Éva Scheuring Vanamee

A view of consecutive binding events from structures of tetrameric endonuclease SfiI bound to DNA.

Many reactions in cells proceed via the sequestration of two DNA molecules in a synaptic complex. SfiI is a member of a growing family of restriction enzymes that can bind and cleave two DNA sites simultaneously. We present here the structures of tetrameric SfiI in complex with cognate DNA. The structures reveal two different binding states of SfiI: one with both DNA‐binding sites fully occupied and the other with fully and partially occupied sites. These two states provide details on how SfiI recognizes and cleaves its target DNA sites, and gives insight into sequential binding events. The SfiI recognition sequence (GGCCNNNN↓NGGCC) is a subset of the recognition sequence of BglI (GCCNNNN↓NGGC), and both enzymes cleave their target DNAs to leave 3‐base 3′ overhangs. We show that even though SfiI is a tetramer and BglI is a dimer, and there is little sequence similarity between the two enzymes, their modes of DNA recognition are unusually similar.

Topology of Type II REases revisited; structural classes and the common conserved core

Type II restriction endonucleases (REases) are deoxyribonucleases that cleave DNA sequences with remarkable specificity. Type II REases are highly divergent in sequence as well as in topology, i.e. the connectivity of secondary structure elements. A widely held assumption is that a structural core of five β-strands flanked by two α-helices is common to these enzymes. We introduce a systematic procedure to enumerate secondary structure elements in an unambiguous and reproducible way, and use it to analyze the currently available X-ray structures of Type II REases. Based on this analysis, we propose an alternative definition of the core, which we term the αβα-core. The αβα-core includes the most frequently observed secondary structure elements and is not a sandwich, as it consists of a five-strand β-sheet and two α-helices on the same face of the β-sheet. We use the αβα-core connectivity as a basis for grouping the Type II REases into distinct structural classes. In these new structural classes, the connectivity correlates with the angles between the secondary structure elements and with the cleavage patterns of the REases. We show that there exists a substructure of the αβα-core, namely a common conserved core, ccc, defined here as one α-helix and four β-strands common to all Type II REase of known structure.

An iron-sulfur cluster in the polymerase domain of yeast DNA polymerase ε.

DNA polymerase ε (Polε) is a multi-subunit polymerase that contributes to genomic stability via its roles in leading strand replication and the repair of damaged DNA. Polε from Saccharomyces cerevisiae is composed of four subunits--Pol2, Dpb2, Dpb3, and Dpb4. Here, we report the presence of a [Fe-S] cluster directly within the active polymerase domain of Pol2 (residues 1-1187). We show that binding of the [Fe-S] cluster is mediated by cysteines in an insertion (Pol2(ins)) that is conserved in Pol2 orthologs but is absent in the polymerase domains of Polα, Polδ, and Polζ. We also show that the [Fe-S] cluster is required for Pol2 polymerase activity but not for its exonuclease activity. Collectively, our work suggests that Polε is perhaps more sensitive than other DNA polymerases to changes in oxidative stress in eukaryotic cells.

HPV vaccines and cancer prevention, science versus activism.

The rationale behind current worldwide human papilloma virus (HPV) vaccination programs starts from two basic premises, 1) that HPV vaccines will prevent cervical cancers and save lives and, 2) have no risk of serious side effects. Therefore, efforts should be made to get as many pre-adolescent girls vaccinated in order to decrease the burden of cervical cancer. Careful analysis of HPV vaccine pre- and post-licensure data shows however that both of these premises are at odds with factual evidence and are largely derived from significant misinterpretation of available data.

Asymmetric DNA recognition by the OkrAI endonuclease, an isoschizomer of BamHI.

Restriction enzymes share little or no sequence homology with the exception of isoschizomers, or enzymes that recognize and cleave the same DNA sequence. We present here the structure of a BamHI isoschizomer, OkrAI, bound to the same DNA sequence (TATGGATCCATA) as that cocrystallized with BamHI. We show that OkrAI is a more minimal version of BamHI, lacking not only the N- and C-terminal helices but also an internal 310 helix and containing β-strands that are shorter than those in BamHI. Despite these structural differences, OkrAI recognizes the DNA in a remarkably similar manner to BamHI, including asymmetric contacts via C-terminal ‘arms’ that appear to ‘compete’ for the minor groove. However, the arms are shorter than in BamHI. We observe similar DNA-binding affinities between OkrAI and BamHI but OkrAI has higher star activity (at 37°C) compared to BamHI. Together, the OkrAI and BamHI structures offer a rare opportunity to compare two restriction enzymes that work on exactly the same DNA substrate.

Crystallization of restriction endonuclease SfiI in complex with DNA

The SfiI endonuclease from Streptomyces fimbriatus (EC 3.1.21.4) is a tetrameric enzyme that binds simultaneously to two recognition sites and cleaves both sites concertedly. It serves as a good model system for studying both specificity and cooperative DNA binding. Crystals of the enzyme were obtained by the hanging-drop vapor-diffusion method in complex with a 21-mer oligonucleotide. The crystals are trigonal, with unit-cell parameters a = b = 85.7, c = 202.6 A, and diffract to 2.6 A resolution on a rotating-anode X-ray generator. Preliminary X-ray analysis reveals the space group to be either P3(1)21 or P3(2)21. Interestingly, the crystals change to space group P6(1)22, with unit-cell parameters a = b = 85.5, c = 419.6 A, when the selenomethionyl (SeMet) derivative of the enzyme is co-crystallized with the same DNA. Phase information is currently being derived from this SeMet SfiI-DNA complex.