Rob Visse

Crystal structure of Escherichia coli UvrB C-terminal domain, and a model for UvrB-UvrC interaction

A crystal structure of the C‐terminal domain of Escherichia coli UvrB (UvrB′) has been solved to 3.0 Å resolution. The domain adopts a helix‐loop‐helix fold which is stabilised by the packing of hydrophobic side‐chains between helices. From the UvrB′ fold, a model for a domain of UvrC (UvrC′) that has high sequence homology with UvrB′ has been made. In the crystal, a dimerisation of UvrB′ domains is seen involving specific hydrophobic and salt bridge interactions between residues in and close to the loop region of the domain. It is proposed that a homologous mode of interaction may occur between UvrB and UvrC. This interaction is likely to be flexible, potentially spanning >50 Å.

The first zinc-binding domain of UvrA is not essential for UvrABC-mediated DNA excision repair

Specific mutations in uvrA were introduced to analyze the role of the zinc-binding domains of the protein in DNA excision repair. Zinc-coordinating cysteines were substituted into non-coordinating serine or glycine residues. Mutations leading to changes in the second zinc-binding domain had a profound effect on UV survival in vivo; however these mutant proteins could not be isolated for in vitro analyses. Amino acid substitutions in the first zinc-binding domain had very little effect on UV survival in vivo. In vitro analyses showed that although this domain no longer coordinates zinc, ATPase activity, helicase activity, DNA binding, incision of damaged DNA and DNA repair synthesis appeared to be normal. Therefore it seems that the first zinc-binding domain of UvrA is not essential for DNA excision repair.

Functional Domains of the E. coli UvrABC Proteins in Nucleotide Excision Repair

Different classes of DNA repair systems exist to counteract the effect of the many forms of DNA damage that are generated by exposure of living cells to ultraviolet or ionising radiation and a variety of chemicals. Of these repair systems the nucleotide excision repair pathway is unique in its ability to recognise and repair a vast array of structurally unrelated DNA lesions (see Van Houten 1990 for review). The principles of nucleotide excision repair are the same in prokaryotic and eukaryotic organisms. After recognition of a DNA lesion, incisions are made in the damaged strand on both sides of the lesion, the oligonucleotide containing the lesion is removed, and the resulting gap is filled by DNA synthesis followed by ligation of the remaining nick. The proteins involved in the prokaryotic system, however, differ from those of the eukaryotic system, indicating that the two repair processes are not evolutionaryly related. Whereas eukaryotic nucleotide excision repair requires about 30 polypeptides (Sancar 1996), just 6 proteins (UvrA, UvrB, UvrC, UvrD, DNA polymerase (Pol) I and ligase) are sufficient for carrying out the repair process in E. coli.

Synthesis and biological evaluation of modified DNA fragments for the study of nucleotide excision repair in E. coli.

Three new cholesterol-containing phosphoramidites where synthesized and used in automated synthesis of modified DNA fragments. These cholesterol lesions are good substrates for the E. coli UvrABC endonuclease. In vitro they are incised from damaged DNA with higher efficiency in respect with the cholesterol lesions previously published.

The in vitro more efficiently repaired cisplatin adduct cis-Pt.GG is in vivo a more mutagenic lesion than the relative slowly repaired cis-Pt.GCG adduct

The toxic effect and the mutagenicity of two differentially repaired site-specific cis-diamminedichloroplatinum(II) (cis-DDP) lesions were investigated. Detailed analysis of the UvrABC-dependent repair of the two lesions in vitro showed a more efficient repair of the cis-Pt.GG adduct compared to that of the cis-Pt.GCG adduct (Visse et al., 1994). Furthermore, previously, a dependency of cis-DDP mutagenesis on UvrA and UvrB, but not on UvrC was found (Brouwer et al., 1988). To possibly relate survival and mutagenesis to repair, plasmids containing the same site-specific cis-DDP lesions as those that were used in the detailed repair studies were transformed into Escherichia coli. The results indicate that both lesions are very efficiently bypassed in vivo. Mutation analysis was performed using a denaturing gradient gel electrophoresis technique, which allows identification of mutations without previous selection. Although the cis-Pt.GG adduct is in vitro more efficiently repaired than the cis-Pt.GCG adduct, it appeared to be more mutagenic. We present a model in which this result is related to the previously observed dependency of the mutagenicity of cis-DDP lesions on the Uvr A and B proteins.

DNA Repair by UvrABC: In Vitro Analysis of the Preincision Complex

The UvrABC endonuclease repairs a variety of structurally different lesions. The preincision complex consisting of UvrB and damaged DNA plays a central role in successful damage recognition by UvrA and UvrB, and is a prerequisite for the subsequent processing in the presence of UvrC, resulting in dual incisions of the DNA. Although there is still an effect on efficiency, the type of lesion seems to have only a minor influence on the mechanism of repair, since incisions occur at fixed distances from each lesion. Therefore, an important aspect of unraveling the molecular mechanisms of UvrABC-dependent DNA repair is to understand how this system is able to process different lesions in a similar way. Attention in this work has been focused on which domains of UvrB are specifically involved in the formation of the UvrB-DNA preincision complex and on the structure of this complex.