Biomedical subjects

Nora Goosen

Publications and source records attributed to Nora Goosen.

9 recordsLinked to original sources

PubMed2006

Functional characterization of the competence protein DprA/Smf in Escherichia coli.

In several bacterial species that show natural transformation, dprA has been described as a competence gene. The DprA protein has been suggested to be involved in the protection of incoming DNA. However, members of the dprA gene family (also called smf) can be detected in virtually all bacterial species, which suggests that their gene products have a more general function. We examined the function of the DprA/Smf homologue of Escherichia coli. Escherichia coli dprA/smf is able to partially restore transformation in a Haemophilus influenzae dprA mutant, which shows that dprA/smf genes from competent and noncompetent species are interchangeable with respect to their involvement in natural transformation. From this, we conclude that natural transformation is probably an additional function of these genes. Subsequently, the dprA/smf gene was deleted in various recombination mutants of E. coli, and the resultant phenotype was tested. All the resultant E. coli dprA/smf mutants did not differ from their parent strains with respect to transformation, Hfr-conjugation, recombination and DNA repair. Therefore, a role of DprA/Smf in DNA recombination could not be established and the basic function of dprA/smf remains unclear.

Bacterial Proteins↗

PubMed2005

Base flipping in nucleotide excision repair.

UvrB, the ultimate damage-binding protein in bacterial nucleotide excision repair is capable of binding a vast array of structurally unrelated lesions. A beta-hairpin structure in the protein plays an important role in damage-specific binding. In this paper we have monitored DNA conformational alterations in the UvrB-DNA complex, using the fluorescent adenine analogue 2-aminopurine. We show that binding of UvrB to a DNA fragment with cholesterol damage moves the base adjacent to the lesion at the 3' side into an extrahelical position. This extrahelical base is not accessible for acrylamide quenching, suggesting that it inserts into a pocket of the UvrB protein. Also the base opposite this flipped base is extruded from the DNA helix. The degree of solvent exposure of both residues varies with the type of cofactor (ADP/ATP) bound by UvrB. Fluorescence of the base adjacent to the damage is higher when UvrB is in the ADP-bound configuration, but concomitantly this UvrB-DNA complex is less stable. In the ATP-bound form the UvrB-DNA complex is very stable and in this configuration the base in the non-damaged strand is more exposed. Hairpin residue Tyr-95 is specifically involved in base flipping in the non-damaged strand. We present evidence that this conformational change in the non-damaged strand is important for 3' incision by UvrC.

2-Aminopurine↗

PubMed2005

Binding of the UvrB dimer to non-damaged and damaged DNA: residues Y92 and Y93 influence the stability of both subunits.

UvrB is the ultimate damage-binding protein in bacterial nucleotide excision repair. Previous AFM experiments have indicated that UvrB binds to a damage as a dimer. In this paper we visualize for the first time a UvrB dimer in a gel retardation assay, with the second subunit (B2) more loosely bound than the subunit (B1) that interacts with the damage. A beta-hairpin motif in UvrB plays an important role in damage specific binding. Alanine substitutions of Y92 or Y93 in the beta-hairpin result in proteins that kill E. coli cells as a consequence of incision in non-damaged DNA. Apparently, both residues are needed to prevent binding of UvrB to non-damaged DNA. The lethality of Y93A results from UvrC-mediated incisions, whereas that of Y92A is due to incisions by Cho. This difference could be ascribed to a difference in stability of the B2 subunit in the mutant UvrB-DNA complexes. We show that for 3' incision UvrC needs to displace this second UvrB subunit from the complex, whereas Cho seems capable to incise the dimer-complex. Footprint analysis of the contacts of UvrB with damaged DNA revealed that the B2 subunit interacts with the flanking DNA at the 3' side of the lesion. The B2 subunit of mutant Y92A appeared to be more firmly associated with the DNA, indicating that even when B1 is bound to a lesion, the B2 subunit probes the adjacent DNA for presence of damage. We propose this to be a reflection of the process that the UvrB dimer uses to find lesions in the DNA. In addition to preventing binding to non-damaged DNA, the Y92 and Y93 residues appear also important for making specific contacts (of B1) with the damaged site. We show that the concerted action of the two tyrosines lead to a conformational change in the DNA surrounding the lesion, which is required for the 3' incision reaction.

Adenosine Triphosphatases↗

PubMed2004

Dual architectural roles of HU: formation of flexible hinges and rigid filaments.

The nucleoid-associated protein HU is one of the most abundant proteins in Escherichia coli and has been suggested to play an important role in bacterial nucleoid organization and regulation. Although the regulatory aspects of HU have been firmly established, much less is understood about the role of HU in shaping the bacterial nucleoid. In both functions (local) modulation of DNA architecture seems an essential feature, but information on the mechanical properties of this type of sequence-independent nucleoprotein complex is scarce. In this study we used magnetic tweezers and atomic force microscopy to quantify HU-induced DNA bending and condensation. Both techniques revealed that HU can have two opposing mechanical effects depending on the protein concentration. At concentrations <100 nM, individual HU dimers induce very flexible bends in DNA that are responsible for DNA compaction up to 50%. At higher HU concentrations, a rigid nucleoprotein filament is formed in which HU appears to arrange helically around the DNA without inducing significant condensation.

Bacterial Proteins↗

PubMed2002

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.

Bacterial Proteins↗

PubMed2002

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 UvrA(2)B(2) 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.

Adenosine Triphosphatases↗

PubMed2002

The C-terminal region of Escherichia coli UvrC contributes to the flexibility of the UvrABC nucleotide excision repair system.

Nucleotide excision repair in Escherichia coli involves formation of the UvrB-DNA complex and subsequent DNA incisions on either site of the damage by UvrC. In this paper, we studied the incision of substrates with different damages in varying sequence contexts. We show that there is not always a correlation between the incision efficiency and the stability of the UvrB-DNA complex. Both stable and unstable UvrB-DNA complexes can be efficiently incised. However some lesions that give rise to stable UvrB-DNA complexes do result in a very low incision. We present evidence that this poor incision is due to sterical hindrance of the damage itself. In its C-terminal region UvrC contains two helix-hairpin-helix (HhH) motifs. Mutational analysis shows that these motifs constitute one functional unit, probably folded as one structural unit; the (HhH)2 domain. This (HhH)2 domain was previously shown to be important for the 5' incision on a substrate containing a (cis-Pt).GG adduct, but not for 3' incision. Here we show that, mainly depending on the sequence context of the lesion, the (HhH)2 domain can be important for 3' and/or 5' incision. We propose that the (HhH)2 domain stabilises specific DNA structures required for the two incisions, thereby contributing to the flexibility of the UvrABC repair system.

Adenosine Triphosphatases↗

PubMed2002

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).

Amino Acid Sequence↗

PubMed2001

Structural basis for H-NS-mediated trapping of RNA polymerase in the open initiation complex at the rrnB P1.

The Escherichia coli H-NS protein is a nucleoid-associated protein involved in both transcription regulation and DNA compaction. Each of these processes involves H-NS-mediated bridge formation between adjacent DNA helices. With respect to transcription regulation, preferential binding sites in the promoter regions of different genes have been reported, and generally these regions are curved. Often H-NS binding sites overlap with promoter core regions or with binding sites of other regulatory factors. Not in all cases, however, transcriptional repression is the result of preferential binding by H-NS to promoter regions leading to occlusion of the RNA polymerase. In the case of the rrnB P1, H-NS actually stimulates open complex formation by forming a ternary RNAP.H-NS.DNA complex, while simultaneously stabilizing it to such an extent that promoter clearance cannot occur. To define the mechanism by which H-NS interferes at this step in the initiation pathway, the architecture of the RNAP.H-NS.DNA complex was analyzed by scanning force microscopy (SFM). The SFM images show that the DNA flanking the RNA polymerase in open initiation complexes is bridged by H-NS. On the basis of these data, we present a model for the specific repression of transcription initiation at the rrnB P1 by H-NS.

Bacterial Proteins↗