Iterative model-building, structure refinement, and density modification with the PHENIX AutoBuild Wizard Thomas C. Terwilliger a* , Ralf W. Grosse-Kunstleve b , Pavel V. Afonine b , Nigel W. Moriarty b , Peter Zwart b , Li-Wei Hung a , Randy J. Read c , Paul D. Adams b* a b Los Alamos National Laboratory, Mailstop M888, Los Alamos, NM 87545, USA Lawrence Berkeley National Laboratory, One Cyclotron Road, Bldg 64R0121, Berkeley, CA 94720, USA. c Department of Haematology, University of Cambridge, Cambridge CB2 0XY, UK. * Email: terwill@lanl.gov or PDAdams@lbl.gov Running title: The PHENIX AutoBuild Wizard Abstract The PHENIX AutoBuild Wizard is a highly automated tool for iterative model- building, structure refinement and density modification using RESOLVE or TEXTAL model- building, RESOLVE statistical density modification, and phenix.refine structure refinement. Recent advances in the AutoBuild Wizard and phenix.refine include automated detection and application of NCS from models as they are built, extensive model completion algorithms, and automated solvent molecule picking. Model completion algorithms in the AutoBuild Wizard include loop-building, crossovers between chains in different models of a structure, and side-chain optimization. The AutoBuild Wizard has been applied to a set of 48 structures at resolutions ranging from 1.1 A to 3.2 A, resulting in a mean R-factor of 0.24 and a mean free R factor of 0.29. The R-factor of the final model is dependent on the quality of the starting electron density, and relatively independent of resolution. Keywords: Model building; model completion; macromolecular models; Protein Data Bank; structure refinement; PHENIX Introduction Iterative model-building and refinement is a powerful approach to obtaining a complete and accurate macromolecular model. The approach consists of cycles of building an atomic model based on an electron density map for a macromolecular structure, refining the structure, using the refined structure as a basis for improving the map, and building a new model. This type of approach has been carried out in a semi-automated fashion for many years, with manual model-building iterating with automated refinement (Jensen, 1997). More recently, with the development first of ARP/wARP (Perrakis et al., 1999), and later other procedures including RESOLVE iterative model-building and refinement (Terwilliger,

Iterative model-building, structure refinement, and density modification with the PHENIX AutoBuild Wizard

Ten measures of experimental electron-density-map quality are examined and the skewness of electron density is found to be the best indicator of actual map quality. A Bayesian approach to estimating map quality is developed and used in the PHENIX AutoSol wizard to make decisions during automated structure solution.

Decision-making in structure solution using Bayesian estimates of map quality: the PHENIX autosol wizard

DNA methylation is an important epigenetic modification(1-3). Ten-eleven translocation (TET) proteins are involved in DNA demethylation through iteratively oxidizing 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC)(4-8). Here we show that human TET1 and TET2 are more active on 5mC-DNA than 5hmC/5fC-DNA substrates. We determine the crystal structures of TET2-5hmC-DNA and TET2-5fC-DNA complexes at 1.80 angstrom and 1.97 angstrom resolution, respectively. The cytosine portion of 5hmC/5fC is specifically recognized by TET2 in a manner similar to that of 5mC in the TET2-5mC-DNA structure(9), and the pyrimidine base of 5mC/5hmC/5fC adopts an almost identical conformation within the catalytic cavity. However, the hydroxyl group of 5hmC and carbonyl group of 5fC face towards the opposite direction because the hydroxymethyl group of 5hmC and formyl group of 5fC adopt restrained conformations through forming hydrogen bonds with the 1-carboxylate of NOG and N4 exocyclic nitrogen of cytosine, respectively. Biochemical analyses indicate that the substrate preference of TET2 results from the different efficiencies of hydrogen abstraction in TET2-mediated oxidation. The restrained conformation of 5hmC and 5fC within the catalytic cavity may prevent their abstractable hydrogen(s) adopting a favourable orientation for hydrogen abstraction and thus result in low catalytic efficiency. Our studies demonstrate that the substrate preference of TET2 results from the intrinsic value of its substrates at their 5mC derivative groups and suggest that 5hmC is relatively stable and less prone to further oxidation by TET proteins. Therefore, TET proteins are evolutionarily tuned to be less reactive towards 5hmC and facilitate the generation of 5hmC as a potentially stable mark for regulatory functions.

Structural insight into substrate preference for TET-mediated oxidation

Oxidatively generated complex DNA damage: tandem and clustered lesions.

The mammalian thymine DNA glycosylase (TDG) is implicated in active DNA demethylation via the base excision repair pathway. TDG excises the mismatched base from G:X mismatches, where X is uracil, thymine or 5-hydroxymethyluracil (5hmU). These are, respectively, the deamination products of cytosine, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). In addition, TDG excises the Tet protein products 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) but not 5hmC and 5mC, when paired with a guanine. Here we present a post-reactive complex structure of the human TDG domain with a 28-base pair DNA containing a G:5hmU mismatch. TDG flips the target nucleotide from the double-stranded DNA, cleaves the N-glycosidic bond and leaves the C1′ hydrolyzed abasic sugar in the flipped state. The cleaved 5hmU base remains in a binding pocket of the enzyme. TDG allows hydrogen-bonding interactions to both T/U-based (5hmU) and C-based (5caC) modifications, thus enabling its activity on a wider range of substrates. We further show that the TDG catalytic domain has higher activity for 5caC at a lower pH (5.5) as compared to the activities at higher pH (7.5 and 8.0) and that the structurally related Escherichia coli mismatch uracil glycosylase can excise 5caC as well. We discuss several possible mechanisms, including the amino-imino tautomerization of the substrate base that may explain how TDG discriminates against 5hmC and 5mC.

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Excision of 5-hydroxymethyluracil and 5-carboxylcytosine by the thymine DNA glycosylase domain: its structural basis and implications for active DNA demethylation

5-Formylcytosine (fC or (5-CHO)dC) and 5-carboxylcytosine (caC or (5-COOH)dC) have recently been identified as constituents of mammalian DNA. The nucleosides are formed from 5-methylcytosine (mC or (5-Me)dC) via 5-hydroxymethylcytosine (hmC or (5-HOMe)dC) and are possible intermediates of an active DNA demethylation process. Here we show efficient syntheses of phosphoramidites which enable the synthesis of DNA strands containing these cytosine modifications based on Pd(0)-catalyzed functionalization of 5-iododeoxycytidine. The first crystal structure of fC reveals the existence of an intramolecular H-bond between the exocyclic amine and the formyl group, which controls the conformation of the formyl substituent. Using a newly designed in vitro mutagenicity assay we show that fC and caC are only marginally mutagenic, which is a prerequisite for the bases to function as epigenetic control units.

Improved Synthesis and Mutagenicity of Oligonucleotides Containing 5‐Hydroxymethylcytosine, 5‐Formylcytosine and 5‐Carboxylcytosine

Nucleotide excision repair (NER) is responsible for the removal of a large variety of structurally diverse DNA lesions. Mutations of the involved proteins cause the xeroderma pigmentosum (XP) cancer predisposition syndrome. Although the general mechanism of the NER process is well studied, the function of the XPA protein, which is of central importance for successful NER, has remained enigmatic. It is known, that XPA binds kinked DNA structures and that it interacts also with DNA duplexes containing certain lesions, but the mechanism of interactions is unknown. Here we present two crystal structures of the DNA binding domain (DBD) of the yeast XPA homolog Rad14 bound to DNA with either a cisplatin lesion (1,2-GG) or an acetylaminofluorene adduct (AAF-dG). In the structures, we see that two Rad14 molecules bind to the duplex, which induces DNA melting of the duplex remote from the lesion. Each monomer interrogates the duplex with a β-hairpin, which creates a 13mer duplex recognition motif additionally characterized by a sharp 70° DNA kink at the position of the lesion. Although the 1,2-GG lesion stabilizes the kink due to the covalent fixation of the crosslinked dG bases at a 90° angle, the AAF-dG fully intercalates into the duplex to stabilize the kinked structure.

https://www.pnas.org/content/pnas/112/27/8272.full.pdf

Structural insights into the recognition of cisplatin and AAF-dG lesion by Rad14 (XPA)

Nucleotide excision repair (NER) is a highly versatile and efficient DNA repair process, which is responsible for the removal of a large number of structurally diverse DNA lesions. Its extreme broad substrate specificity ranges from DNA damages formed upon exposure to ultraviolet radiation to numerous bulky DNA adducts induced by mutagenic environmental chemicals and cytotoxic drugs used in chemotherapy. Defective NER leads to serious diseases, such as xeroderma pigmentosum (XP). Eight XP complementation groups are known of which seven (XPA-XPG) are caused by mutations in genes involved in the NER process. The eighth gene, XPV, codes for the DNA polymerase ɳ, which replicates through DNA lesions in a process called translesion synthesis (TLS). Over the past decade, detailed structural information of these DNA repair proteins involved in eukaryotic NER and TLS have emerged. These structures allow us now to understand the molecular mechanism of the NER and TLS processes in quite some detail and we have begun to understand the broad substrate specificity of NER. In this review, we aim to highlight recent advances in the process of damage recognition and repair as well as damage tolerance by the XP proteins.

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Molecular mechanisms of xeroderma pigmentosum (XP) proteins

Oxidative degradation of DNA is a major mutagenic process. Reactive oxygen species (ROS) produced in the course of oxidative phosphorylation or by exogenous factors are known to attack preferentially deoxyguanosine. The latter decomposes to give mutagenic lesions, which under physiological conditions are efficiently repaired by specialized maintenance systems in the cell. Although many intermediates of the degradation pathway are today well-known, we report in this study the discovery of a new intermediate with an interesting guanidinoformimine structure. The structure elucidation of the new lesion was possible by using HPLC–MS techniques and organic synthesis. Finally we report the mutagenic potential of the new lesion in comparison to the known lesions imidazolone and oxazolone using primer extension and pyrosequencing experiments.

Sandra C. Koch

Papers

Improved Synthesis and Mutagenicity of Oligonucleotides Containing 5‐Hydroxymethylcytosine, 5‐Formylcytosine and 5‐Carboxylcytosine

Structural insights into the recognition of cisplatin and AAF-dG lesion by Rad14 (XPA)

Molecular mechanisms of xeroderma pigmentosum (XP) proteins

Discovery and mutagenicity of a guanidinoformimine lesion as a new intermediate of the oxidative deoxyguanosine degradation pathway.