PCNA Binding Research Articles

Two independent DNA repair pathways cause mutagenesis in template switching deficient Saccharomyces cerevisiae.

Upon DNA replication stress, cells utilize the post-replication repair pathway to repair single-stranded DNA and maintain genome integrity. Post-replication repair is divided into two branches: error-prone translesion synthesis, signaled by PCNA mono-ubiquitination, and error-free template switching, signaled by PCNA poly-ubiquitination. In Saccharomyces cerevisiae, Rad5 is involved in both branches of repair during DNA replication stress. When the PCNA poly-ubiquitination function of Rad5 is disrupted, Rad5 recruits translesion synthesis polymerases to stalled replication forks, resulting in mutagenic repair. Details of how mutagenic repair is carried out, as well as the relationship between Rad5-mediated mutagenic repair and the canonical PCNA-mediated mutagenic repair, remain to be understood. We find that Rad5-mediated mutagenic repair requires the translesion synthesis polymerase ζ but does not require other yeast translesion polymerase activities. Furthermore, we show that Rad5-mediated mutagenic repair is independent of PCNA binding by Rev1 and so is separable from canonical mutagenic repair. In the absence of error-free template switching, both modes of mutagenic repair contribute additively to replication stress response in a replication timing-independent manner. Cellular contexts where error-free template switching is compromised are not simply laboratory phenomena, as we find that a natural variant in RAD5 is defective in PCNA poly-ubiquitination and therefore defective in error-free repair, resulting in Rad5- and PCNA-mediated mutagenic repair. Our results highlight the importance of Rad5 in regulating spontaneous mutagenesis and genetic diversity in S. cerevisiae through different modes of post-replication repair.

CAF-1 deposits newly synthesized histones during DNA replication using distinct mechanisms on the leading and lagging strands.

During every cell cycle, both the genome and the associated chromatin must be accurately replicated. Chromatin Assembly Factor-1 (CAF-1) is a key regulator of chromatin replication, but how CAF-1 functions in relation to the DNA replication machinery is unknown. Here, we reveal that this crosstalk differs between the leading and lagging strand at replication forks. Using biochemical reconstitutions, we show that DNA and histones promote CAF-1 recruitment to its binding partner PCNA and reveal that two CAF-1 complexes are required for efficient nucleosome assembly under these conditions. Remarkably, in the context of the replisome, CAF-1 competes with the leading strand DNA polymerase epsilon (Polϵ) for PCNA binding. However, CAF-1 does not affect the activity of the lagging strand DNA polymerase Delta (Polδ). Yet, in cells, CAF-1 deposits newly synthesized histones equally on both daughter strands. Thus, on the leading strand, chromatin assembly by CAF-1 cannot occur simultaneously to DNA synthesis, while on the lagging strand these processes may be coupled. We propose that these differences may facilitate distinct parental histone recycling mechanisms and accommodate the inherent asymmetry of DNA replication.

EP117 - IMAGe syndrome: further delineation of the clinical phenotype of PCNA binding domain CDKN1C variants

eP117 - IMAGe syndrome: further delineation of the clinical phenotype of PCNA binding domain CDKN1C variants

Deep exploration of a CDKN1C mutation causing a mixture of Beckwith-Wiedemann and IMAGe syndromes revealed a novel transcript associated with developmental delay

BackgroundLoss-of-function mutations in CDKN1C cause overgrowth, that is, Beckwith-Wiedemann syndrome (BWS), while gain-of-function variants in the gene’s PCNA binding motif cause a growth-restricted condition called IMAGe syndrome. We report on...

Macromolecular crowding induces compaction and DNA binding in the disordered N-terminal domain of hUNG2

Many human DNA repair proteins have disordered domains at their N- or C-termini with poorly defined biological functions. We recently reported that the partially structured N-terminal domain (NTD) of human uracil DNA glycosylase 2 (hUNG2), functions to enhance DNA translocation in crowded environments and also targets the enzyme to single-stranded/double-stranded DNA junctions. To understand the structural basis for these effects we now report high-resolution heteronuclear NMR studies of the isolated NTD in the presence and absence of an inert macromolecular crowding agent (PEG8K). Compared to dilute buffer, we find that crowding reduces the degrees of freedom for the structural ensemble, increases the order of a PCNA binding motif and dramatically promotes binding of the NTD for DNA through a conformational selection mechanism. These findings shed new light on the function of this disordered domain in the context of the crowded nuclear environment.

Nucleolar residence of the seckel syndrome protein TRAIP is coupled to ribosomal DNA transcription.

The RING finger protein TRAIP protects genome integrity and its mutation causes Seckel syndrome. TRAIP encodes a nucleolar protein that migrates to UV-induced DNA lesions via a direct interaction with the DNA replication clamp PCNA. Thus far, mechanistically how UV mobilizes TRAIP from the nucleoli remains unknown. We found that PCNA binding is dispensable for the nucleolus-nucleoplasm shuttling of TRAIP following cell exposure to UV irradiation, and that its redistribution did not rely on the master DNA damage kinases ATM and ATR. Interestingly, I-PpoI-induced ribosomal DNA damage led to TRAIP exclusion from the nucleoli, raising the possibility that active ribosomal DNA transcription may underlie TRAIP retention in the nuclear sub-compartments. Accordingly, chemical inhibition of RNA polymerase I activity led to TRAIP diffusion into the nucleoplasm, and was coupled with marked reduction of DNA/RNA hybrids in the nucleoli, suggesting that TRAIP may be sequestered via binding to nucleic acid structures in the nucleoli. Consistently, cell pre-treatment with DNase/RNase effectively released TRAIP from the nucleoli. Taken together, our study defines a bipartite mechanism that drives TRAIP trafficking in response to UV damage, and highlights the nucleolus as a stress sensor that contributes to orchestrating DNA damage responses.

Characterization of the dual function of ATXR5 PIP motif in PCNA and nucleosome binding

Characterization of the dual function of ATXR5 PIP motif in PCNA and nucleosome binding

Rational Design of a 310 -Helical PIP-Box Mimetic Targeting PCNA, the Human Sliding Clamp.

The human sliding clamp (PCNA) controls access to DNA for many proteins involved in DNA replication and repair. Proteins are recruited to the PCNA surface by means of a short, conserved peptide motif known as the PCNA-interacting protein box (PIP-box). Inhibitors of these essential protein-protein interactions may be useful as cancer therapeutics by disrupting DNA replication and repair in these highly proliferative cells. PIP-box peptide mimetics have been identified as a potentially rapid route to potent PCNA inhibitors. Here we describe the rational design and synthesis of the first PCNA peptidomimetic ligands, based on the high affinity PIP-box sequence from the natural PCNA inhibitor p21. These mimetics incorporate covalent i,i+4 side-chain/side-chain lactam linkages of different lengths, designed to constrain the peptides into the 310 -helical structure required for PCNA binding. NMR studies confirmed that while the unmodified p21 peptide had little defined structure in solution, mimetic ACR2 pre-organized into 310 -helical structure prior to interaction with PCNA. ACR2 displayed higher affinity binding than most known PIP-box peptides, and retains the native PCNA binding mode, as observed in the co-crystal structure of ACR2 bound to PCNA. This study offers a promising new strategy for PCNA inhibitor design for use as anti-cancer therapeutics.

Abstract 2307: Differential effects of N-terminal vs C-terminal truncating CDKN1A mutations on cisplatin resistance in bladder cancer

Abstract Introduction Approximately 15% of muscle invasive bladder cancers harbor mutations in the CDKN1A gene, which encodes the p53 target gene p21, with the majority of alterations truncating the peptide. We previously found that proximal truncation of p21 sensitized cells to cisplatin, while distal truncations made them more resistant. We hypothesized that truncations preserving the CDKi domain but ablating the PCNA binding domain would abrogate p21's ability to sequester PCNA but maintain cell cycle arrest capacities, enhancing the interaction between mono-ubiquitiniated-PCNA (ub-PCNA) and DNA polymerase eta (Pol η), resulting in increased TLS and DNA damage tolerance. Methods We used SW780 cell line for this study based on its resistance to cisplatin and p53 and p21 WT status. CRISPR sgRNAs designed to target amino acids 12 and 109 (SW780-sg12 and SW780-sg109) were used to introduce frameshifts and disrupt the majority of the peptide or only the PCNA the binding domain, respectively. sgRNA designed to target GFP was used as a control (sgGFP). We treated the cells with GI50 dose of cisplatin and analyzed post-cisplatin cell-cycle profile over 96 hours using PI-FACS and immunoblot. We treated all three cell types with increasing doses (0-40 μM) of cisplatin for 24 hours and measured the levels of ub-PCNA (K164) and Pol η using immunoblot. Results SW780-sgGFP arrested in S-phase at 48 and 72 hours but overcame this arrest at 96 hours and began to cycle again. SW780-sg12 were unable to arrest in S-phase, whereas SW780-sg109 were able to arrest in S-phase at 48 to 96 hours. This pattern of S-phase arrest was further confirmed by prolonged upregulation of cyclin D1 in SW780-sg109 and brief upregulation in SW780-sgGFP. ub-PCNA and Pol η accumulated in a dose responsive manner in all three cell types at 24 hours after cisplatin treatment. ub-PCNA increased abruptly at higher cisplatin doses in SW780-sgGFP, whereas the increase was gradual in CDKN1A-edited cells. Interestingly, in SW780-sg109, Pol η induction was strongest at all doses. Conclusion Cisplatin causes S-phase arrest in SW780-sgGFP cells, possibly by inducing replicative stress. However, this arrest is eventually bypassed and the cells progress through the cell cycle. S-phase arrest is not possible in the absence of a functioning p21, indicating that p21 plays a vital role in bringing about post-cisplatin cell cycle arrest. Interestingly, truncated p21 with intact CDKi domain is capable of causing stronger and more prolonged S-phase arrest compared to the full-length peptide. TLS may play a role in DNA damage tolerance in SW780 cells as indicated by elevated ub-PCNA and pol η. Stronger accumulation of Pol η in SW780-sg109, possibly enhances TLS and thus cisplatin resistance. Moving forward, we would like to confirm the anticipated competition between p21 and Pol η for PCNA and further verify this phenomenon in other bladder cancer cell lines. Citation Format: Rahmat K. Sikder, Wafik S. El-Deiry, Philip H. Abbosh. Differential effects of N-terminal vs C-terminal truncating CDKN1A mutations on cisplatin resistance in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2307.

Identification of PCNA Binding Sites on Chromatin Assembly Factor 1

Identification of PCNA Binding Sites on Chromatin Assembly Factor 1

KDM6A addiction of cervical carcinoma cell lines is triggered by E7 and mediated by p21CIP1 suppression of replication stress.

Expression of E7 proteins encoded by carcinogenic, high-risk human papillomaviruses (HPVs) triggers increased expression of the histone H3 lysine 27 demethylase KDM6A. KDM6A expression is necessary for survival of high-risk HPV E7 expressing cells, including several cervical cancer lines. Here we show that increased KDM6A in response to high-risk HPV E7 expression causes epigenetic de-repression of the cell cycle and DNA replication inhibitor p21CIP1, and p21CIP1 expression is necessary for survival of high-risk HPV E7 expressing cells. The requirement for KDM6A and p21CIP1 expression for survival of high-risk HPV E7 expressing cells is based on p21CIP1’s ability to inhibit DNA replication through PCNA binding. We show that ectopic expression of cellular replication factors can rescue the loss of cell viability in response to p21CIP1 and KDM6A depletion. Moreover, we discovered that nucleoside supplementation will override the loss of cell viability in response to p21CIP1 depletion, suggesting that p21CIP1 depletion causes lethal replication stress. This model is further supported by increased double strand DNA breaks upon KDM6A or p21CIP1 depletion and DNA combing experiments that show aberrant re-replication upon KDM6A or p21CIP1 depletion in high-risk HPV E7 expressing cells. Therefore, KDM6A and p21CIP1 expression are essential to curb E7 induced replication stress to levels that do not markedly interfere with cell viability.

Abstract 2356: Functional characterization of CDKN1A loss in bladder cancer and effects on cisplatin sensitivity

Abstract Introduction Muscle-invasive bladder cancer (MIBC) is most optimally treated with cisplatin-containing combination chemotherapy, and if clinically localized, bladder removal. With recent discoveries delineating aberrant driver genes involved in MIBC, it is now possible to personalize treatment approaches. Development of a therapeutic rationale for personalized therapies which is based on mutational landscape or other characteristic of each tumor may prospectively identify patients prone to chemoresponse, thus maximizing therapeutic index. Inactivating mutations in CDKN1A, the gene encoding cyclin dependent kinase inhibitor p21, occur in about 14% of MIBCs, with a majority of these truncating the peptide. We hypothesized that after DNA-damaging events, cells deficient in p21 would be unable to halt the cell cycle to repair the damage, and subsequently proceed down an apoptotic pathway, potentially serving as a therapeutic vulnerability. Methods RT4 and SW780 cell lines were chosen for study based on chemoresistance to cisplatin and WT status of CDKN1A and TP53. Cell-cycle arrest and apoptotic pathways were evaluated with Western blot after treatment with IC50 doses. CRISPR sgRNAs were generated to introduce frameshifts 5’ to the regions encoding the cyclin binding domain (CDI), within the CDI, and 5’ to the PCNA binding domains. Knock-outs were confirmed by immunoblot. MTT was used to assess cipslatin response. Results p53 increased in both cell lines 24 hours after a 3h pulse of cisplatin exposure. However, p53 peaked at 24 hours and then diminished in RT4 but continued to rise in SW780. p21 was basally expressed in both cell lines, decreased at 24 hours, then increasing at 72 hours. In RT4, peak Rb phosphorylation (pRb) occurred at 24 hours, indicating cell cycle arrest, and gradually decreased at 72 hours. In SW780 pRb remained unchanged during the course of the treatment. In RT4, PARP cleavage was observed at 24 hours and increased at 48 hours indicating apoptosis. RT4 cells harbored extensive DNA damage as indicated by persistent phosphorylated γH2AX. In contrast a smaller fraction of SW780 cells underwent PARP cleavage at 48 and 72 hours and there was less phosphorylated γH2AX. Single cell CDKN1A knockout clones were generated and confirmed by immunoblotting. sgRNAs targeting amino acids 12 and 59 completely abolished p21 detection, and an sgRNA targeting amino acid 109 generated a truncated peptide, confirming p21’s apparent stability. CDKN1A disruption at any of the three sites did not reverse chemorensitance to cisplatin in either cell line. Conclusion Cisplatin resistant cell lines use a combination of arrest and repair to survive an apoptotic insult. Although p21 loss did not reverse cisplatin resistance, it may impact sensitivity to other agents. Other mechanisms of chemo- and radio-sensitization are being explored. Citation Format: Philip H. Abbosh, Rahmat K. Sikder, Wafik S. El-Deiry. Functional characterization of CDKN1A loss in bladder cancer and effects on cisplatin sensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2356. doi:10.1158/1538-7445.AM2017-2356

Srs2 promotes synthesis-dependent strand annealing by disrupting DNA polymerase δ-extending D-loops.

Synthesis-dependent strand annealing (SDSA) is the preferred mode of homologous recombination in somatic cells leading to an obligatory non-crossover outcome, thus avoiding the potential for chromosomal rearrangements and loss of heterozygosity. Genetic analysis identified the Srs2 helicase as a prime candidate to promote SDSA. Here, we demonstrate that Srs2 disrupts D-loops in an ATP-dependent fashion and with a distinct polarity. Specifically, we partly reconstitute the SDSA pathway using Rad51, Rad54, RPA, RFC, DNA Polymerase δ with different forms of PCNA. Consistent with genetic data showing the requirement for SUMO and PCNA binding for the SDSA role of Srs2, Srs2 displays a slight but significant preference to disrupt extending D-loops over unextended D-loops when SUMOylated PCNA is present, compared to unmodified PCNA or monoubiquitinated PCNA. Our data establish a biochemical mechanism for the role of Srs2 in crossover suppression by promoting SDSA through disruption of extended D-loops.

Differential requirement of Srs2 helicase and Rad51 displacement activities in replication of hairpin-forming CAG/CTG repeats.

Trinucleotide repeats are a source of genome instability, causing replication fork stalling, chromosome fragility, and impaired repair. Specialized helicases play an important role in unwinding DNA structures to maintain genome stability. The Srs2 helicase unwinds DNA hairpins, facilitates replication, and prevents repeat instability and fragility. However, since Srs2 is a multifunctional protein with helicase activity and the ability to displace Rad51 recombinase, it was unclear which functions were required for its various protective roles. Here, using SRS2 separation-of-function alleles, we show that in the absence of Srs2 recruitment to PCNA or in helicase-deficient mutants, breakage at a CAG/CTG repeat increases. We conclude that Srs2 interaction with PCNA allows the helicase activity to unwind fork-blocking CAG/CTG hairpin structures to prevent breaks. Independently of PCNA binding, Srs2 also displaces Rad51 from nascent strands to prevent recombination-dependent repeat expansions and contractions. By 2D gel electrophoresis, we detect two different kinds of structured intermediates or joint molecules (JMs). Some JMs are Rad51-independent and exhibit properties of reversed forks, including being processed by the Exo1 nuclease. In addition, in a helicase-deficient mutant, Rad51-dependent JMs are detected, probably corresponding to recombination between sisters. These results clarify the many roles of Srs2 in facilitating replication through fork-blocking hairpin lesions.

Distinct and overlapping DNMT1 interactions with multiple transcription factors in erythroid cells: Evidence for co-repressor functions

DNMT1 is the maintenance DNA methyltransferase shown to be essential for embryonic development and cellular growth and differentiation in many somatic tissues in mammals. Increasing evidence has also suggested a role for DNMT1 in repressing gene expression through interactions with specific transcription factors. Previously, we identified DNMT1 as an interacting partner of the TR2/TR4 nuclear receptor heterodimer in erythroid cells, implicated in the developmental silencing of fetal β-type globin genes in the adult stage of human erythropoiesis. Here, we extended this work by using a biotinylation tagging approach to characterize DNMT1 protein complexes in mouse erythroleukemic cells. We identified novel DNMT1 interactions with several hematopoietic transcription factors with essential roles in erythroid differentiation, including GATA1, GFI-1b and FOG-1. We provide evidence for DNMT1 forming distinct protein subcomplexes with specific transcription factors and propose the existence of a “core” DNMT1 complex with the transcription factors ZBP-89 and ZNF143, which is also present in non-hematopoietic cells. Furthermore, we identified the short (17a.a.) PCNA Binding Domain (PBD) located near the N-terminus of DNMT1 as being necessary for mediating interactions with the transcription factors described herein. Lastly, we provide evidence for DNMT1 serving as a co-repressor of ZBP-89 and GATA1 acting through upstream regulatory elements of the PU.1 and GATA1 gene loci.

A DNA binding winged helix domain in CAF-1 functions with PCNA to stabilize CAF-1 at replication forks.

Chromatin assembly factor 1 (CAF-1) is a histone H3–H4 chaperone that deposits newly synthesized histone (H3–H4)2 tetramers during replication-coupled nucleosome assembly. However, how CAF-1 functions in this process is not yet well understood. Here, we report the crystal structure of C terminus of Cac1 (Cac1C), a subunit of yeast CAF-1, and the function of this domain in stabilizing CAF-1 at replication forks. We show that Cac1C forms a winged helix domain (WHD) and binds DNA in a sequence-independent manner. Mutations in Cac1C that abolish DNA binding result in defects in transcriptional silencing and increased sensitivity to DNA damaging agents, and these defects are exacerbated when combined with Cac1 mutations deficient in PCNA binding. Similar phenotypes are observed for corresponding mutations in mouse CAF-1. These results reveal a mechanism conserved in eukaryotic cells whereby the ability of CAF-1 to bind DNA is important for its association with the DNA replication forks and subsequent nucleosome assembly.

PDIP46 (DNA polymerase δ interacting protein 46) is an activating factor for human DNA polymerase δ.

PDIP46 (SKAR, POLDIP3) was discovered through its interaction with the p50 subunit of human DNA polymerase δ (Pol δ). Its functions in DNA replication are unknown. PDIP46 associates with Pol δ in cell extracts both by immunochemical and protein separation methods, as well as by ChIP analyses. PDIP46 also interacts with PCNA via multiple copies of a novel PCNA binding motif, the APIMs (AlkB homologue-2 PCNA-Interacting Motif). Sites for both p50 and PCNA binding were mapped to the N-terminal region containing the APIMs. Functional assays for the effects of PDIP46 on Pol δ activity on singly primed ssM13 DNA templates revealed that it is a novel and potent activator of Pol δ. The effects of PDIP46 on Pol δ in primer extension, strand displacement and synthesis through simple hairpin structures reveal a mechanism where PDIP46 facilitates Pol δ4 synthesis through regions of secondary structure on complex templates. In addition, evidence was obtained that PDIP46 is also capable of exerting its effects by a direct interaction with Pol δ, independent of PCNA. Mutation of the Pol δ and PCNA binding region resulted in a loss of PDIP46 functions. These studies support the view that PDIP46 is a novel accessory protein for Pol δ that is involved in cellular DNA replication. This raises the possibility that altered expression of PDIP46 or its mutation may affect Pol δ functions in vivo, and thereby be a nexus for altered genomic stability.

P21 Exploits Residue Tyr151 as a Tether for High-Affinity PCNA Binding.

Proliferating cell nuclear antigen (PCNA, processivity factor, sliding clamp) is a ring-shaped protein that tethers proteins to DNA in processes, including DNA replication, DNA repair, and cell-cycle control. Often used as a marker for cell proliferation, PCNA is overexpressed in cancer cells, making it an appealing pharmaceutical target. PCNA interacts with proteins through a PCNA interacting protein (PIP)-box, an eight-amino acid consensus sequence; different binding partners display a wide range of affinities based on function. Of all biological PIP-boxes, p21 has the highest known affinity for PCNA, allowing for inhibition of DNA replication and cell growth under cellular stress. As p21 is one of the few PIP-box sequences to contain a tyrosine rather than a phenylalanine in the eighth conserved position, we probed the significance of the hydroxyl group at this position using a mutational approach. Here we present the cocrystal structure of PCNA bound to a mutant p21 PIP-box peptide, p21Tyr151Phe, with associated isothermal titration calorimetry data. The p21Tyr151Phe peptide showed a 3-fold difference in affinity, as well as differences in entropy and enthalpy of binding. These differences can be attributed to a loss of hydrogen bonding capacity, as well as structural plasticity in the PCNA interdomain connector loop and the hydrophobic cavity of PCNA to which p21 binds. Thus, the hydroxyl group of Tyr151 in p21 acts as a tethering point for ideal packing and surface recognition of the peptide interface, increasing the binding affinity of p21 for PCNA.

PDIP46 – A Pol δ and PCNA Binding Protein that Stimulates Human Pol δ Activity

PDIP46 (poldip3) is a 46 kDa protein which interacts with human DNA polymerase δ (Pol δ) via the p50 subunit, and whose biochemical effects on Pol δ are unknown. The objectives of this study were to characterize the effects of PDIP46 on Pol δ activity. The interaction sites between the p50 subunit of Pol δ and PDIP46 were mapped. PDIP46 was also found to interact with PCNA, which it does via multiple copies of a novel PCNA binding motif (APIM ‐ AlkB homologue 2 PCNA‐Interacting Motif). Both the p50 and PCNA interaction regions are located in the N‐terminus. PDIP46 potently activates Pol δ activity (ca. 10‐fold with an apparent KD of ca. 34 nM) on singly primed 7 kb ssM13 DNA in an assay which requires highly processive synthesis. Further dissection of the effects of PDIP46 using model oligonucleotide templates showed that it stimulated primer extension and strand displacement reactions in the presence of PCNA. PDIP46 also stimulated primer extension in the absence of PCNA, indicating that it has the ability to exert a direct effect on Pol δ. PDIP46 was associated with Pol δ on DNA as shown by chromatin immunoprecipitation using antibody against the Pol δ p125 subunit. These in vitro studies show that PDIP46 has the biochemical attributes to function as a novel accessory protein for Pol δ that accelerates its activity, and thus is a candidate for a role as a cellular regulator of Pol δ.

Structure and specificity of the bacterial cysteine methyltransferase effector NleE suggests a novel substrate in human DNA repair pathway.

Enteropathogenic E. coli (EPEC) and related enterobacteria rely on a type III secretion system (T3SS) effector NleE to block host NF-κB signaling. NleE is a first in class, novel S-adenosyl-L-methionine (SAM)-dependent methyltransferase that methylates a zinc-coordinating cysteine in the Npl4-like Zinc Finger (NZF) domains in TAB2/3 adaptors in the NF-κB pathway, but its mechanism of action and other human substrates are unknown. Here we solve crystal structure of NleE-SAM complex, which reveals a methyltransferase fold different from those of known ones. The SAM, cradled snugly at the bottom of a deep and narrow cavity, adopts a unique conformation ready for nucleophilic attack by the methyl acceptor. The substrate NZF domain can be well docked into the cavity, and molecular dynamic simulation indicates that Cys673 in TAB2-NZF is spatially and energetically favorable for attacking the SAM. We further identify a new NleE substrate, ZRANB3, that functions in PCNA binding and remodeling of stalled replication forks at the DNA damage sites. Specific inactivation of the NZF domain in ZRANB3 by NleE offers a unique opportunity to suggest that ZRANB3-NZF domain functions in DNA repair processes other than ZRANB3 recruitment to DNA damage sites. Our analyses suggest a novel and unexpected link between EPEC infection, virulence proteins and genome integrity.