Repair Protein Complex Research Articles

Impact of polβ/XRCC1 Interaction Variants on the Efficiency of Nick Sealing by DNA Ligase IIIα in the Base Excision Repair Pathway

Base excision repair (BER) requires a coordination from gap filling by DNA polymerase (pol) β to subsequent nick sealing by DNA ligase (LIG) IIIα at downstream steps of the repair pathway. X-ray cross-complementing protein 1 (XRCC1), a non-enzymatic scaffolding protein, forms repair complexes with polβ and LIGIIIα. Yet, the impact of the polβ mutations that affect XRCC1 interaction and protein stability on the repair pathway coordination during nick sealing by LIGIIIα remains unknown. Our results show that the polβ colon cancer-associated variant T304 exhibits a reduced interaction with XRCC1 and the mutations in the interaction interface of V303 loop (L301R/V303R/V306R) and at the lysine residues (K206A/K244A) that prevent ubiquitin-mediated degradation of the protein exhibit a diminished repair protein complex formation with XRCC1. Furthermore, we demonstrate no significant effect on gap and nick DNA binding affinity of wild-type polβ by these mutations. Finally, our results reveal that XRCC1 leads to an efficient channeling of nick repair products after nucleotide incorporation by polβ variants to LIGIIIα, which is compromised by the L301R/V303R/V306R and K206A/K244A mutations. Overall, our findings provide insight into how the mutations in the polβ/XRCC1 interface and the regions affecting protein stability could dictate accurate BER pathway coordination at the downstream steps involving nick sealing by LIGIIIα.

Abstract 6093: IDE161, a potential first-in-class clinical candidate PARG inhibitor, selectively targets homologous-recombination-deficient and PARP inhibitor resistant breast and ovarian tumors

Abstract Poly (ADP-ribose) polymerase inhibitors (PARPi) were the first clinically approved drugs designed to exploit synthetic lethality. Despite initial responses, patients harboring homologous recombination (HR) alterations that receive PARPi develop treatment resistance, which has generated a need for additional therapies targeting homologous recombination deficient (HRD) tumors. Poly (ADP-Ribose) glycohydrolase (PARG) plays a key role in the resolution of PARP1/2-dependent DNA damage repair through hydrolysis of Poly (ADP-ribose) (PAR) chains and consequent release of DNA repair protein complexes from chromatin. In culture, the accumulation of PAR chains in the absence of a functional PARG enzyme causes delayed repair of DNA breaks resulting in increased sensitivity to alkylating agents and ionizing radiation. Notably, some HRD cell models have shown differential sensitivity to PARGi vs PARPi, suggesting that PARGi may overcome some PARPi resistance mechanisms. The ability of PARGi to exacerbate replication deficiencies nominates it as a potential therapeutic target for a broad range of cancer types with genomic instability. IDE161 is an orally bioavailable small molecule inhibitor of PARG. Biochemical and cellular assays demonstrate that IDE161 is a potent inhibitor of PAR chain hydrolysis and has anti-proliferative activity in breast and ovarian HRD cancer cell lines with both inherent and acquired PARPi resistance. IDE161 anti-proliferative effects were associated with induction of mitotic arrest and activation of the DNA damage response pathway, suggesting the position of PARG in the DNA repair cycle remains essential in the context of some PARPi resistance mechanisms. Furthermore, profiling of IDE161 across a panel of 264 molecularly characterized cancer cell lines indicated that PARGi may show benefit beyond HRD and in indications other than ovarian and breast. Nonclinical studies in cell line and patient derived xenograft models of breast, ovarian, and gastric tumor types harboring defects in the HR pathway demonstrated anti-tumor activity in response to IDE161. Consistent with in vitro findings, we observed IDE161 antitumor activity in PARPi resistant xenograft models. Moreover, studies in cell lines, tumors and tissues revealed that dose and time-dependent accumulation of PAR chains serves as a robust proximal pharmacodynamic biomarker indicative of PARG target engagement. IDE161 is a novel targeted therapy that exploits the synthetic lethal relationship between PARG and genomic instability, thus leading to selective anti-proliferative effects in tumors harboring defects in the HR pathway. Citation Format: Monah Abed, Diana Muñoz, Vidya Seshadri, Steve Federowicz, Arjun A. Rao, Deepthi Bhupathi, Marya Liimatta, Rita Ousterhout, Firoz Jaipuri, Claire Neilan, Mark Lackner, Mike White, Zineb Mounir. IDE161, a potential first-in-class clinical candidate PARG inhibitor, selectively targets homologous-recombination-deficient and PARP inhibitor resistant breast and ovarian tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6093.

Multi-Faceted Roles of ERCC1-XPF Nuclease in Processing Non-B DNA Structures

Genetic instability can result from increases in DNA damage and/or alterations in DNA repair proteins and can contribute to disease development. Both exogenous and endogenous sources of DNA damage and/or alterations in DNA structure (e.g., non-B DNA) can impact genome stability. Multiple repair mechanisms exist to counteract DNA damage. One key DNA repair protein complex is ERCC1-XPF, a structure-specific endonuclease that participates in a variety of DNA repair processes. ERCC1-XPF is involved in nucleotide excision repair (NER), repair of DNA interstrand crosslinks (ICLs), and DNA double-strand break (DSB) repair via homologous recombination. In addition, ERCC1-XPF contributes to the processing of various alternative (i.e., non-B) DNA structures. This review will focus on the processing of alternative DNA structures by ERCC1-XPF.

Live Cell Detection of Poly(ADP-Ribose) for Use in Genetic and Genotoxic Compound Screens.

Simple SummaryPoly(ADP-ribose) (PAR) functions in genome repair, cell metabolism, and cancer treatment; however, in vivo molecular tools to assess PAR levels are not readily available. Here, we describe two methods to measure PAR with high sensitivity in real time in vivo using the PAR binding domain (termed WWE) of Ring Finger Protein 146 (RNF146). In response to genotoxic stress, the WWE-EGFP fusion protein, termed LivePAR, displays nuclear enrichment enabling quantitative and visual detection of PAR formation. Additionally, a split luciferase assay using the same WWE domain facilitates rapid PAR quantitation in a plate format and provides a tool for chemotherapeutic candidate compound screening. Both tools can be used to identify genetic determinants of PAR formation and help in identifying compounds that alter PAR formation or degradation.Poly(ADP-ribose) (PAR) is a molecular scaffold that aids in the formation of DNA repair protein complexes. Tools to sensitively quantify PAR in live cells have been lacking. We recently described the LivePAR probe (EGFP fused to the RNF146-encoded WWE PAR binding domain) to measure PAR formation at sites of laser micro-irradiation in live cells. Here, we present two methods that expand on the use of LivePAR and its WWE domain. First, LivePAR enriches in the nucleus of cells following genotoxic challenge. Image quantitation can identify single-cell PAR formation following genotoxic stress at concentrations lower than PAR ELISA or PAR immunoblot, with greater sensitivity to genotoxic stress than CometChip. In a second approach, we used the RNF146-encoded WWE domain to develop a split luciferase probe for analysis in a 96-well plate assay. We then applied these PAR analysis tools to demonstrate their broad applicability. First, we show that both approaches can identify genetic modifications that alter PARylation levels, such as hyper-PARylation in BRCA2-deficient cancer cells. Second, we demonstrate the utility of the WWE split luciferase assay to characterize the cellular response of genotoxins, PARP inhibitors, and PARG inhibitors, thereby providing a screening method to identify PAR modulating compounds.

MRG Proteins Are Shared by Multiple Protein Complexes With Distinct Functions

MRG15/MORF4L1 is a highly conserved protein in eukaryotes that contains a chromodomain (CHD) recognizing methylation of lysine 36 on histone H3 (H3K36me3) in chromatin. Intriguingly, it has been reported in the literature to interact with several different factors involved in chromatin modifications, gene regulation, alternative mRNA splicing, and DNA repair by homologous recombination. To get a complete and reliable picture of associations in physiological conditions, we used genome editing and tandem affinity purification to analyze the stable native interactome of human MRG15, its paralog MRGX/MORF4L2 that lacks the CHD, and MRGBP (MRG-binding protein) in isogenic K562 cells. We found stable interchangeable association of MRG15 and MRGX with the NuA4/TIP60 histone acetyltransferase/chromatin remodeler, Sin3B histone deacetylase/demethylase, ASH1L histone methyltransferase, and PALB2–BRCA2 DNA repair protein complexes. These associations were further confirmed and analyzed by CRISPR tagging of endogenous proteins and comparison of expressed isoforms. Importantly, based on structural information, point mutations could be introduced that specifically disrupt MRG15 association with some complexes but not others. Most interestingly, we also identified a new abundant native complex formed by MRG15/X-MRGBP-BRD8-EP400NL (EP400 N-terminal like) that is functionally similar to the yeast TINTIN (Trimer Independent of NuA4 for Transcription Interactions with Nucleosomes) complex. Our results show that EP400NL, being homologous to the N-terminal region of NuA4/TIP60 subunit EP400, creates TINTIN by competing for BRD8 association. Functional genomics indicate that human TINTIN plays a role in transcription of specific genes. This is most likely linked to the H4ac-binding bromodomain of BRD8 along the H3K36me3–binding CHD of MRG15 on the coding region of transcribed genes. Taken together, our data provide a complete detailed picture of human MRG proteins–associated protein complexes, which are essential to understand and correlate their diverse biological functions in chromatin-based nuclear processes.

DNA repair proteins cooperate with SOX2 in regulating the transition of human embryonic stem cells to neural progenitor cells

SOX2, a well-established pluripotency factor supporting the self-renewal of pluripotent stem cells (PSCs), is also a crucial factor for maintaining the properties and functionalities of neural progenitor cells (NPCs). It regulates the transcription of target genes by forming complexes with its partner factors, but systematic comparison of SOX2 binding partners in human PSCs versus NPCs is lacking. Here, by deciphering and comparing the SOX2-protein interactomes in human embryonic stem cells (hESCs) versus the NPCs derived from them, we identified 23 proteins with high reproducibility that are most differentially associated with SOX2, of which 9 are DNA repair proteins (PARP1, PARP2, PRKDC, XRCC1, XRCC5, XRCC6, RPA1, LIG3, DDB1). Genetic knocking-down or pharmacological inhibiting two of the DNA repair proteins (PARP1 and PRKDC) significantly up-regulated certain NPC or ectodermal biomarkers that are transcriptionally-suppressed by the SOX2/DNA repair protein complexes. These findings point to a crucial role of DNA repair proteins in pluripotent state transition and neural induction.

Temporal dynamics of base excision/single-strand break repair protein complex assembly/disassembly are modulated by the PARP/NAD+/SIRT6 axis.

SUMMARYAssembly and disassembly of DNA repair protein complexes at DNA damage sites are essential for maintaining genomic integrity. Investigating factors coordinating assembly of the base excision repair (BER) proteins DNA polymerase β (Polβ) and XRCC1 to DNA lesion sites identifies a role for Polβ in regulating XRCC1 disassembly from DNA repair complexes and, conversely, demonstrates Polβ’s dependence on XRCC1 for complex assembly. LivePAR, a genetically encoded probe for live-cell imaging of poly(ADP-ribose) (PAR), reveals that Polβ and XRCC1 require PAR for repair-complex assembly, with PARP1 and PARP2 playing unique roles in complex dynamics. Further, BER complex assembly is modulated by attenuation/augmentation of NAD+ biosynthesis. Finally, SIRT6 does not modulate PARP1 or PARP2 activation but does regulate XRCC1 recruitment, leading to diminished Polβ abundance at sites of DNA damage. These findings highlight coordinated yet independent roles for PARP1, PARP2, and SIRT6 and their regulation by NAD+ bioavailability to facilitate BER.

FANCM regulates repair pathway choice at stalled replication forks

Repair pathway "choice" at stalled mammalian replication forks is an important determinant of genome stability; however, the underlying mechanisms are poorly understood. FANCM encodes a multi-domain scaffolding and motor protein that interacts with several distinct repair protein complexes at stalled forks. Here, we use defined mutations engineered within endogenous Fancm in mouse embryonic stem cells to study how Fancm regulates stalled fork repair. We find that distinct FANCM repair functions are enacted by molecularly separable scaffolding domains. These findings define FANCM as a key mediator of repair pathway choice at stalled replication forks and reveal its molecular mechanism. Notably, mutations that inactivate FANCM ATPase function disable all its repair functions and "trap" FANCM at stalled forks. We find that Brca1 hypomorphic mutants are synthetic lethal with Fancm null or Fancm ATPase-defective mutants. The ATPase function of FANCM may therefore represent a promising "druggable" target for therapy of BRCA1-linked cancer.

LncRNA H19 Suppresses Osteosarcomagenesis by Regulating snoRNAs and DNA Repair Protein Complexes.

Osteosarcoma is one of the most frequent common primary malignant tumors in childhood and adolescence. Long non-coding RNAs (lncRNAs) have been reported to regulate the initiation and progression of tumors. However, the exact molecular mechanisms involving lncRNA in osteosarcomagenesis remain largely unknown. Li-Fraumeni syndrome (LFS) is a familial cancer syndrome caused by germline p53 mutation. We investigated the tumor suppressor function of lncRNA H19 in LFS-associated osteosarcoma. Analyzing H19-induced transcriptome alterations in LFS induced pluripotent stem cell (iPSC)-derived osteoblasts, we unexpectedly discovered a large group of snoRNAs whose expression was significantly affected by H19. We identified SNORA7A among the H19-suppressed snoRNAs. SNORA7A restoration impairs H19-mediated osteogenesis and tumor suppression, indicating an oncogenic role of SNORA7A. TCGA analysis indicated that SNORA7A expression is associated with activation of oncogenic signaling and poor survival in cancer patients. Using an optimized streptavidin-binding RNA aptamer designed from H19 lncRNA, we revealed that H19-tethered protein complexes include proteins critical for DNA damage response and repair, confirming H19's tumor suppressor role. In summary, our findings demonstrate a critical role of H19-modulated SNORA7A expression in LFS-associated osteosarcomas.

XRCC1 Prevents Toxic PARP1 Trapping During DNA Base Excision Repair

Mammalian DNA base excision repair (BER) is an essential pathway comprised of damaged base excision by a DNA glycosylase, incision by AP endonuclease-1 (APE1), end processing and gap filling by DNA polymerase β (POLβ), and DNA ligation by DNA ligase I (LIG1) or DNA ligase III (LIG3). In mammals, BER additionally employs PARP1 and/or PARP2 and the scaffold protein XRCC1 to accelerate and coordinate the overall process. Whereas PARP1 and PARP2 are sensor proteins that detect unrepaired DNA single-strand breaks, the essential role of XRCC1 during BER is unknown. Here, we have identified this role. We show that the DNA repair protein complexes that are assembled by XRCC1 compete with and prevent excessive PARP1 engagement during BER, which otherwise leads to PARP1 ‘trapping’ on BER intermediates in a manner reminiscent of that induced by clinical PARP inhibitors. We demonstrate that this elevated engagement and trapping of PARP1 collectively renders BER intermediates inaccessible to POLβ and impedes their repair. Consequently, PARP1 deletion rescues both the accessibility and repair of BER intermediates in XRCC1-/- cells, and also their cellular resistance to DNA base damage. These data demonstrate that PARP1 trapping is an endogenous threat to genome integrity, and identify XRCC1 as an “anti-trapper” that prevents the toxic binding of PARP1 to BER intermediates to ensure their efficient repair.

Loss of MDC1 in Endometrial Carcinoma Is Associated With Loss of MRN Complex and MMR Deficiency.

To evaluate the frequency of loss of mediator of DNA damage checkpoint protein 1 (MDC1) protein expression in endometrial cancer (EC) and to determine whether loss of MDC1 is associated with certain clinicopathological parameters. MDC1 expression was examined in 426 samples of EC. The nuclear immunoreactivity of the protein was defined as: negative, weak, moderate and strong. Loss of MDC1 expression (defined as negative nuclear staining) was found in 8.9% (38/426) of ECs and was significantly associated with the loss of MRE11 homolog, double-strand break repair nuclease, RAD50 double-strand break repair protein and nibrin complex components. In addition, loss of expression of MDC1 showed a significant correlation with any mismatch repair deficiency, with endometrioid histological subtype and low tumour grading. Based on these findings, we suggest that MDC1 loss frequently occurs in ECs with microsatellite instability. Due to deficient homologous recombination DNA repair, MDC1-negative ECs might show an increased sensitivity to poly(ADP-ribose) polymerase-inhibitory therapy.

TGF beta promotes repair of bulky DNA damage through increased ERCC1/XPF and ERCC1/XPA interaction.

Transforming growth factor beta (TGFβ) is multifunctional cytokine that is involved in the coordination and regulation of many cellular homeostatic processes. Compromised TGFβ activity has been attributed to promotion of human cancers. Recent studies have identified a role for TGFβ in response to radiation-induced DNA damage, suggesting a link between TGFβ and the DNA damage response with implications for cancer development. In this study, the effects of TGFβ on promoting the repair of bulky DNA damage, through modulation of nucleotide excision repair (NER), were investigated. We show that treatment of cells with exogenous TGFβ leads to enhanced repair of DNA damage formed by polycyclic aromatic hydrocarbons and ultraviolet-C radiation; similarly, cells with constitutively activated endogenous TGFβ signaling show comparable responses. This effect of TGFβ is independent of the cell cycle. The response to TGFβ is decreased in cells that have compromised TGFβ signaling through RNA interference of Smad4 and is decreased in NER-deficient cells and cells with compromised NER through RNA interference of excision repair cross-complementing group 1 (ERCC1). Increased interaction and nuclear localization of ERCC1/xeroderma pigmentosum (XP) F and ERCC1/XPA proteins is observed after TGFβ treatment. Our study represents the first experimental evidence of a role for TGFβ in the repair of bulky DNA damage resulting from promotion of the interaction and localization of repair protein complexes involved in the incision step of NER.

Analyzing Protein Architectures and Protein-Ligand Complexes by Integrative Structural Mass Spectrometry

Proteins are an important class of biological macromolecules that play many key roles in cellular functions including gene expression, catalyzing metabolic reactions, DNA repair and replication. Therefore, a detailed understanding of these processes provides critical information on how cells function. Integrative structural MS methods offer structural and dynamical information on protein complex assembly, complex connectivity, subunit stoichiometry, protein oligomerization and ligand binding. Recent advances in integrative structural MS have allowed for the characterization of challenging biological systems including large DNA binding proteins and membrane proteins. This protocol describes how to integrate diverse MS data such as native MS and ion mobility-mass spectrometry (IM-MS) with molecular dynamics simulations to gain insights into a helicase-nuclease DNA repair protein complex. The resulting approach provides a framework for detailed studies of ligand binding to other protein complexes involved in important biological processes.

Analyzing Protein Architectures and Protein-Ligand Complexes by Integrative Structural Mass Spectrometry.

Proteins are an important class of biological macromolecules that play many key roles in cellular functions including gene expression, catalyzing metabolic reactions, DNA repair and replication. Therefore, a detailed understanding of these processes provides critical information on how cells function. Integrative structural MS methods offer structural and dynamical information on protein complex assembly, complex connectivity, subunit stoichiometry, protein oligomerization and ligand binding. Recent advances in integrative structural MS have allowed for the characterization of challenging biological systems including large DNA binding proteins and membrane proteins. This protocol describes how to integrate diverse MS data such as native MS and ion mobility-mass spectrometry (IM-MS) with molecular dynamics simulations to gain insights into a helicase-nuclease DNA repair protein complex. The resulting approach provides a framework for detailed studies of ligand binding to other protein complexes involved in important biological processes.

Fusobacterium nucleatum Infection in Colorectal Cancer: Linking Inflammation, DNA Mismatch Repair and Genetic and Epigenetic Alterations.

It has been recently reported that the population of Fusobacterium, particularly Fusobacterium nucleatum (Fn), is overrepresented in colorectal cancers (CRCs) and adenomas. The promoting effects of Fn infection on adenoma and/or carcinoma formation have been shown in ApcMin/+mice. Characteristics of Fn-associated CRC were identified through studies using human CRC cohorts and include right-sided colon location, CpG island methylation phenotype (CIMP)-high, high level of microsatellite instability (MSI-H), and poor patient prognosis. A subset of Fn-associated CRC exhibits a low level of microsatellite instability (MSI-L) and elevated microsatellite alterations in selected tetra-nucleotide repeats (EMAST) induced by translocation of MSH3 from the nucleus to the cytoplasm in response to oxidative DNA damage or inflammatory signals. The association between CIMP/MSI-H and Fn infection can be explained by the role of the mismatch repair protein complex formed between MSH2 and MSH6 (MutSα) to repair aberrant bases generated by reactive oxygen species (ROS) to form 7,8-dihydro-8-oxo-guanine (8-oxoG). Clustered 8-oxoGs formed at CpG-rich regions including promoters by ROS is refractory to base excision repair. Under these conditions, MutSα initiates repair in cooperation with DNA methyltransferases (DNMTs) and the polycomb repressive complex 4. DNMTs at damaged sites methylate CpG islands to repress transcription of target genes and promote repair reactions. Thus, continuous generation of ROS through chronic Fn infection may initiate (1) CIMP-positive adenoma and carcinoma in an MSH2/MSH6-dependent manner and/or (2) MSI-L/EMAST CRC in an MSH3-dependent manner. The poor prognosis of Fn-associated CRC can be explained by Fn-induced immune-evasion and/or chemoresistance.

Visualizing correlated motion with HDBSCAN clustering.

Correlated motion analysis provides a method for understanding communication between and dynamic similarities of biopolymer residues and domains. The typical equal-time correlation matrices-frequently visualized with pseudo-colorings or heat maps-quickly convey large regions of highly correlated motion but hide more subtle similarities of motion. Here we propose a complementary method for visualizing correlations within proteins (or general biopolymers) that quickly conveys intuition about which residues have a similar dynamic behavior. For grouping residues, we use the recently developed non-parametric clustering algorithm HDBSCAN. Although the method we propose here can be used to group residues using correlation as a similarity matrix-the most straightforward and intuitive method-it can also be used to more generally determine groups of residues which have similar dynamic properties. We term these latter groups "Dynamic Domains", as they are based not on spatial closeness but rather closeness in the column space of a correlation matrix. We provide examples of this method across three human proteins of varying size and function-the Nf-Kappa-Beta essential modulator, the clotting promoter Thrombin and the mismatch repair protein (dimer) complex MutS-alpha. Although the examples presented here are from all-atom molecular dynamics simulations, this visualization technique can also be used on correlations matrices built from any ensembles of conformations from experiment or computation.

STING is an essential mediator of the Ku70-mediated production of IFN-λ1 in response to exogenous DNA.

We previously identified Ku70, a subunit of a DNA repair protein complex, as a cytosolic DNA sensor that induces the production of interferon-λ1 (IFN-λ1) by human primary cells and cell lines. IFN-λ1 is a type III IFN and has similar antiviral activity to that of the type I IFNs (IFN-α and IFN-β). We observed that human embryonic kidney (HEK) 293T cells, which are deficient in the innate immune adaptor protein STING (stimulator of IFN genes), did not produce IFN-λ1 in response to DNA unless they were reconstituted with STING. Conversely, parental HEK 293 cells produced IFN-λ1 after they were exposed to exogenous DNA; however, when STING was knocked out in the HEK 293 cells through the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 genome editing system, they lost this response. Through confocal microscopy, we demonstrated that endogenous Ku70 was located in the nucleus and then translocated to the cytoplasm upon DNA exposure to form a complex with STING. Additionally, the DNA binding domain of Ku70 was essential for formation of the Ku70-STING complex. Knocking down STING in primary human macrophages inhibited their ability to produce IFN-λ1 in response to transfection with DNA or infection with the DNA virus HSV-2 (herpes simplex virus-2). Together, these data suggest that STING mediates the Ku70-mediated IFN-λ1 innate immune response to exogenous DNA or DNA virus infection.

Single-stranded DNA Binding by the Helix-Hairpin-Helix Domain of XPF Protein Contributes to the Substrate Specificity of the ERCC1-XPF Protein Complex

The nucleotide excision repair protein complex ERCC1-XPF is required for incision of DNA upstream of DNA damage. Functional studies have provided insights into the binding of ERCC1-XPF to various DNA substrates. However, because no structure for the ERCC1-XPF-DNA complex has been determined, the mechanism of substrate recognition remains elusive. Here we biochemically characterize the substrate preferences of the helix-hairpin-helix (HhH) domains of XPF and ERCC-XPF and show that the binding to single-stranded DNA (ssDNA)/dsDNA junctions is dependent on joint binding to the DNA binding domain of ERCC1 and XPF. We reveal that the homodimeric XPF is able to bind various ssDNA sequences but with a clear preference for guanine-containing substrates. NMR titration experiments and in vitro DNA binding assays also show that, within the heterodimeric ERCC1-XPF complex, XPF specifically recognizes ssDNA. On the other hand, the HhH domain of ERCC1 preferentially binds dsDNA through the hairpin region. The two separate non-overlapping DNA binding domains in the ERCC1-XPF heterodimer jointly bind to an ssDNA/dsDNA substrate and, thereby, at least partially dictate the incision position during damage removal. Based on structural models, NMR titrations, DNA-binding studies, site-directed mutagenesis, charge distribution, and sequence conservation, we propose that the HhH domain of ERCC1 binds to dsDNA upstream of the damage, and XPF binds to the non-damaged strand within a repair bubble.

Imaging and structural studies of DNA-protein complexes and membrane ion channels.

In bio-imaging by electron microscopy, damage of the sample and limited contrast are the two main hurdles for reaching high image quality. We extend a new preparation method based on nanofabrication and super-hydrophobicity to the imaging and structural studies of nucleic acids, nucleic acid-protein complexes (DNA/Rad51 repair protein complex) and neuronal ion channels (gap-junction, K+ and GABAA channels) as paradigms of biological significance and increasing complexity. The preparation method is based on the liquid phase and is compatible with physiological conditions. Only in the very last stage, samples are dried for TEM analysis. Conventional TEM and high-resolution TEM (HRTEM) were used to achieve a resolution of 3.3 and 1.5 Å, respectively. The EM dataset quality allows the determination of relevant structural and metrological information on the DNA structure, DNA-protein interactions and ion channels, allowing the identification of specific macromolecules and their structure.

Bloom syndrome complex promotes FANCM recruitment to stalled replication forks and facilitates both repair and traverse of DNA interstrand crosslinks.

The recruitment of FANCM, a conserved DNA translocase and key component of several DNA repair protein complexes, to replication forks stalled by DNA interstrand crosslinks (ICLs) is a step upstream of the Fanconi anemia (FA) repair and replication traverse pathways of ICLs. However, detection of the FANCM recruitment has been technically challenging so that its mechanism remains exclusive. Here, we successfully observed recruitment of FANCM at stalled forks using a newly developed protocol. We report that the FANCM recruitment depends upon its intrinsic DNA translocase activity, and its DNA-binding partner FAAP24. Moreover, it is dependent on the replication checkpoint kinase, ATR; but is independent of the FA core and FANCD2–FANCI complexes, two essential components of the FA pathway, indicating that the FANCM recruitment occurs downstream of ATR but upstream of the FA pathway. Interestingly, the recruitment of FANCM requires its direct interaction with Bloom syndrome complex composed of BLM helicase, Topoisomerase 3α, RMI1 and RMI2; as well as the helicase activity of BLM. We further show that the FANCM–BLM complex interaction is critical for replication stress-induced FANCM hyperphosphorylation, for normal activation of the FA pathway in response to ICLs, and for efficient traverse of ICLs by the replication machinery. Epistasis studies demonstrate that FANCM and BLM work in the same pathway to promote replication traverse of ICLs. We conclude that FANCM and BLM complex work together at stalled forks to promote both FA repair and replication traverse pathways of ICLs.