Steven M. Shell

Checkpoint Kinase ATR Promotes Nucleotide Excision Repair of UV-induced DNA Damage via Physical Interaction with Xeroderma Pigmentosum Group A

In response to DNA damage, eukaryotic cells activate a series of DNA damage-dependent pathways that serve to arrest cell cycle progression and remove DNA damage. Coordination of cell cycle arrest and damage repair is critical for maintenance of genomic stability. However, this process is still poorly understood. Nucleotide excision repair (NER) and the ATR-dependent cell cycle checkpoint are the major pathways responsible for repair of UV-induced DNA damage. Here we show that ATR physically interacts with the NER factor Xeroderma pigmentosum group A (XPA). Using a mass spectrometry-based protein footprinting method, we found that ATR interacts with a helix-turn-helix motif in the minimal DNA-binding domain of XPA where an ATR phosphorylation site (serine 196) is located. XPA-deficient cells complemented with XPA containing a point mutation of S196A displayed a reduced repair efficiency of cyclobutane pyrimidine dimers as compared with cells complemented with wild-type XPA, although no effect was observed for repair of (6-4) photoproducts. This suggests that the ATR-dependent phosphorylation of XPA may promote NER repair of persistent DNA damage. In addition, a K188A point mutation of XPA that disrupts the ATR-XPA interaction inhibits the nuclear import of XPA after UV irradiation and, thus, significantly reduced DNA repair efficiency. By contrast, the S196A mutation has no effect on XPA nuclear translocation. Taken together, our results suggest that the ATR-XPA interaction mediated by the helix-turn-helix motif of XPA plays an important role in DNA-damage responses to promote cell survival and genomic stability after UV irradiation.

Xeroderma pigmentosum complementation group C protein (XPC) serves as a general sensor of damaged DNA

The Xeroderma pigmentosum complementation group C protein (XPC) serves as the primary initiating factor in the global genome nucleotide excision repair pathway (GG-NER). Recent reports suggest XPC also stimulates repair of oxidative lesions by base excision repair. However, whether XPC distinguishes among various types of DNA lesions remains unclear. Although the DNA binding properties of XPC have been studied by several groups, there is a lack of consensus over whether XPC discriminates between DNA damaged by lesions associated with NER activity versus those that are not. In this study we report a high-throughput fluorescence anisotropy assay used to measure the DNA binding affinity of XPC for a panel of DNA substrates containing a range of chemical lesions in a common sequence. Our results demonstrate that while XPC displays a preference for binding damaged DNA, the identity of the lesion has little effect on the binding affinity of XPC. Moreover, XPC was equally capable of binding to DNA substrates containing lesions not repaired by GG-NER. Our results suggest XPC may act as a general sensor of damaged DNA that is capable of recognizing DNA containing lesions not repaired by NER.

Redefining the DNA-binding domain of human XPA.

Xeroderma pigmentosum complementation group A (XPA) protein plays a critical role in the repair of DNA damage via the nucleotide excision repair (NER) pathway. XPA serves as a scaffold for NER, interacting with several other NER proteins as well as the DNA substrate. The critical importance of XPA is underscored by its association with the most severe clinical phenotypes of the genetic disorder Xeroderma pigmentosum. Many of these disease-associated mutations map to the XPA98–219 DNA-binding domain (DBD) first reported ∼20 years ago. Although multiple solution NMR structures of XPA98–219 have been determined, the molecular basis for the interaction of this domain with DNA is only poorly characterized. In this report, we demonstrate using a fluorescence anisotropy DNA-binding assay that the previously reported XPA DBD binds DNA with substantially weaker affinity than the full-length protein. In-depth analysis of the XPA sequence suggested that the original DBD construct lacks critical basic charge and helical elements at its C-terminus. Generation and analysis of a series of C-terminal extensions beyond residue 219 yielded a stable, soluble human XPA98–239 construct that binds to a Y-shaped ssDNA–dsDNA junction and other substrates with the same affinity as the full-length protein. Two-dimensional 15N–1H NMR suggested XPA98–239 contains the same globular core as XPA98–219 and likely undergoes a conformational change upon binding DNA. Together, our results demonstrate that the XPA DBD should be redefined and that XPA98–239 is a suitable model to examine the DNA binding activity of human XPA.

Other Proteins Interacting with XP Proteins

Genetic defects in Nucleotide excision repair (NER) lead to the clinical disorder xeroderma pigmentosum (XP) in humans which is characterized by dramatically increased sensitivity to UV light and a predisposition to development of skin cancers.1,2 NER is a major mechanism of DNA repair in cells for the removal of a large variety of bulky DNA lesions induced by environmental genotoxic agents and chemicals. The molecular basis of XP has been attributed to mutations in any of the eight XP genes, XPA through G whose products are required for NER-mediated removal of DNA damage and XP-variant (XPV). The XP proteins involved in NER can be divided into three groups based on their activity in the NER process. XPA, XPC and XPE are required for sensing DNA damage and initiating the repair process. XPB and XPD, components of the basal transcription factor TFIIH, are helicases that create a DNA strand opening surrounding the adducted base(s) during NER. XPG and XPF are the endonucleases that perform the dual incisions to release the damaged strand and allow resynthesis using the nondamaged strand as a template.3, 4, 5 Protein-protein interactions are integral for the correct assembly of the pre-incision complex and for the positioning of the nucleases prior to incision. However, these proteins have been found to form complexes with other proteins not directly involved in the NER mechanism. This chapter describes these proteins and their interactions and discusses their effects on the XP proteins, DNA repair, and genome stability.

A new structural insight into XPA-DNA interactions.

XPA (xeroderma pigmentosum group A) protein is an essential factor for NER (nucleotide excision repair) which is believed to be involved in DNA damage recognition/verification, NER factor recruiting and stabilization of repair intermediates. Past studies on the structure of XPA have focused primarily on XPA interaction with damaged DNA. However, how XPA interacts with other DNA structures remains unknown though recent evidence suggest that these structures could be important for its roles in both NER and non-NER activities. Previously, we reported that XPA recognizes undamaged DNA ds/ssDNA (double-strand/single-strandDNA) junctions with a binding affinity much higher than its ability to bind bulky DNA damage. To understand how this interaction occurs biochemically we implemented a structural determination of the interaction using a MS-based protein footprinting method and limited proteolysis. By monitoring surface accessibility of XPA lysines to NHS-biotin modification in the free protein and the DNA junction-bound complex we show that XPA physically interacts with the DNA junctions via two lysines, K168 and K179, located in the previously known XPA(98–219) DBD (DNA-binding domain). Importantly, we also uncovered new lysine residues, outside of the known DBD, involved in the binding. We found that residues K221, K222, K224 and K236 in the C-terminal domain are involved in DNA binding. Limited proteolysis analysis of XPA–DNA interactions further confirmed this observation. Structural modelling with these data suggests a clamp-like DBD for the XPA binding to ds/ssDNA junctions. Our results provide a novel structure-function view of XPA–DNA junction interactions.

Dimerization and phosphatase activity of calcyclin-binding protein/Siah-1 interacting protein: the influence of oxidative stress

CacyBP/SIP [calcyclin‐binding protein/Siah‐1 [seven in absentia homolog 1 (Siah E3 ubiquitin protein ligase 1)] interacting protein] is a multifunctional protein whose activity includes acting as an ERK1/2 phosphatase. We analyzed dimerization of mouse CacyBP/SIP in vitro and in mouse neuroblastoma cell line (NB2a) cells, as well as the structure of a full‐length protein. Moreover, we searched for the CacyBP/SIP domain important for dimerization and dephosphorylation of ERK2, and we analyzed the role of dimerization in ERK1/2 signaling in NB2a cells. Cell‐based assays showed that CacyBP/SIP forms a homodimer in NB2a cell lysate, and biophysical methods demonstrated that CacyBP/SIP forms a stable dimer in vitro. Data obtained using small‐angle X‐ray scattering supported a model in which CacyBP/SIP occupies an anti‐parallel orientation mediated by the N‐terminal dimerization domain. Site‐directed mutagenesis established that the N‐terminal domain is indispensable for full phosphatase activity of CacyBP/SIP. We also demonstrated that the oligomerization state of CacyBP/SIP as well as the level of post‐translational modifications and subcellular distribution of CacyBP/SIP change after activation of the ERK1/2 pathway in NB2a cells due to oxidative stress. Together, our results suggest that dimerization is important for controlling phosphatase activity of CacyBP/SIP and for regulating the ERK1/2 signaling pathway.—Topolska‐Woś, A. M., Shell, S. M., Kilańczyk, E., Szczepanowski, R. H., Chazin, W. J., Filipek, A. Dimerization and phosphatase activity of calcyclin‐binding protein/Siah‐1 interacting protein: the influence of oxidative stress. FASEB J. 29, 1711‐1724 (2015). www.fasebj.org

XPF-ERCC1: On the Bubble

In this issue of Structure, Das et al. report the structure of the helix-hairpin-helix dimerization domain of XPF bound to ssDNA. These results provide insight into the architecture of nucleotide excision repair machinery and how it interacts with damaged DNA substrates.

Protein Modification by Adenine Propenal

Base propenals are products of the reaction of DNA with oxidants such as peroxynitrite and bleomycin. The most reactive base propenal, adenine propenal, is mutagenic in Escherichia coli and reacts with DNA to form covalent adducts; however, the reaction of adenine propenal with protein has not yet been investigated. A survey of the reaction of adenine propenal with amino acids revealed that lysine and cysteine form adducts, whereas histidine and arginine do not. Nε-Oxopropenyllysine, a lysine–lysine cross-link, and S-oxopropenyl cysteine are the major products. Comprehensive profiling of the reaction of adenine propenal with human serum albumin and the DNA repair protein, XPA, revealed that the only stable adduct is Nε-oxopropenyllysine. The most reactive sites for modification in human albumin are K190 and K351. Three sites of modification of XPA are in the DNA-binding domain, and two sites are subject to regulatory acetylation. Modification by adenine propenal dramatically reduces XPA’s ability to bind to a DNA substrate.