Nora Goosen

The regulation of transcription initiation by integration host factor

Integration host factor (IHF) of Escherichia coli is an asymmetric histone‐like protein that binds and bends the DNA at specific sequences. IHF functions as an accessory factor in a wide variety of processes including replication, site‐specific recombination and transcription. In many of these processes IHF was shown to act as an architectural element which helps the formation of nucleo‐protein complexes by bending of the DNA at specific sites. This MicroReview shows how such a structural role of IHF can influence the initiation of transcription. In addition, it summarizes the evidence indicating that IHF can stimulate transcription via a direct interaction with RNA polymerase and explores the possibility that the asymmetry of the IHF protein might reflect such an interaction

HU: promoting or counteracting DNA compaction?

The role of HU in Escherichia coli as both a protein involved in DNA compaction and as a protein with regulatory function seems to be firmly established. However, a critical look at the available data reveals that this is not true for each of the proposed roles of this protein. The role of HU as a regulatory or accessory protein in a number of systems has been thoroughly investigated and in many cases has been largely elucidated. However, almost 30 years after its discovery, convincing evidence for the proposed role of HU in DNA compaction is still lacking. Here we present an extensive literature survey of the available data which, in combination with novel microscopic insights, suggests that the role of HU could be the opposite as well. The protein is likely to play an architectural role, but instead of being responsible for DNA compaction it could be involved in antagonising compaction by other proteins such as H‐NS.

Structural basis for preferential binding of H-NS to curved DNA.

The Escherichia coli H-NS protein is a nucleoid-associated protein involved in transcription regulation and DNA compaction. H-NS exerts its role in DNA condensation by non-specific interactions with DNA. With respect to transcription regulation preferential binding sites in the promoter regions of different genes have been reported. In this paper we describe the analysis of H-NS-DNA complexes on a preferred H-NS binding site by atomic force microscopy. On the basis of these data we present a model for the specific recognition of DNA by H-NS as a function of DNA curvature.

DNA inversions in phages and bacteria

In certain phages and bacteria, there is a recombination system that specifically promotes the inversion of a DNA fragment. These inversion events appear to act as genetic switches allowing the alternate expression of different sets of genes which in general code for surface proteins. The mechanism of inversion in one class of inversion systems (Gin/Hin) has been studied in detail. It involves the formation of a highly specific nucleoprotein complex in which not only the two recombination sites and the DNA invertase participate but also a recombinational enhancer to which the DNA-bending protein Fis is bound.

The presence of two UvrB subunits in the UvrAB complex ensures damage detection in both DNA strands

It is generally accepted that the damage recognition complex of nucleotide excision repair in Escherichia coli consists of two UvrA and one UvrB molecule, and that in the preincision complex UvrB binds to the damage as a monomer. Using scanning force microscopy, we show here that the damage recognition complex consists of two UvrA and two UvrB subunits, with the DNA wrapped around one of the UvrB monomers. Upon binding the damage and release of the UvrA subunits, UvrB remains a dimer in the preincision complex. After association with the UvrC protein, one of the UvrB monomers is released. We propose a model in which the presence of two UvrB subunits ensures damage recognition in both DNA strands. Upon binding of the UvrA2B2 complex to a putative damaged site, the DNA wraps around one of the UvrB monomers, which will subsequently probe one of the DNA strands for the presence of a lesion. When no damage is found, the DNA will wrap around the second UvrB subunit, which will check the other strand for aberrations.

Cho, a second endonuclease involved in Escherichia coli nucleotide excision repair

Nucleotide excision repair removes damages from the DNA by incising the damaged strand on the 3′ and 5′ sides of the lesion. In Escherichia coli, the two incisions are made by the UvrC protein, which consists of two functional halves. The N-terminal half contains the catalytic site for 3′ incision and the C-terminal half contains the residues involved in 5′ incision. The genome of E. coli contains an SOS-inducible gene (ydjQ) encoding a protein that is homologous to the N-terminal half of UvrC. In this paper we show that this protein, which we refer to as Cho (UvrC homologue), can incise the DNA at the 3′ side of a lesion during nucleotide excision repair. The incision site of Cho is located 4 nt further away from the damage compared with the 3′ incision site of UvrC. Cho and UvrC bind to different domains of UvrB, which is probably the reason of the shift in incision position. Some damaged substrates that are poorly incised by UvrC are very efficiently incised by Cho. We propose that E. coli uses Cho for repair of such damages in vivo. Initially, most of the lesions in the cell will be repaired by the action of UvrC alone. Remaining damages, that for structural reasons obstruct the 3′ incision by UvrC, will be repaired by the combined action of Cho (for 3′ incision) and UvrC (for 5′ incision).

Architecture of nucleotide excision repair complexes: DNA is wrapped by UvrB before and after damage recognition

Nucleotide excision repair (NER) is a major DNA repair mechanism that recognizes a broad range of DNA damages. In Escherichia coli, damage recognition in NER is accomplished by the UvrA and UvrB proteins. We have analysed the structural properties of the different protein–DNA complexes formed by UvrA, UvrB and (damaged) DNA using atomic force microscopy. Analysis of the UvrA2B complex in search of damage revealed the DNA to be wrapped around the UvrB protein, comprising a region of about seven helical turns. In the UvrB–DNA pre‐incision complex the DNA is wrapped in a similar way and this DNA configuration is dependent on ATP binding. Based on these results, a role for DNA wrapping in damage recognition is proposed. Evidence is presented that DNA wrapping in the pre‐incision complex also stimulates the rate of incision by UvrC.

Cloning, characterization and DNA sequencing of the gene encoding the Mr50000 quinoprotein glucose dehydrogenase fromAcinetobacter calcoaceticus

SummaryRecently we described the cloning of the gene coding for a Mr 87000 glucose dehydrogenase (GDH-A) fromAcinetobacter calcoaceticus. In this report we describe the cloning of a gene coding for a second GDH (GDH-B) with a Mr of 50000 from the same organism. This gene was isolated using a 20-mer synthetic oligonucleotide, derived from the N-terminal amino acid sequence of purified GDH-B as a probe to screen a genomic bank. From the DNA sequence of thegdhB gene, a protein can be derived of Mr 52772 with a 24 amino acid signal peptide which is removed, resulting in the mature protein with a Mr 50231. In vitro transcription-translation of thegdhB clone shows the mature and the precursor protien. The derived amino acid sequence has no obvious homology with GDH-A ofA. calcoaceticus. We show that disaccharides are specific GDH-B substrates and that 2-deoxyglucose is specific for GDH-A.

Clue to damage recognition by UvrB: residues in the β‐hairpin structure prevent binding to non‐damaged DNA

UvrB, the ultimate damage‐recognizing component of bacterial nucleotide excision repair, contains a flexible β‐hairpin rich in hydrophobic residues. We describe the properties of UvrB mutants in which these residues have been mutated. The results show that Y101 and F108 in the tip of the hairpin are important for the strand‐separating activity of UvrB, supporting the model that the β‐hairpin inserts between the two DNA strands during the search for DNA damage. Residues Y95 and Y96 at the base of the hairpin have a direct role in damage recognition and are positioned close to the damage in the UvrB–DNA complex. Strikingly, substituting Y92 and Y93 results in a protein that is lethal to the cell. The mutant protein forms pre‐ incision complexes on non‐damaged DNA, indicating that Y92 and Y93 function in damage recognition by preventing UvrB binding to non‐damaged sites. We propose a model for damage recognition by UvrB in which, stabilized by the four tyrosines at the base of the hairpin, the damaged nucleotide is flipped out of the DNA helix.

Role of ATP hydrolysis by UvrA and UvrB during nucleotide excision repair

Nucleotide excision repair in eubacteria is a process that repairs DNA damages by the removal of a 12-13-mer oligonucleotide containing the lesion. Recognition and cleavage of the damaged DNA is a multistep ATP-dependent reaction that requires the UvrA, UvrB and UvrC proteins. Both UvrA and UvrB are ATPases, with UvrA having two ATP binding sites which have the characteristic signature of the family of ABC proteins and UvrB having one ATP binding site that is structurally related to that of helicases.