Pierre Aller

Clustering procedures for the optimal selection of data sets from multiple crystals in macromolecular crystallography.

A systematic approach to the scaling and merging of data from multiple crystals in macromolecular crystallography is introduced and explained.

A structural rationale for stalling of a replicative DNA polymerase at the most common oxidative thymine lesion, thymine glycol

Thymine glycol (Tg) is a common product of oxidation and ionizing radiation, including that used for cancer treatment. Although Tg is a poor mutagenic lesion, it has been shown to present a strong block to both repair and replicative DNA polymerases. The 2.65-Å crystal structure of a binary complex of the replicative RB69 DNA polymerase with DNA shows that the templating Tg is intrahelical and forms a regular Watson–Crick base pair with the incorporated A. The C5 methyl group protrudes axially from the ring of the damaged pyrimidine and hinders stacking of the adjacent 5′ template guanine. The position of the displaced 5′ template guanine is such that the next incoming nucleotide cannot be incorporated into the growing primer strand, and it explains why primer extension past the lesion is prohibited even though DNA polymerases can readily incorporate an A across from the Tg lesion.

Structural and Biochemical Investigation of the Role in Proofreading of a β Hairpin Loop Found in the Exonuclease Domain of a Replicative DNA Polymerase of the B Family

Replicative DNA polymerases, as exemplified by the B family polymerases from bacteriophages T4 and RB69, not only replicate DNA but also have the ability to proofread misincorporated nucleotides. Because the two activities reside in separate protein domains, polymerases must employ a mechanism that allows for efficient switching of the primer strand between the two active sites to achieve fast and accurate replication. Prior mutational and structural studies suggested that a β hairpin structure located in the exonuclease domain of family B polymerases might play an important role in active site switching in the event of a nucleotide misincorporation. We show that deleting the β hairpin loop in RB69 gp43 affects neither polymerase nor exonuclease activities. Single binding event studies with mismatched primer termini, however, show that the β hairpin plays a role in maintaining the stability of the polymerase/DNA interactions during the binding of the primer DNA in the exonuclease active site but not on the return of the corrected primer to the polymerase active site. In addition, the deletion variant showed a more stable incorporation of a nucleotide opposite an abasic site. Moreover, in the 2.4Å crystal structure of the β hairpin deletion variant incorporating an A opposite a templating furan, all four molecules in the crystal asymmetric unit have DNA in the polymerase active site, despite the presence of DNA distortions because of the misincorporation, confirming that the primer strand is not stably bound within the exonuclease active site in the absence of the β hairpin loop.

Human DNA polymerase θ grasps the primer terminus to mediate DNA repair

DNA polymerase θ protects against genomic instability via an alternative end-joining repair pathway for DNA double-strand breaks. Polymerase θ is overexpressed in breast, lung and oral cancers, and reduction of its activity in mammalian cells increases sensitivity to double-strand break–inducing agents, including ionizing radiation. Reported here are crystal structures of the C-terminal polymerase domain from human polymerase θ, illustrating two potential modes of dimerization. One structure depicts insertion of ddATP opposite an abasic-site analog during translesion DNA synthesis. The second structure describes a cognate ddGTP complex. Polymerase θ uses a specialized thumb subdomain to establish unique upstream contacts to the primer DNA strand, including an interaction with the 3′-terminal phosphate from one of five distinctive insertion loops. These observations demonstrate how polymerase θ grasps the primer to bypass DNA lesions or extend poorly annealed DNA termini to mediate end-joining.

Crystal structure of a replicative DNA polymerase bound to the oxidized guanine lesion guanidinohydantoin.

The oxidation of guanine generates one of the most common DNA lesions, 8-oxo-7,8-dihydroguanine (8-oxoG). The further oxidation of 8-oxoG can produce either guanidinohydantoin (Gh) in duplex DNA or spiroiminodihydantoin (Sp) in nucleosides and ssDNA. Although Gh can be a strong block for replicative DNA polymerases such as RB69 DNA polymerase, this lesion is also mutagenic: DNA polymerases bypass Gh by preferentially incorporating a purine with a slight preference for adenine, which results in G.C --> T.A or G.C --> C.G transversions. The 2.15 A crystal structure of the replicative RB69 DNA polymerase in complex with DNA containing Gh reveals that Gh is extrahelical and rotated toward the major groove. In this conformation Gh is no longer in position to serve as a templating base for the incorporation of an incoming nucleotide. This work also constitutes the first crystallographic structure of Gh, which is stabilized in the R configuration in the two polymerase/DNA complexes present in the crystal asymmetric unit. In contrast to 8-oxoG, Gh is found in a high syn conformation in the DNA duplex and therefore presents the same hydrogen bond donor and acceptor pattern as thymine, which explains the propensity of DNA polymerases to incorporate a purine opposite Gh when bypass occurs.

5-Hydroxy-5-methylhydantoin DNA lesion, a molecular trap for DNA glycosylases

DNA base-damage recognition in the base excision repair (BER) is a process operating on a wide variety of alkylated, oxidized and degraded bases. DNA glycosylases are the key enzymes which initiate the BER pathway by recognizing and excising the base damages guiding the damaged DNA through repair synthesis. We report here biochemical and structural evidence for the irreversible entrapment of DNA glycosylases by 5-hydroxy-5-methylhydantoin, an oxidized thymine lesion. The first crystal structure of a suicide complex between DNA glycosylase and unrepaired DNA has been solved. In this structure, the formamidopyrimidine-(Fapy) DNA glycosylase from Lactococcus lactis (LlFpg/LlMutM) is covalently bound to the hydantoin carbanucleoside-containing DNA. Coupling a structural approach by solving also the crystal structure of the non-covalent complex with site directed mutagenesis, this atypical suicide reaction mechanism was elucidated. It results from the nucleophilic attack of the catalytic N-terminal proline of LlFpg on the C5-carbon of the base moiety of the hydantoin lesion. The biological significance of this finding is discussed.

Applications of thin-film sandwich crystallization platforms

Crystallization via sandwiches of thin polymer films is presented and discussed.

S-Adenosyl-S-carboxymethyl-L-homocysteine: a novel cofactor found in the putative tRNA-modifying enzyme CmoA.

The putative methyltransferase CmoA is involved in the nucleoside modification of transfer RNA. X-ray crystallography and mass spectrometry are used to show that it contains a novel SAM derivative, S-adenosyl-S-carboxymethyl-l-homocysteine, in which the donor methyl group is replaced by a carboxymethyl group.

Using high-throughput in situ plate screening to evaluate the effect of dehydration on protein crystals.

Crystal dehydration is a post-crystallization technique that can potentially improve the diffraction of macromolecular crystals. There are currently several ways of undertaking this process; however, dehydration experiments are often limited in their throughput and require prior manipulation of the samples. In the present study, a novel method is proposed that uses in situ plate screening to assess the effect of dehydration by combining the throughput of 96-well crystallization plates with direct X-ray feedback on crystal diffraction quality.

Transmembrane helix packing of ErbB/Neu receptor in membrane environment: a molecular dynamics study.

Abstract Dimerization or oligomerization of the ErbB/Neu receptors are necessary but not sufficient for initiation of receptor signaling. The two intracellular domains must be properly oriented for the juxtaposition of the kinase domains allowing trans-phosphorylation. This suggests that the transmembrane (TM) domain acts as a guide for defining the proper orientation of the intracellular domains. Two structural models, with the two helices either in left-handed or in right-handed coiling have been proposed as the TM domain structure of the active receptor. Because experimental data do not distinguish clearly helix-helix packing, molecular dynamics (MD) simulations are used to investigate the energetic factors that drive Neu TM-TM interactions of the wild and the oncogenic receptor (Val664/Glu mutation) in DMPC or in POPC environments. MD results indicate that helix-lipid interactions in the bilayer core are extremely similar in the two environments and raise the role of the juxtamembrane residues in helix insertion and helix-helix packing. The TM domain shows a greater propensity to adopt a left-handed structure in DMPC, with helices in optimal position for strong inter-helical Hbonds induced by the Glu mutation. In POPC, the right-handed structure is preferentially formed with the participation of water in inter-helical Hbonds. The two structural arrangements of the NeuTM helices both with GG4 residue motif in close contact at the interface are permissible in the membrane environment. According to the hypothesis of a monomer-dimer equilibrium of the proteins it is likely that the bilayer imposes structural constraints that favor dimerization- competent structure responsible of the proper topology necessary for receptor activation.