Mechanism of DNA translocation in a replicative hexameric helicase

The E1 protein of papillomavirus is a hexameric ring helicase belonging to the AAA + family. The mechanism that couples the ATP cycle to DNA translocation has been unclear. Here we present the crystal structure of the E1 hexamer with single-stranded DNA discretely bound within the hexamer channel and nucleotides at the subunit interfaces. This structure demonstrates that only one strand of DNA passes through the hexamer channel and that the DNA-binding hairpins of each subunit form a spiral 'staircase' that sequentially tracks the oligonucleotide backbone. Consecutively grouped ATP, ADP and apo configurations correlate with the height of the hairpin, suggesting a straightforward DNA translocation mechanism. Each subunit sequentially progresses through ATP, ADP and apo states while the associated DNA-binding hairpin travels from the top staircase position to the bottom, escorting one nucleotide of single-stranded DNA through the channel. These events permute sequentially around the ring from one subunit to the next.     

Structure of the repressor-operator complex of bacteriophage 434

The crystal structure of a specific complex between the DNA-binding domain of phage 434 repressor and a synthetic 434 operator DNA shows interactions that determine sequence-dependent affinity. The repressor recognizes its operators by its complementarity to a particular DNA conformation as well as by direct interaction with base pairs in the major groove.     

Structure of a phage 434 Cro/DNA complex

Comparison of the crystal structure of a complex of the phage 434 Cro protein and a synthetic DNA operator with the complex of the same operator and the 434 repressor DNA-binding domain shows different DNA conformations in the two structures. Binding of the protein determines the precise conformation of the DNA in each case.     

3-A resolution structure of a protein with histone-like properties in prokaryotes

The 3-A structure of DNA-binding protein II, which exhibits histone-like properties in bacteria, has been determined. The molecule is dimeric and appears to bind to the phosphate backbone of DNA through two symmetry-related arms. A mechanism by which the protein induces DNA supercoiling is proposed.     

The SRA domain of UHRF1 flips 5-methylcytosine out of the DNA helix

Maintenance methylation of hemimethylated CpG dinucleotides at DNA replication forks is the key to faithful mitotic inheritance of genomic methylation patterns. UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) is required for maintenance methylation by interacting with DNA nucleotide methyltransferase 1 (DNMT1), the maintenance methyltransferase, and with hemimethylated CpG, the substrate for DNMT1 (refs 1 and 2). Here we present the crystal structure of the SET and RING-associated (SRA) domain of mouse UHRF1 in complex with DNA containing a hemimethylated CpG site. The DNA is contacted in both the major and minor grooves by two loops that penetrate into the middle of the DNA helix. The 5-methylcytosine has flipped completely out of the DNA helix and is positioned in a binding pocket with planar stacking contacts, Watson-Crick polar hydrogen bonds and van der Waals interactions specific for 5-methylcytosine. Hence, UHRF1 contains a previously unknown DNA-binding module and is the first example of a non-enzymatic, sequence-specific DNA-binding protein domain to use the base flipping mechanism to interact with DNA.     

A phage repressor-operator complex at 7 A resolution

The crystal structure of a complex between the DNA-binding domain of phage 434 repressor and a synthetic 434 operator shows that the protein, very similar in conformation to gamma repressor, binds to B-form DNA with the second alpha-helix of a helix-turn-helix motif lying in the major groove.     

Human DNA ligase I completely encircles and partially unwinds nicked DNA

The end-joining reaction catalysed by DNA ligases is required by all organisms and serves as the ultimate step of DNA replication, repair and recombination processes. One of three well characterized mammalian DNA ligases, DNA ligase I, joins Okazaki fragments during DNA replication. Here we report the crystal structure of human DNA ligase I (residues 233 to 919) in complex with a nicked, 5' adenylated DNA intermediate. The structure shows that the enzyme redirects the path of the double helix to expose the nick termini for the strand-joining reaction. It also reveals a unique feature of mammalian ligases: a DNA-binding domain that allows ligase I to encircle its DNA substrate, stabilizes the DNA in a distorted structure, and positions the catalytic core on the nick. Similarities in the toroidal shape and dimensions of DNA ligase I and the proliferating cell nuclear antigen sliding clamp are suggestive of an extensive protein-protein interface that may coordinate the joining of Okazaki fragments.     

The crystal structure of DNA mismatch repair protein MutS binding to a G x T mismatch

DNA mismatch repair ensures genomic integrity on DNA replication. Recognition of a DNA mismatch by a dimeric MutS protein initiates a cascade of reactions and results in repair of the newly synthesized strand; however, details of the molecular mechanism remain controversial. Here we present the crystal structure at 2.2 A of MutS from Escherichia coli bound to a G x T mismatch. The two MutS monomers have different conformations and form a heterodimer at the structural level. Only one monomer recognizes the mismatch specifically and has ADP bound. Mismatch recognition occurs by extensive minor groove interactions causing unusual base pairing and kinking of the DNA. Nonspecific major groove DNA-binding domains from both monomers embrace the DNA in a clamp-like structure. The interleaved nucleotide-binding sites are located far from the DNA. Mutations in human MutS alpha (MSH2/MSH6) that lead to hereditary predisposition for cancer, such as hereditary non-polyposis colorectal cancer, can be mapped to this crystal structure.     

The structure of the E. coli recA protein monomer and polymer.

The crystal structure of the recA protein from Escherichia coli at 2.3-A resolution reveals a major domain that binds ADP and probably single- and double-stranded DNA. Two smaller subdomains at the N and C termini protrude from the protein and respectively stabilize a 6(1) helical polymer of protein subunits and interpolymer bundles. This polymer structure closely resembles that of recA/DNA filaments determined by electron microscopy. Mutations in recA protein that enhance coprotease, DNA-binding and/or strand-exchange activity can be explained if the interpolymer interactions in the crystal reflect a regulatory mechanism in vivo.     

A novel steroid thyroid hormone receptor-related gene inappropriately expressed in human hepatocellular carcinoma

We have previously isolated from a human hepatocellular carcinoma a hepatitis B virus integration in a 147-base-pair cellular DNA fragment, similar to steroid- and c-erb-A/thyroid-hormone receptor genes. We have now cloned the corresponding complementary DNA from a human-liver cDNA library. Nucleotide sequence analysis revealed that the overall structure of the cellular gene, which we have named hap, is similar to that of the DNA-binding hormone receptors. That is, it displays two highly conserved regions identified as the putative DNA-binding and hormone-binding domains of the c-erb A/steroid receptors. Six out of seven hepatoma and hepatoma-derived cell-lines express a 2.5-kilobase (kb) hap messenger RNA species which is undetectable in normal adult and fetal livers but present in all non-hepatic tissues analysed. The data suggest that the hap gene product may be a novel ligand-responsive regulatory protein whose inappropriate expression in liver may relate to the hepatocellular carcinogenesis.     

Structure of Arc repressor in solution: evidence for a family of beta-sheet DNA-binding proteins

The Arc repressor, which is involved in the switch between lysis and lysogeny of Salmonella bacteriophage P22, does not belong to any of the known classes of DNA-binding proteins. Mutagenesis studies show that the DNA-binding region is located in the 15 N-terminal amino-acid residues. We have now determined the three-dimensional structure of the Arc dimer from an extensive set of interproton-distance data obtained from 1H NMR spectroscopy. A priori, intra- and inter-monomer nuclear Overhauser effects (NOEs) cannot be distinguished for a symmetric dimer. But by using the homology with the Escherichia coli Met repressor we could interpret the NOEs unambiguously in an iterative structure refinement procedure. The final structure satisfies a large set of NOE constraints (1,352 for the dimer). It shows a strongly intertwined dimer, in which residues 8-14 of different monomers form an antiparallel beta-sheet. A model for the Arc repressor-operator complex can account for all available biochemical and genetic data. In this model two Arc dimers bind with their beta-sheet regions in successive major grooves on one side of the DNA helix, similar to the Met repressor interaction. Thus, Arc and Met repressors are members of the same family of proteins, which contain an antiparallel beta-sheet as the DNA-binding motif.     

Structural basis for gate-DNA recognition and bending by type IIA topoisomerases

Type II topoisomerases disentangle DNA to facilitate chromosome segregation, and represent a major class of therapeutic targets. Although these enzymes have been studied extensively, a molecular understanding of DNA binding has been lacking. Here we present the structure of a complex between the DNA-binding and cleavage core of Saccharomyces cerevisiae Topo II (also known as Top2) and a gate-DNA segment. The structure reveals that the enzyme enforces a 150 degrees DNA bend through a mechanism similar to that of remodelling proteins such as integration host factor. Large protein conformational changes accompany DNA deformation, creating a bipartite catalytic site that positions the DNA backbone near a reactive tyrosine and a coordinated magnesium ion. This configuration closely resembles the catalytic site of type IA topoisomerases, reinforcing an evolutionary link between these structurally and functionally distinct enzymes. Binding of DNA facilitates opening of an enzyme dimerization interface, providing visual evidence for a key step in DNA transport.     

6-位脂肪胺取代的萘酰亚胺衍生物与DNA的相互作用

利用紫外可见光谱、荧光光谱、圆二色谱和粘度测试,考察了8个6-位脂肪胺取代的萘酰亚胺衍生物与小牛胸腺DNA的相互作用.研究结果表明:小牛胸腺DNA对与其结合能力强的化合物3a～3d有荧光猝灭作用,对与其结合能力弱的3e～3h有荧光增强效应;圆二色谱显示这些萘酰亚胺化合物能引起DNA二级结构发生不同的变化.在紫外-可见吸收光谱和粘度测试中,这些化合物与DNA的相互作用没有差别.综合光谱学和粘度测试的结果,推断这些化合物是通过嵌插方式与DNA结合,由于结构不同,它们与DNA之间作用程度存在差异.     

The structure of trp pseudorepressor at 1.65A shows why indole propionate acts as a trp 'inducer'

The trp repressor is a small dimeric regulatory protein which controls the expression of three operons in Escherichia coli. The inactive aporepressor protein must bind two molecules of L-tryptophan to form the active repressor. If desamino analogues of L-tryptophan such as indole propionate (IPA) are substituted for L-tryptophan, an inactive pseudorepressor is formed. Because the desamino analogues thus cause derepression of operons under control of the trp repressor, they appear to be 'inducers'. We have determined the crystal structure of the pseudorepressor and refined it to 1.65 A. The molecular structure was compared to that of the nearly isomorphous orthorhombic form of the repressor. Surprisingly, the indole ring of IPA is in the same position as the indole ring of L-tryptophan in the repressor, but is 'flipped over'. As a result, the carboxyl group of IPA is oriented toward the DNA-binding surface of the protein and is in a position where it sterically and electrostatically repels the phosphate backbone of both operator and non-operator DNA. This explains why IPA acts as an apparent trp inducer.