Structural Insights into the Mechanism of Base Excision by MBD4

DNA glycosylases remove damaged or modified nucleobases by cleaving the N-glycosyl bond and the correct nucleotide is restored through subsequent base excision repair. In addition to excising threatening lesions, DNA glycosylases contribute to epigenetic regulation by mediating DNA demethylation and perform other important functions. However, the catalytic mechanism remains poorly defined for many glycosylases, including MBD4 (methyl-CpG binding domain IV), a member of the helix-hairpin-helix (HhH) superfamily. MBD4 excises thymine from G·T mispairs, suppressing mutations caused by deamination of 5-methylcytosine, and it removes uracil and modified uracils (e.g., 5-hydroxymethyluracil) mispaired with guanine. To investigate the mechanism of MBD4 we solved high-resolution structures of enzyme-DNA complexes at three stages of catalysis. Using a non-cleavable substrate analog, 2′-deoxy-pseudouridine, we determined the first structure of an enzyme-substrate complex for wild-type MBD4, which confirms interactions that mediate lesion recognition and suggests that a catalytic Asp, highly conserved in HhH enzymes, binds the putative nucleophilic water molecule and stabilizes the transition state. Observation that mutating the Asp (to Gly) reduces activity by 2700-fold indicates an important role in catalysis, but probably not one as the nucleophile in a double-displacement reaction, as previously suggested. Consistent with direct-displacement hydrolysis, a structure of the enzyme-product complex indicates a reaction leading to inversion of configuration. A structure with DNA containing 1-azadeoxyribose models a potential oxacarbenium-ion intermediate and suggests the Asp could facilitate migration of the electrophile towards the nucleophilic water. Finally, the structures provide detailed snapshots of the HhH motif, informing how these ubiquitous metal-binding elements mediate DNA binding.     

Dynamic Processing of a Common Oxidative DNA Lesion by the First Two Enzymes of the Base Excision Repair Pathway

Base excision repair (BER) is the primary pathway by which eukaryotic cells resolve single base damage. One common example of single base damage is 8-oxo-7,8-dihydro-2ʹ-deoxoguanine (8-oxoG). High incidence and mutagenic potential of 8-oxoG necessitate rapid and efficient DNA repair. How BER enzymes coordinate their activities to resolve 8-oxoG damage while limiting cytotoxic BER intermediates from propagating genomic instability remains unclear. Here we use single-molecule Förster resonance energy transfer (smFRET) and ensemble-level techniques to characterize the activities and interactions of consecutive BER enzymes important for repair of 8-oxoG. In addition to characterizing the damage searching and processing mechanisms of human 8-oxoguanine glycosylase 1 (hOGG1), our data support the existence of a ternary complex between hOGG1, the damaged DNA substrate, and human AP endonuclease 1 (APE1). Our results indicate that hOGG1 is actively displaced from its abasic site containing product by protein–protein interactions with APE1 to ensure timely repair of damaged DNA.     

Expression and purification of NEIL3, a human DNA glycosylase homolog

The base excision repair (BER) pathway is mainly responsible for the repair of a vast number of non-bulky lesions produced by alkylation, oxidation or deamination of bases. DNA glycosylases are the key enzymes that recognize damaged bases and initiate BER by catalyzing the cleavage of the N-glycosylic bond between the base and the sugar. Many of the mammalian DNA glycosylases have been identified by a combination of biochemical and bioinformatics analysis. Thus, a mammalian family of three proteins (NEIL1, NEIL2 and NEIL3) that showed homology to the Escherichia coli Fpg/Nei DNA glycosylases was identified. Two of the proteins, NEIL1 and NEIL2 have been thoroughly characterized and shown to initiate BER of a diverse number of oxidized lesions. However, much less is known about NEIL3. The biochemical properties of NEIL3 have not been elucidated. This is mainly due to the difficulty in the expression and purification of NEIL3. Here, we describe the expression and partial purification of full-length human NEIL3 and the expression, purification and characterization of a truncated human core-NEIL3 (amino acids 1–301) that contains the complete E. coli Fpg/Nei-like domain but lacks the C-terminal region.     

NEIL2-initiated, APE-independent repair of oxidized bases in DNA: Evidence for a repair complex in human cells

DNA glycosylases/AP lyases initiate repair of oxidized bases in the genomes of all organisms by excising these lesions and then cleaving the DNA strand at the resulting abasic (AP) sites and generate 3' phospho alpha,beta-unsaturated aldehyde (3' PUA) or 3' phosphate (3' P) terminus. In Escherichia coli, the AP-endonucleases (APEs) hydrolyze both 3' blocking groups (3' PUA and 3' P) to generate the 3'-OH termini needed for repair synthesis. In mammalian cells, the previously characterized DNA glycosylases, NTH1 and OGG1, produce 3' PUA, which is removed by the only AP-endonuclease, APE1. However, APE1 is barely active in removing 3' phosphate generated by the recently discovered mammalian DNA glycosylases NEIL1 and NEIL2. We showed earlier that the 3' phosphate generated by NEIL1 is efficiently removed by polynucleotide kinase (PNK) and not APE1. Here we show that the NEIL2-initiated repair of 5-hydroxyuracil (5-OHU) similarly requires PNK. We have also observed stable interaction between NEIL2 and other BER proteins DNA polymerase beta (Pol beta), DNA ligase IIIalpha (Lig IIIalpha) and XRCC1. In spite of their limited sequence homology, NEIL1 and NEIL2 interact with the same domains of Pol beta and Lig IIIalpha. Surprisingly, while the catalytically dispensable C-terminal region of NEIL1 is the common interacting domain, the essential N-terminal segment of NEIL2 is involved in analogous interaction. The BER proteins including NEIL2, PNK, Pol beta, Lig IIIalpha and XRCC1 (but not APE1) could be isolated as a complex from human cells, competent for repair of 5-OHU in plasmid DNA.     

Non-Canonical Allostery in Cyclic Nucleotide Dependent Kinases

The cAMP- and cGMP-dependent protein kinases (PKA and PKG) are canonically activated by the corresponding cyclic nucleotides. However, both systems are also sensitive to a wide range of non-canonical allosteric effectors, such as reactive oxygen species, which induce the formation of regulatory inter- and intra-molecular disulfide bridges, and disease-related mutations (DRMs). Here, we present a combined analysis of representative non-canonical allosteric effectors for PKA and PKG, and we identify common molecular mechanisms underlying non-canonical allostery in these kinases, from shifts in dynamical regulatory equilibria to modulation of inter-protomer interactions. In addition, mutations may also drive oligomerization beyond dimerization, and possibly phase transitions, causing loss of kinase inhibitory function and amplifying the allosteric effects of DRMs. Hence non-canonical allosteric stimuli often result in constitutive kinase activation underlying either physiological control of downstream signaling pathways or pathological outcomes, from aortic aneurisms to cancer predisposition. Overall, PKA and PKG emerge as “pan-sensors” going well beyond canonical cyclic nucleotide activation, revealing their versatile roles as central signaling hubs.     

The N-terminal Tails of Histones H2A and H2B Adopt Two Distinct Conformations in the Nucleosome with Contact and Reduced Contact to DNA

The nucleosome comprises two histone dimers of H2A-H2B and one histone tetramer of (H3-H4)₂, wrapped around by ~145 bp of DNA. Detailed core structures of nucleosomes have been established by X-ray and cryo-EM, however, histone tails have not been visualized. Here, we have examined the dynamic structures of the H2A and H2B tails in 145-bp and 193-bp nucleosomes using NMR, and have compared them with those of the H2A and H2B tail peptides unbound and bound to DNA. Whereas the H2A C-tail adopts a single but different conformation in both nucleosomes, the N-tails of H2A and H2B adopt two distinct conformations in each nucleosome. To clarify these conformations, we conducted molecular dynamics (MD) simulations, which suggest that the H2A N-tail can locate stably in either the major or minor grooves of nucleosomal DNA. While the H2B N-tail, which sticks out between two DNA gyres in the nucleosome, was considered to adopt two different orientations, one toward the entry/exit side and one on the opposite side. Then, the H2A N-tail minor groove conformation was obtained in the H2B opposite side and the H2B N-tail interacts with DNA similarly in both sides, though more varied conformations are obtained in the entry/exit side. Collectively, the NMR findings and MD simulations suggest that the minor groove conformer of the H2A N-tail is likely to contact DNA more strongly than the major groove conformer, and the H2A N-tail reduces contact with DNA in the major groove when the H2B N-tail is located in the entry/exit side.     

Structural Studies Reveal Unique Non-canonical Regulators of G Protein Signaling Homology (RH) Domains in Sorting Nexins

As a subgroup of sorting nexins (SNXs) that contain regulator of G protein signaling homology (RH) domain, SNX-RH proteins, including SNX13, SNX14 and SNX25, were proposed to play bifunctional roles in protein sorting and GPCR signaling regulation. However, mechanistic details of SNX-RH proteins functioning via RH domain remain to be illustrated. Here, we delineate crystal structures of the RH domains of SNX13 and SNX25, revealing a homodimer of SNX13 RH domain mediated by unique extended α4 and α5 helices, and a thiol modulated homodimer of SNX25-RH triggered by a unique cysteine on α6 helix. Further studies showed that RH domains of SNX-RH do not possess binding capacity toward Gα subunits, owing to the lack of critical residues for interaction. Thus, this study identifies a group of novel non-canonical RH domains that can act as a dimerization module in sorting nexins, which provides structural basis for mechanism studies on SNX-RH protein functions.     

Structure Basis for Shaping the Nse4 Protein by the Nse1 and Nse3 Dimer within the Smc5/6 Complex

The Smc5/6 complex facilitates chromosome replication and DNA break repair. Within this complex, a subcomplex composed of Nse1, Nse3 and Nse4 is thought to play multiple roles through DNA binding and regulating ATP-dependent activities of the complex. However, how the Nse1-Nse3-Nse4 subcomplex carries out these multiple functions remain unclear. To address this question, we determine the crystal structure of the Xenopus laevis Nse1-Nse3-Nse4 subcomplex at 1.7 Å resolution and examine how it interacts with DNA. Our structural analyses show that the Nse1-Nse3 dimer adopts a closed conformation and forms three interfaces with a segment of Nse4, forcing it into a Z-shaped conformation. The Nse1-Nse3-Nse4 structure provides an explanation for how the lung disease immunodeficiency and chromosome breakage syndrome-causing mutations could dislodge Nse4 from Nse1-Nse3. Our DNA binding and mutational analyses reveal that the N-terminal and the middle region of Nse4 contribute to DNA interaction and cell viability. Integrating our data with previous crosslink mass spectrometry data, we propose potential roles of the Nse1-Nse3-Nse4 complex in binding DNA within the Smc5/6 complex.     

Structural investigation of a viral ortholog of human NEIL2/3 DNA glycosylases

Assault to DNA that leads to oxidative base damage is repaired by the base excision repair (BER) pathway with specialized enzymes called DNA glycosylases catalyzing the first step of this pathway. These glycosylases can be categorized into two families: the HhH superfamily, which includes endonuclease III (or Nth), and the Fpg/Nei family, which comprises formamidopyrimidine DNA glycosylase (or Fpg) and endonuclease VIII (or Nei). In humans there are three Nei-like (NEIL) glycosylases: NEIL1, 2, and 3. Here we present the first crystal structure of a viral ortholog of the human NEIL2/NEIL3 proteins, Mimivirus Nei2 (MvNei2), determined at 2.04 Å resolution. The C-terminal region of the MvNei2 enzyme comprises two conserved DNA binding motifs: the helix-two-turns-helix (H2TH) motif and a C-H-C-C type zinc-finger similar to that of human NEIL2. The N-terminal region of MvNei2 is most closely related to NEIL3. Like NEIL3, MvNei2 bears a valine at position 2 instead of the usual proline and it lacks two of the three conserved void-filling residues present in other members of the Fpg/Nei family. Mutational analysis of the only conserved void-filling residue methionine 72 to alanine yields an MvNei2 variant with impaired glycosylase activity. Mutation of the adjacent His73 causes the enzyme to be more productive thereby suggesting a plausible role for this residue in the DNA lesion search process.     

Transient Tertiary Structures of Disordered Dynein Intermediate Chain Regulate its Interactions with Multiple Partners

The N-terminal domain of dynein intermediate chain (N–IC) is central to the cytoplasmic dynein ‘cargo attachment subcomplex’ and regulation of motor activity. It is a prototypical intrinsically disordered protein (IDP), serving as a primarily disordered polybivalent molecular scaffold for numerous binding partners, including three dimeric dynein light chains and coiled coil domains of dynein partners dynactin p150^Glued and NudE. At the very N-terminus, a 40 amino acid single alpha helix (SAH) forms the major binding site for both p150^Glued and NudE, while a shorter nascent helix (H2) separated from SAH by a disordered linker, is necessary for tight binding to dynactin p150^Glued but not to NudE. Here we demonstrate that transient tertiary interactions in this highly dynamic protein underlie the differences in its interactions with p150^Glued and NudE. NMR paramagnetic relaxation enhancement experiments and restrained molecular dynamics simulations identify interactions between the two non-contiguous SAH and H2 helical regions, the extent of which correlates with the length and stability of H2, showing clearly that tertiary and secondary structure formation are coupled in IDPs. These interactions are significantly attenuated when N–IC is bound to NudE, suggesting that NudE binding shifts the conformational ensemble to one that is more extended and with less structure in H2. While the intrinsic disorder and flexibility in N–IC modulate its ability to serve as a binding platform for numerous partners, deviations of this protein from random-coil behavior provide a process for regulating these binding interactions and potentially the dynein motor.     

Eukaryotic endonuclease VIII-Like proteins: New components of the base excision DNA repair system

Base excision DNA repair is necessary for removal of damaged nucleobases from the genome and their replacement with normal nucleobases. Base excision repair is initiated by DNA glycosylases, the enzymes that cleave the N-glycosidic bonds of damaged deoxynucleotides. Until recently, only eight DNA glycosylases with different substrate specificity were known in human cells. In 2002, three new human DNA glycosylases (NEIL1, NEIL2, and NEIL3) were discovered, all homologous to endonuclease VIII, a bacterial protein, which also participates in DNA repair. The role of these enzymes remains mostly unknown. In this review we discuss recent data on the substrate specificity of the NEIL enzymes, their catalytic mechanism, structure, interactions with other components of DNA repair system, and possible biological role in preventing diseases associated with DNA damage.     

Solution Structure of the C-terminal Domain of A20, the Missing Brick for the Characterization of the Interface between Vaccinia Virus DNA Polymerase and its Processivity Factor

Poxviruses are enveloped viruses with a linear, double-stranded DNA genome. Viral DNA synthesis is achieved by a functional DNA polymerase holoenzyme composed of three essential proteins. For vaccinia virus (VACV) these are E9, the catalytic subunit, a family B DNA polymerase, and the heterodimeric processivity factor formed by D4 and A20. The A20 protein links D4 to the catalytic subunit. High-resolution structures have been obtained for the VACV D4 protein in complex with an N-terminal fragment of A20 as well as for E9. In addition, biochemical studies provided evidence that a poxvirus-specific insertion (insert 3) in E9 interacts with the C-terminal residues of A20. Here, we provide solution structures of two different VACV A20 C-terminal constructs containing residues 304–426, fused at their C-terminus to either a BAP (Biotin Acceptor Peptide)-tag or a short peptide containing the helix of E9 insert 3. Together with results from titration studies, these structures shed light on the molecular interface between the catalytic subunit and the processivity factor component A20. The interface comprises hydrophobic residues conserved within the Chordopoxvirinae subfamily. Finally, we constructed a HADDOCK model of the VACV A20_304-426-E9 complex, which is in excellent accordance with previous experimental data.