N Jamin, F. Toma/ Progress in Nuclear Magnetic Resonance Spectroscopy 38 (2001)83-114 3.4. Recognition using B-sheet 3. 1. Tn916 integrase 3. 4.2. GCC-box binding domain 4. Perspectives 112 References 1. Introduction bound to a 14-mer duplex DNA containing the bs site [1] and the lac repressor headpiece(residues 1 Understanding at a molecular level, the mechan- 56, HP56)complexed with a 11-mer operator [2] isms for the control of genetic information and its This review will describe the use of nmr to obtain replication, packaging and repair necessitates the information on complexes of proteins with their speci elucidation of the detailed interactions between fic DNA targets. Most of the NMR techniques used to proteins and DNA. The last ten years have produced study protein-DNA interactions are also employed a large amount of structural information about for other type of protein complexes. Therefore, for a protein-DNA complexes from both X-ray crystallo- detailed description of the NMr techniques, the graphy and NMR. These data reveal the complexity of reader is referred to recent reviews [3-5]or to specific the DNA recognition process. The absence of a papers referenced in the text recognition code' is particularly evident among the divided in three parts. The first part is hree zinc fingers of the transcription factor TFIIIA as an overview of the NMR techniques commonly used homologue residues in different complexes do not to get information on protein-DNA interactions. It always contact corresponding base pairs. Direct inter- includes a brief description of DNA labeling techni- action between protein side-chains and DNa bases ques, the use of chemical shift or hydrogen exchange not only involve secondary structures like a-helix or changes to find the binding site, the use of hydrogen B-sheet but also flexible loops and arms. Moreover exchange or relaxation data to get dynamics informa residues not involved in specific interactions such as tion on the binding process, the use of the main the linker residues of the three zinc fingers domain of isotope filtering and editing techniques as well as TFIIA can be as important for the protein-DNA transverse relaxation-optimized spectroscopy to interaction as residues making contact with DNA assign the NMR signals, and newly developed tech- bases niques to deal with large complexes or to obtain long NMR makes its unique contribution to the under- range distance restraints. The second part comprises standing of protein-DNA interactions by highlighting applications of these techniques to different protein the dynamic aspects of protein-DNA interactions: DNA complexes. Protein-DNA complexes are clas dynamics of disorder-to-order transitions upon DNa sified according to the protein recognition motif: binding, dynamics at the protein-DNA interface, helix-turn-helix(HT), zinc finger, minor groove dynamics of opening and closing of base-pairs and, binding motif and B-sheet. Finally, the third part measurements of lifetimes of water molecules at th presents the future perspectives that can be inferred protein-DNA interface. from the emerging NMR techniques During the last 10 years, more than 20 structures of specific protein-DNA complexes and numerous data on protein-DNA interactions have been obtained by 2. Overview of techniques NMR thanks to the developments in protein and nucleic acid synthesis, in isotopic labeling techniques Protein-nucleic acids complexes are large entities and in heteronuclear magnetic resonance spectro- and the availability ofC-andN-labeled proteins copy. The first 3D NMR structures of a protein has made the determination of their solution structures DNA complex were obtained in 1993: the Drosophila attainable. Double and triple resonance spectroscopy antennapedia mutant homeodomain(Antp(C39S) facilitates the resonance assignments, the measurement3.4. Recognition using b-sheet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 3.4.1. Tn916 integrase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 3.4.2. GCC-box binding domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 4. Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 1. Introduction Understanding at a molecular level, the mechan￾isms for the control of genetic information and its replication, packaging and repair necessitates the elucidation of the detailed interactions between proteins and DNA. The last ten years have produced a large amount of structural information about protein±DNA complexes from both X-ray crystallo￾graphy and NMR. These data reveal the complexity of the DNA recognition process. The absence of a `recognition code' is particularly evident among the three zinc ®ngers of the transcription factor TFIIIA as homologue residues in different complexes do not always contact corresponding base pairs. Direct inter￾action between protein side-chains and DNA bases not only involve secondary structures like a-helix or b-sheet but also ¯exible loops and arms. Moreover residues not involved in speci®c interactions such as the linker residues of the three zinc ®ngers domain of TFIIIA can be as important for the protein±DNA interaction as residues making contact with DNA bases. NMR makes its unique contribution to the under￾standing of protein±DNA interactions by highlighting the dynamic aspects of protein±DNA interactions: dynamics of disorder-to-order transitions upon DNA binding, dynamics at the protein±DNA interface, dynamics of opening and closing of base-pairs and, measurements of lifetimes of water molecules at the protein±DNA interface. During the last 10 years, more than 20 structures of speci®c protein±DNA complexes and numerous data on protein±DNA interactions have been obtained by NMR thanks to the developments in protein and nucleic acid synthesis, in isotopic labeling techniques and in heteronuclear magnetic resonance spectro￾scopy. The ®rst 3D NMR structures of a protein± DNA complex were obtained in 1993: the Drosophila antennapedia mutant homeodomain (Antp(C39S)) bound to a 14-mer duplex DNA containing the BS2 site [1] and the lac repressor headpiece (residues 1± 56, HP56) complexed with a 11-mer operator [2]. This review will describe the use of NMR to obtain information on complexes of proteins with their speci- ®c DNA targets. Most of the NMR techniques used to study protein±DNA interactions are also employed for other type of protein complexes. Therefore, for a detailed description of the NMR techniques, the reader is referred to recent reviews [3±5] or to speci®c papers referenced in the text. This review is divided in three parts. The ®rst part is an overview of the NMR techniques commonly used to get information on protein±DNA interactions. It includes a brief description of DNA labeling techni￾ques, the use of chemical shift or hydrogen exchange changes to ®nd the binding site, the use of hydrogen exchange or relaxation data to get dynamics informa￾tion on the binding process, the use of the main isotope ®ltering and editing techniques as well as transverse relaxation-optimized spectroscopy to assign the NMR signals, and newly developed tech￾niques to deal with large complexes or to obtain long￾range distance restraints. The second part comprises applications of these techniques to different protein± DNA complexes. Protein±DNA complexes are clas￾si®ed according to the protein recognition motif: helix-turn-helix (HTH), zinc ®nger, minor groove binding motif and b-sheet. Finally, the third part presents the future perspectives that can be inferred from the emerging NMR techniques. 2. Overview of techniques Protein±nucleic acids complexes are large entities and the availability of 13C- and 15N-labeled proteins has made the determination of their solution structures attainable. Double and triple resonance spectroscopy facilitates the resonance assignments, the measurement 84 N. Jamin, F. Toma / Progress in Nuclear Magnetic Resonance Spectroscopy 38 (2001) 83±114