Clue to damage recognition by UvrB: residues in the beta-hairpin structure prevent binding to non-damaged DNA.

UvrB, the ultimate damage-recognizing component of bacterial nucleotide excision repair, contains a flexible beta-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 beta-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.

Solution structure, hydrodynamics and thermodynamics of the UvrB C-terminal domain.

The solution structure, thermodynamic stability and hydrodynamic properties of the 55-residue C-terminal domain of UvrB that interacts with UvrC during excision repair in E. coli have been determined using a combination of high resolution NMR, ultracentrifugation, 15N NMR relaxation, gel permeation, NMR diffusion, circular dichroism and differential scanning calorimetry. The subunit molecular weight is 7,438 kDa., compared with 14.5+/-1.0 kDa. determined by equilibrium sedimentation, indicating a dimeric structure. The structure determined from NMR showed a stable dimer of anti-parallel helical hairpins that associate in an unusual manner, with a small and hydrophobic interface. The Stokes radius of the protein decreases from a high plateau value (ca. 22 A) at protein concentrations greater than 4 microM to about 18 A at concentrations less than 0.1 microM. The concentration and temperature-dependence of the far UV circular dichroism show that the protein is thermally stable (Tm ca. 71.5 degrees C at 36 microM). The simplest model consistent with these data was a dimer dissociating into folded monomers that then unfolds co-operatively. The van't Hoff enthalpy and dissociation constant for both transition was derived by fitting, with deltaH1=23 kJ mol(-1). K1(298)=0.4 microM and deltaH2= 184 kJ mol(-1). This is in good agreement with direct calorimetric analysis of the thermal unfolding of the protein, which gave a calorimetric enthalpy change of 181 kJ mol(-1) and a van't Hoff enthalpy change of 354 kJ mol(-1), confirming the dimer to monomer unfolding. The thermodynamic data can be reconciled with the observed mode of dimerisation. 15N NMR relaxation measurements at 14.1 T and 11.75 T confirmed that the protein behaves as an asymmetric dimer at mM concentrations, with a flexible N-terminal linker for attachment to the remainder of the UvrB protein. The role of dimerisation of this domain in the excision repair mechanism is discussed.

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.

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 UvrA(2)B 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.

Role of the Escherichia coli nucleotide excision repair proteins in DNA replication.

DNA polymerase I (PolI) functions both in nucleotide excision repair (NER) and in the processing of Okazaki fragments that are generated on the lagging strand during DNA replication. Escherichia coli cells completely lacking the PolI enzyme are viable as long as they are grown on minimal medium. Here we show that viability is fully dependent on the presence of functional UvrA, UvrB, and UvrD (helicase II) proteins but does not require UvrC. In contrast, delta polA cells grow even better when the uvrC gene has been deleted. Apparently UvrA, UvrB, and UvrD are needed in a replication backup system that replaces the PolI function, and UvrC interferes with this alternative replication pathway. With specific mutants of UvrC we could show that the inhibitory effect of this protein is related to its catalytic activity that on damaged DNA is responsible for the 3' incision reaction. Specific mutants of UvrA and UvrB were also studied for their capacity to support the PolI-independent replication. Deletion of the UvrC-binding domain of UvrB resulted in a phenotype similar to that caused by deletion of the uvrC gene, showing that the inhibitory incision activity of UvrC is mediated via binding to UvrB. A mutation in the N-terminal zinc finger domain of UvrA does not affect NER in vivo or in vitro. The same mutation, however, does give inviability in combination with the delta polA mutation. Apparently the N-terminal zinc-binding domain of UvrA has specifically evolved for a function outside DNA repair. A model for the function of the UvrA, UvrB, and UvrD proteins in the alternative replication pathway is discussed.

The effect of the DNA flanking the lesion on formation of the UvrB-DNA preincision complex. Mechanism for the UvrA-mediated loading of UvrB onto a DNA damaged site.

The UvrB-DNA preincision complex plays a key role in nucleotide excision repair in Escherichia coli. To study the formation of this complex, derivatives of a DNA substrate containing a cholesterol adduct were constructed. Introduction of a single strand nick into either the top or the bottom strand at the 3' side of the adduct stabilized the UvrB-DNA complex, most likely by the release of local stress in the DNA. Removal of both DNA strands up to the 3' incision site still allowed formation of the preincision complex. Similar modifications at the 5' side of the damage, however, gave different results. The introduction of a single strand nick at the 5' incision site completely abolished the UvrA-mediated formation of the UvrB-DNA complex. Deletion of both DNA strands up to the 5' incision site also prevented the UvrA-mediated loading of UvrB onto the damaged site, but UvrB by itself could bind very efficiently. This demonstrates that the UvrB protein is capable of recognizing damage without the matchmaker function of the UvrA protein. Our results also indicate that the UvrA-mediated loading of the UvrB protein is an asymmetric process, which starts at the 5' side of the damage.

The role of ATP binding and hydrolysis by UvrB during nucleotide excision repair.

We have isolated UvrB-DNA complexes by capture of biotinylated damaged DNA substrates on streptavidin-coated magnetic beads. With this method the UvrB-DNA preincision complex remains stable even in the absence of ATP. For the binding of UvrC to the UvrB-DNA complex no cofactor is needed. The subsequent induction of 3' incision does require ATP binding by UvrB but not hydrolysis. This ATP binding induces a conformational change in the DNA, resulting in the appearance of the DNase I-hypersensitive site at the 5' side of the damage. In contrast, the 5' incision is not dependent on ATP binding because it occurs with the same efficiency with ADP. We show with competition experiments that both incision reactions are induced by the binding of the same UvrC molecule. A DNA substrate containing damage close to the 5' end of the damaged strand is specifically bound by UvrB in the absence of UvrA and ATP (Moolenaar, G. F., Monaco, V., van der Marel, G. A., van Boom, J. H., Visse, R., and Goosen, N. (2000) J. Biol. Chem. 275, 8038-8043). To initiate the formation of an active UvrBC-DNA incision complex, however, UvrB first needs to hydrolyze ATP, and subsequently a new ATP molecule must be bound. Implications of these findings for the mechanism of the UvrA-mediated formation of the UvrB-DNA preincision complex will be discussed.

Catalytic sites for 3' and 5' incision of Escherichia coli nucleotide excision repair are both located in UvrC.

Nucleotide excision repair in Escherichia coli is a multistep process in which DNA damage is removed by incision of the DNA on both sides of the damage, followed by removal of the oligonucleotide containing the lesion. The two incision reactions take place in a complex of damaged DNA with UvrB and UvrC. It has been shown (Lin, J. -J., and Sancar, A. (1992) J. Biol. Chem. 267, 17688-17692) that the catalytic site for incision on the 5' side of the damage is located in the UvrC protein. Here we show that the catalytic site for incision on the 3' side is in this protein as well, because substitution R42A abolishes 3' incision, whereas formation of the UvrBC-DNA complex and the 5' incision reaction are unaffected. Arg(42) is part of a region that is homologous to the catalytic domain of the homing endonuclease I-TevI. We propose that the UvrC protein consists of two functional parts, with the N-terminal half for the 3' incision reaction and the C-terminal half containing all the determinants for the 5' incision reaction.

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.

NMR assignments and secondary structure of the UvrC binding domain of UvrB.

The 55 residue C-terminal domain of UvrB that interacts with UvrC during excision repair in Escherichia coli has been expressed and purified as a (His)6 fusion construct. The fragment forms a stable folded domain in solution. Heteronuclear NMR experiments were used to obtain extensive 15N, 13C and 1H NMR assignments. NOESY and chemical shift data showed that the protein comprises two helices from residues 630 to 648 and from 652 to 670. 15N relaxation data also show that the first 11 and last three residues are unstructured. The effective rotational correlation time within the structured region is not consistent with a monomer. This oligomerisation may be relevant to the mode of dimerisation of UvrB with the homologous domain of UvrC.

Characterization of the Escherichia coli damage-independent UvrBC endonuclease activity.

Incision of damaged DNA templates by UvrBC in Escherichia coli depends on UvrA, which loads UvrB on the site of the damage. A 50-base pair 3' prenicked DNA substrate containing a cholesterol lesion is incised by UvrABC at two positions 5' to the lesion, the first incision at the eighth and the second at the 15th phosphodiester bond. Analysis of a 5' prenicked cholesterol substrate revealed that the second 5' incision is efficiently produced by UvrBC independent of UvrA. This UvrBC incision was also found on the same substrate without a lesion and, with an even higher efficiency, on a DNA substrate containing a 5' single strand overhang. Incision occurred in the presence of ATP or ADP but not in the absence of cofactor. We could show an interaction between UvrB and UvrC in solution and subsequent binding of this complex to the substrate with a 5' single strand overhang. Analysis of mutant UvrB and UvrC proteins revealed that the damage-independent nuclease activity requires the protein-protein interaction domains, which are exclusively needed for the 3' incision on damaged substrates. However, the UvrBC incision uses the catalytic site in UvrC which makes the 5' incision on damaged DNA substrates.

The C-terminal region of the Escherichia coli UvrC protein, which is homologous to the C-terminal region of the human ERCC1 protein, is involved in DNA binding and 5'-incision.

The incisions in the DNA at the 3'- and 5'-side of a DNA damage during nucleotide excision repair in Escherichia coli occur in a complex consisting of damaged DNA, UvrB and UvrC. The exact requirements for the two incision events, however, are different. It has previously been shown that the 3'-incision requires the interaction between the C-terminal domain of UvrB and a homologous region in UvrC. This interaction, however, is dispensable for the 5'-incision. Here we show that the C-terminal domain of the UvrC protein is essential for the 5'-incision, whereas this domain can be deleted without affecting the 3'-incision. The C-terminal domain of UvrC is homologous with the C-terminal part of the ERCC1 protein which, in a complex with XPF, is responsible for the 5'-incision reaction in human nucleotide excision repair. Both in the UvrC and the ERCC1 domain a Helix-hairpin-Helix (HhH) motif can be indicated, albeit at different positions. Such a motif also has been found in a large variety of DNA binding proteins and it has been suggested to form a structure involved in non-sequence-specific DNA binding. In contrast to the full length UvrC protein, a truncated UvrC protein (UvrC554) lacking the entire ERCC1 homology including the HhH motif no longer binds to ssDNA. Analysis of protein-DNA complexes using bandshift experiments showed that this putative DNA binding domain of UvrC is required for stabilisation of the UvrBC-DNA complex after the 3'-incision has taken place. We propose that after the initial 3'-incision the HhH motif recognises a specific DNA structure, thereby positioning the catalytic site for the subsequent 5'-incision reaction.

Function of the homologous regions of the Escherichia coli DNA excision repair proteins UvrB and UvrC in stabilization of the UvrBC-DNA complex and in 3'-incision.

The nicking of damaged DNA during the nucleotide excision repair reaction in E. coli, is the result of a multi-step process involving three enzymes, UvrA, UvrB and UvrC. The UvrB protein is loaded on the site of the damage by UvrA, forming a stable UvrB-DNA complex. This complex is recognized by UvrC and in the resulting UvrBC-DNA complex dual incision takes place, first on the 3'-side and next on the 5'-side of the damaged nucleotide. A domain in the C-terminal part of UvrB has been identified to be essential for formation of the specific UvrBC-DNA complex that induces the 3'-incision [1]. The N-terminal half of UvrC contains a region that is homologous to this C-terminal domain of UvrB. Using site-directed mutagenesis of a conserved phenylalanine in the homologous regions of UvrB and UvrC two mutants were constructed, UvrB(F652L) and UvrC(F223L). Both proteins were tested in vitro using a DNA substrate with a defined cisplatin lesion. The protein-DNA and protein-protein interactions were studied using bandshift assays and DNAse I footprinting. We show that both domains are important for the binding of UvrC to the UvrB-DNA complex.

The C-terminal region of the UvrB protein of Escherichia coli contains an important determinant for UvrC binding to the preincision complex but not the catalytic site for 3'-incision.

The UvrABC endonuclease from Escherichia coli repairs damage in the DNA by dual incision of the damaged strand on both sides of the lesion. The incisions are in an ordered fashion, first on the 3'-side and next on the 5'-side of the damage, and they are the result of binding of UvrC to the UvrB-DNA preincision complex. In this paper, we show that at least the C-terminal 24 amino acids of UvrB are involved in interaction with UvrC and that this binding is important for the 3'-incision. The C-terminal region of UvrB, which shows homology with a domain of the UvrC protein, is part of a region that is predicted to be able to form a coiled-coil. We therefore propose that UvrB and UvrC interact through the formation of such a structure. The C-terminal region of UvrB only interacts with UvrC when present in the preincision complex, indicating that the conformational change in UvrB accompanying the formation of this complex exposes the UvrC binding domain. Binding of UvrC to the C-terminal region of UvrB is not important for the 5'-incision, suggesting that for this incision a different interaction of UvrC with the UvrB-DNA complex is required. Truncated UvrB mutants that lack up to 99 amino acids from the C terminus still give rise to significant incision (1-2%), indicating that this C-terminal region of UvrB does not participate in the formation of the active site for 3'-incision. This region, however, contains the residue (Glu-640) that was proposed to be involved in 3'-catalysis, since a mutation of the residue (E640A) fails to promote 3'-incision (Lin, J.J., Phillips, A.M., Hearst, J.E., and Sancar, A. (1992) J. Biol. Chem. 267, 17693-17700). We have isolated a mutant UvrB with the same E640A substitution, but this protein shows normal UvrC binding and incision in vitro and also results in normal survival after UV irradiation in vivo. As a consequence of these results, it is still an open question as to whether the catalytic site for 3'-incision is located in UvrB, in UvrC, or is formed by both proteins.

Protein-DNA interactions and alterations in the DNA structure upon UvrB-DNA preincision complex formation during nucleotide excision repair in Escherichia coli.

The UvrB-DNA preincision complex is a key intermediate in the repair of damaged DNA by the UvrABC endonuclease from Escherichia coli. DNaseI footprinting of this complex on DNA with a cis-[Pt(NH3)2[d(GpG)-N7(1),N7(2)]] adduct provided global information on the protein binding site on this substrate [Visse, R., et al. (1991) J. Biol. Chem. 266, 7609-7617]. By applying a method developed by Fairall and Rhodes [Fairall, L., & Rhodes, D. (1992) Nucleic Acids Res. 20, 4727-4731], who have used the size and shape of DNasI for the interpretation of a footprint, we were able to define in more detail the region where UvrB-DNA interactions in the preincision complex occur. The potential interactions with phosphate groups could be reduced to less then 14 in the damaged and to 12 in the nondamaged strand. The main UvrB-DNA interactions seem restricted to the major groove on both sides of the lesion. As a consequence UvrB crosses the minor groove just downstream of the damage. Such a binding of UvrB orients the protein away from the damage. The more detailed interpretation of UvrB-DNA interactions was supported by methylation protection experiments. The structure of the DNA in the preincision complex formed on cis-[Pt(NH3)2[GpG-N7(1),N7(2)]] is altered as could be shown diethylpyrocarbonate sensitivity of adenines just downstream of the lesion. However the adenines just downstream of another cisplatin adduct, cis-[Pt(NH3)2[d(GpCpG)-N7(1),N7(3)]], did not become diethylpyrocarbonate sensitive in the preincision complex although this complex is incision proficient.(ABSTRACT TRUNCATED AT 250 WORDS)

Helicase motifs V and VI of the Escherichia coli UvrB protein of the UvrABC endonuclease are essential for the formation of the preincision complex.

The UvrB protein is a subunit of the UvrABC endonuclease which is involved in the repair of a large variety of DNA lesions. We have 91 isolated random uvrB mutants which are impaired in the repair of UV-damage in vivo. These mutants were classified on the basis of the ability to form normal levels of protein and the position of the mutations in the gene. The amino acid substitutions in the N-terminal part or in the C-terminal part of the UvrB protein are exclusively found in the conserved boxes of the so-called "helicase motifs" present in these parts of the protein, indicating that these motifs are essential for UvrB function. The proteins of four C-terminal mutants were purified: two mutants in motif V (E514K and G509S), one mutant in motif VI (R544H) and a double mutant in both motifs (E514K + R541H). In vitro experiments with these mutant proteins show that the helicase motifs V and VI are involved in the induction of ATP hydrolysis in the presence of (damaged) DNA and in the strand-displacement activity of the UvrA2B complex as is observed in a helicase assay. Furthermore, our results suggest that this strand-displacement activity is correlated to a local unwinding, which seems to be used to form the UvrB-DNA preincision complex.

The actual incision determines the efficiency of repair of cisplatin-damaged DNA by the Escherichia coli UvrABC endonuclease.

The UvrABC endonuclease from Escherichia coli repairs a broad spectrum of DNA lesions with variable efficiencies. The effectiveness of repair is influenced by the nature of the lesion, the local DNA sequence, and/or the topology of the DNA. To get a better understanding of the aspects of this multistep repair reaction that determine the effectiveness of repair, we compared the incision efficiencies of linear DNA fragments containing either a site-specific cis-[Pt(NH3)2(d(GpG)-N7(1),-N7(2)]] or a cis- Pt(NH3)2[d(GpCpG)-N7(1),-N7(3)]] adduct. Overall the DNA with the cis-PtGG adduct was incised about 3.5 times more efficiently than the cis-Pt.GCG-containing DNA. The rate of UvrB-DNA preincision complex formation for both lesions was similar and high in relation to the incision. DNase I footprints, however, showed that the local structure of the two preincision complexes is different. An assay was developed to measure the binding of UvrC to the preincision complexes and it was found that the binding rate of UvrC to the more slowly incised cis-Pt.GCG preincision complex was higher than to the cis-Pt.GG preincision complex. This most likely reflects a qualitative difference in preincision complex structures. For both lesions the binding of UvrC to the preincision complex was fast compared to the kinetics of actual incision. Apparently, direct incision of cisplatin damage requires an additional conformational change after the binding of UvrC.

Analysis of UvrABC endonuclease reaction intermediates on cisplatin-damaged DNA using mobility shift gel electrophoresis.

One of the least understood steps in the UvrABC mediated excision repair process is the recognition of lesions in the DNA. The isolation of different reaction intermediates is of vital importance for the unraveling of the mechanism. A mobility shift gel electrophoresis assay is described which visualizes such intermediates. After incubation of a DNA substrate containing a specific cisplatin adduct with UvrA alone or with UvrA and UvrB, UvrA.DNA, UvrAB.DNA and UvrB.DNA complexes were observed which could be identified using specific antibodies. At low UvrA concentrations in the presence of UvrB only the UvrB.DNA complex is observed. Bands corresponding to the UvrAB.DNA complex and also other nonspecific bands are found at relatively high UvrA concentrations. The DNase-I footprint for the UvrAB.- and UvrB.DNA complex are very similar and protect about 20 bases. Both complexes are incised in the presence of UvrC with comparable efficiency. The UvrAB.- and the UvrB.DNA complex were both incised at the 8th phosphodiester bond 5' to a specific cisplatin adduct. In addition the UvrAB.DNA complex could also be incised at the 15th phosphodiesterbond 5' to the damaged site. The results suggest that the UvrB.DNA complex is the natural substrate for UvrC-induced incision.

Regulation of the Escherichia coli excision repair gene uvrC. Overlap between the uvrC structural gene and the region coding for a 24 kD protein.

The UvrA, UvrB and UvrC proteins of E. coli are subunits of a DNA repair enzyme, the ABC exonuclease. In this paper we study the uvrC regulatory region. The uvrC structural gene is preceded by an open reading frame encoding a 24 kD protein. A uvrC promoter has been mapped within this gene. The transcription start of a second promoter located 5' of the 24 kD gene is mapped in vivo. We show that transcription from both promoters on the chromosome is not inducible by UV damage. The possible translation start codons of the UvrC and of the 24 kD protein are determined. Sequences encoding the N-terminal part of the UvrC protein overlap with sequences encoding the C-terminal part of the 24 kD protein. To examine a possible function of the 24 kD gene in repair, a 24 kD insertion mutant was created in the chromosome. The mutant however only slightly affects the UV sensitivity of the cell. Transcription of P3 alone provides sufficient UvrC protein for the normal repair of UV lesions.