The Actual Incision Determines the Efficiency of Repair of Cisplatin-Damaged DNA by the Escherichia coli UvrABC Endonuclease

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.Nucleotide excision repair in Escherichia coli is initiated by the binding of the UvrA 2 B complex to DNA containing damage. Following this, UvrB is loaded onto the site of the damage, and the UvrA protein is released. The resulting UvrB-DNA preincision complex is bound by the UvrC protein, leading to incision of the DNA at the 4 th or 5 th phosphodiester bond on the 3Ј side of the damage. This 3Ј incision is immediately followed by hydrolysis of the 8 th phosphodiester bond at the 5Ј side of the damage. The repair reaction is completed by the action of the UvrD, polymerase I, and ligase proteins, which replace the damaged oligo with a newly synthesized strand (for reviews see Refs. 1 and 2).ATP binding and hydrolysis play important roles throughout the repair reaction. The function of this cofactor is quite complex, which is illustrated by the presence of five ATP-binding sites in a single UvrA 2 B complex, two in each UvrA subunit and one in UvrB. The dimerization of UvrA is stimulated by ATP binding but not hydrolysis (3). The formation of an (active) UvrA 2 B complex in solution requires the hydrolysis of ATP by UvrA (4). The ATP hydrolysis by UvrA is also an important factor in discriminating between damaged and nondamaged DNA (5). In solution the UvrB protein on its own does not hydrolyze ATP, but as part of the UvrA 2 B complex it displays a DNA damage-dependent ATPase activity (6). This ATPase activity is associated with a limited DNA unwinding activity (7), which was shown to be important for loading the UvrB protein onto the site of the damage (8). Finally it has been shown that binding of ATP to the UvrBC-DNA complex is important for incision (9). In the latter experiments incision was monitored by the conversion of UV-irradiated supercoiled DNA substrate to the relaxed form, and therefore it could not be determined whether ATP binding is needed for 3Ј incision alone or for both incision reactions.In this paper we take a closer look at the function of ATP binding and hydrolysis in formation of the UvrB-DNA preincision complex and formation of the UvrBC-DNA incision complex and in the two incision reactions. For this purpose we have constructed biotinylated damaged DNA substrates, which are used to capture repair intermediates on streptavidin-coated magnetic beads. EXPERIMENTAL PROCEDURESProtein Purifications-The UvrA, UvrB, and UvrC proteins were purified as described (10). Mutant proteins UvrC (D466A) (11), UvrC(L221PϩF223L) (12), UvrB(G509S), and UvrB(R544H) (8) have been described and were purified according to the same procedure as the wild type proteins.Construction of DNA Substrates-The DNA substrates used in this study are schematically shown in Fig. 1. The cholesterol lesion was ...

Section: Discussionmentioning

confidence: 84%

“…Protein Purifications-The UvrA, UvrB, and UvrC proteins were purified as described (10). Mutant proteins UvrC (D466A) (11), UvrC(L221PϩF223L) (12), UvrB(G509S), and UvrB(R544H) (8) have been described and were purified according to the same procedure as the wild type proteins.…”

Section: Methodsmentioning

confidence: 99%

The Role of ATP Binding and Hydrolysis by UvrB during Nucleotide Excision Repair

Moolenaar

Herron

Monaco

et al. 2000

“…All these results suggest that the UvrAB recognition and UvrC incision steps might obey different binding criteria. The recognition would work for bent DNA structures as well as for any type of adducts, particularly with bulky hydrophobic groups, whereas incision would be more sensitive to the DNA sequence and the torsional constraint (39).…”

Section: Discussionmentioning

confidence: 99%

Binding of the Escherichia coli UvrAB Proteins to the DNA Mono- and Diadducts of cis-[N-2-Amino-N-2-methylamino-2,2,1-bicycloheptane]dichloroplatinum(II) and Cisplatin

Lambert

Jestin

Brehin

et al. 1995

The interactions of the Escherichia coli endonuclease UvrAB proteins with the DNA mono-and diadducts of both the cis-racemic exo-[N-2-amino-N-2-methylamino-2,2,1-bicycloheptane]dichloroplatinum(II) (complex 1) and cisplatin (cis-diamminedichloroplatinum(II) (cis-DDP)), have been studied. Complex 1 reacts faster with DNA than cis-DDP and gives monoadducts with a longer lifetime (8 h 20 min chelation t1 ⁄2 compared with 2 h 40 min for cis-DDP). Using pSP65 plasmid [ 3 H]DNA, the filter binding assay was associated with the analysis of the nucleoprotein complexes to characterize the UvrAB recognition of the platinum adducts and to demonstrate the occurrence of platinum-mediated DNA-protein cross-linking. First, it is shown that the UvrAB proteins recognize the complex 1 mono-and diadducts with a higher affinity than those of cis-DDP. Fifteen times more cis-DDP adducts per plasmid are required than complex 1 adducts, to lead to similar UvrAB binding. However, the UvrAB proteins recognize monoadducts and diadducts of each complex with a similar affinity. Second, it is shown that UvrB is the protein involved in the nucleoprotein complexes formed from mono-and diadducts of complex 1 and cis-DDP. This protein is also partly crosslinked to DNA with a similar efficiency by monoadducts derived from complex 1 and cis-DDP. However, as UvrB has a greater affinity for the DNA adducts of complex 1 than for those of cis-DDP, more UvrB-platinum-DNA cross-links are formed with complex 1 than with cis-DDP. This study, using a bacterial repair system as a model, points to a possible strategy for making new cytotoxic platinum complexes for mammalian cells. cis-Diamminedichloroplatinum(II) (cis-DDP)1 is one of the most widely used anticancer chemotherapeutic agents. It is thought that this compound exerts its cytotoxic effects through damage to DNA by activating a programmed cell death (1, 2). Several experimental arguments suggest that the cis-DDP biological properties might result from the specific recognition, by high affinity proteins, of the DNA structural motif induced by intrastrand chelation. Such proteins have been isolated and characterized (3-5). It was shown that HMG1 and several other cellular proteins like SSRP1 which contain the same sequence motif, called HMG box, were implied in such recognition. HMG1 or HMG2 proteins are implied in transcription processes (6, 7). This led the authors to suggest that intrastrand diadducts could induce a DNA structural motif which would mimic the natural binding site of such proteins. According to this hypothesis, cis-DDP activity would result from two nonmutually exclusive events due to the binding of HMG box containing proteins to the intrastrand diadducts: (i) the trapping of these proteins causing cell death by perturbating the transcription of critical genes, (ii) the inhibition of the binding of repair proteins preventing the excision of lesions. All these observations suggest that platinum derivatives able to form intrastrand diadducts should elicit pharmacological properties.Se...

“…UvrA, wild type UvrB, UvrBK45A, and SSB were overexpressed and purified as described (33,(53)(54)(55).…”

Section: Methodsmentioning

confidence: 99%

Stimulation of UvrD Helicase by UvrAB

Atkinson

Guy

Cadman

et al. 2009

Helicases play critical roles in all aspects of nucleic acid metabolism by catalyzing the remodeling of DNA and RNA structures. UvrD is an abundant helicase in Escherichia coli with well characterized functions in mismatch and nucleotide excision repair and a possible role in displacement of proteins such as RecA from single-stranded DNA. Helicases and translocases use the energy derived from NTP hydrolysis to translocate along single-stranded or doublestranded nucleic acids to remodel nucleic acid structures. Many of these enzymes have RecA-like motor domains, reflecting close similarities in translocation mechanisms (1-3). Specificity is often, therefore, conferred by additional domains within these motor enzymes (4). It is also becoming apparent that helicases and translocases often function as part of larger multisubunit complexes rather than in isolation and that such interactions impact on motor function and specificity. The complex interactions between replicative helicases and other proteins acting at the replication fork have long been known to be critical for replisome function (5-7). Many helicases also interact with, and their activities modulated by, ssDNA 2 -binding proteins (8 -13), while helicase/translocase interactions with RNA polymerases are emerging (14, 15).The Escherichia coli 3Ј-5Ј helicase UvrD (16) has roles in mismatch (17) and nucleotide excision repair (18) and may also act to displace proteins such as RecA at replication forks or ssDNA gaps in duplex DNA (19 -21). UvrD is likely the most abundant helicase in E. coli (22). There is also a second helicase in E. coli, Rep, that shares 40% identity with UvrD but has no known role in mismatch or nucleotide excision repair or in protein displacement in vivo. Employment of UvrD, but not Rep, in a diversity of roles in vivo might, therefore, demand specific physical or functional interactions between UvrD and other proteins in each system. However, specific interaction of UvrD has only been documented with a component of the mismatch repair system, MutL (23). This interaction appears to be essential in allowing a motor with limited dsDNA processivity to unwind the large tracts of DNA necessary during mismatch repair (23)(24)(25)(26).Little is known concerning the protein displacement function of UvrD in vivo. Lack of UvrD leads to a hyperrecombination phenotype (27), whereas UvrD can both promote (20,21) and inhibit (20) RecA-catalyzed strand exchange in vitro, depending on reaction conditions. UvrD might, therefore, promote turnover of RecA-ssDNA complexes at blocked forks and gaps in duplex DNA. Inhibition of RecA function at blocked forks might facilitate other pathways of fork repair that do not rely on strand exchange, possibly minimizing the risks to genome stability that blocked forks present (28). However, whether abortion of strand exchange by UvrD in vivo requires other proteins is unknown. In contrast, the role of UvrD in nucleotide excision repair is well characterized. Nucleotide excision repair is needed to remove and replace ...