from humans and lower organisms. Our results demonstrate that single-stranded templates containing four telomeric GGG runs fold into intramolecular G-quadruplex structures. These intramolecular G quadruplexes are somewhat dynamic in nature and stabilized by increasing KCl concentrations and decreasing temperatures. Furthermore, the presence of these intramolecular G-quadruplexes in the template dramatically inhibits DNA synthesis by various DNA polymerases, including the human polymerase δ employed during lagging strand replication of G-rich telomeric strands and several human translesion DNA polymerases potentially recruited to sites of replication blockage. Notably, misincorporation of nucleotides is observed when certain translesion polymerases are employed on substrates containing intramolecular G-quadruplexes, as is extension of the resulting mismatched base pairs upon dynamic unfolding of this secondary structure. These findings reveal the potential for blockage of DNA replication and genetic changes related to sequences capable of forming intramolecular G-quadruplexes.

Structurally Distinct Complexes of Ubiquitin and Sumo-Modified PCNA Lead to Distinct DNA Damage Response Pathways

Ubiquitination of proliferating cell nuclear antigen (PCNA) in response to DNA damage leads to the recruitment of specialized translesion polymerases to the damage locus. This constitutes the initial step in translesion synthesis (TLS) - a critical pathway for cell survival and genome stability. By contrast, PCNA sumoylation leads to suppression of homologous recombination through recruitment of the antirecombinogenic helicase SRS2. How do these similar posttranslational modifications effect such vastly different functional outcomes? We modeled PCNA covalently modified by Ub and SUMO using a multiscale protocol (conjugated docking with the Rosetta package, molecular dynamics and minimal ensemble searches). Our models rationalized the differences in solution scattering data (SAXS) from the PCNA-K164Ub, PCNA-K107Ub and PCNA-K164SUMO complexes. Our results suggest a structural basis for the different functional outcomes of Ub vs. SUMO modification of PCNA.View Large Image | View Hi-Res Image | Download PowerPoint Slide