Specific Binding of Human MSH2·MSH6 Mismatch-Repair Protein Heterodimers to DNA Incorporating Thymine- or Uracil-containing UV Light Photoproducts Opposite Mismatched Bases

The DNA mismatch repair (MMR) system plays a critical role in sensitizing both prokaryotic and eukaryotic cells to the clinically potent anticancer drug cisplatin. It is thought to mediate cytotoxicity through recognition of cisplatin DNA lesions. This drug generates a range of lesions that may also give rise to compound lesions resulting from the misincorporation of a base during translesion synthesis. Using gel mobility shift competition assays and surface plasmon resonance, we have analyzed the interaction of Escherichia coli MutS protein with site-specifically modified DNA oligonucleotides containing each of the four cisplatin cross-links or a set of compound lesions. The major 1,2-d(GpG) cisplatin intrastrand cross-link was recognized with only a 1.5-fold specificity, whereas a 47-fold specificity was found with a natural G/T containing DNA substrate. The rate of association, k on, for binding to the 1,2-d(GpG) adduct was 3.1 ؋ 10 4 M ؊1 s ؊1 and the specificity of binding was essentially dependent on k off . DNA duplexes containing a single 1,2-d(ApG), 1,3-d(GpCpG) adduct, and an interstrand cross-link of cisplatin were not preferentially recognized. Among 12 DNA substrates, each containing a different cisplatin compound lesion derived from replicative misincorporation of one base opposite either of the 1,2-intrastrand adducts, 10 were specifically recognized including those that are more likely formed in vivo based on cisplatin mutation spectra. Moreover, among these lesions, two compound lesions formed when an adenine was misincorporated opposite a 1,2-d(GpG) adduct were not substrates for the MutYdependent mismatch repair pathway. The ability of MutS to sense differentially various platinated DNA substrates suggests that cisplatin compound lesions formed during misincorporation of a base opposite either adducted base of both 1,2-intrastrand cross-links are more plausible critical lesions for MMR-mediated cisplatin cytotoxicity.cis-Diamminedichloroplatinum(II) (cisplatin) 1 is among the most widely used anticancer chemotherapeutic agents in the treatment of many human tumors, particularly testicular and ovarian tumors (1). In the reaction between DNA and cisplatin, different types of bifunctional adducts are formed that are thought to be the key toxic lesions (2-4). Although these adducts are responsible for a variety of cellular responses including replication and/or transcription inhibition, there is not yet a clear understanding of the molecular mechanisms linking the formation of adducts and cisplatin-induced apoptosis (5, 6). Cisplatin reacts preferentially with the N-7 atoms of purine residues in DNA. The major adducts (90%) are 1,2-intrastrand cross-links at d(GpG) and d(ApG) sites and the minor adducts correspond to 1,3-intrastrand cross-links at d(GpNpG) (N being a nucleotide residue) and interstrand cross-links formed at d(GpC/GpC) sites (7-10). Once formed, cisplatin lesions such as the major 1,2-intrastrand cross-links can undergo replication bypass in cells treated with cisplatin (11,12). Be...

Section: Discussionmentioning

confidence: 41%

Section: Methodsmentioning

confidence: 99%

Binding Discrimination of MutS to a Set of Lesions and Compound Lesions (Base Damage and Mismatch) Reveals Its Potential Role as a Cisplatin-damaged DNA Sensing Protein

Fourrier

Brooks²,

Malinge³

2003

“…Purification of hMutS␣ and Analysis of Electrophoretic Mobility Shift-Human hMutS␣ protein heterodimers were prepared essentially as described (17). Briefly, the 30 -65% ammonium sulfate fraction from HeLaS3 nuclear extracts was passed through a single-stranded DNA cellulose column and fractions were subsequently eluted with 1 mM ATP, then chromatographed on a 1-ml Pharmacia HR 5/5 Mono Q column.…”

Section: Methodsmentioning

confidence: 99%

Mismatch Repair in Human Nuclear Extracts

Wang

Hays

2003

Self Cite

DNA mismatch repair (MMR) couples recognition of base mispairs by MSH2⅐MSH6 heterodimers to initiation, hundreds of nucleotides away, of nascent strand 3 -5 or 5 -3 excision through the mispair. Mismatchrecognition complexes have been hypothesized to move along DNA to excision-initiation signals, in eukaryotes, perhaps ends of nascent DNA, or to remain at mismatches and search through space for initiation signals. Subsequent MMR excision, whether simple processive digestion of the targeted strand or tracking of an excision complex, remains poorly understood. In human cell-free extracts, we analyzed correction of a mismatch in a 2.2-kilobase pair (kbp) circular plasmid containing a pre-existing excision-initiation nick for initiation, and measured MMR excision (in the absence of exogenous dNTPs) at specific locations. Excision specificities were ϳ100:1 for nicked versus continuous strands, 80:1 for mismatched versus homoduplex DNA, and 30:1 for shorter (0.3-kbp) versus longer (1.9-kbp) nick-mispair paths. To test models for recognition-excision coupling and excision progress, we inserted potential blockades, 20-bp hairpins, into nick-mispair paths, using a novel technique to first generate gapped plasmid. Continuous strand longer-path hairpins did not affect mismatch correction, but shorter-path hairpins reduced correction 4-fold, and both together eliminated it. Shorter-path hairpins had little effect on initiation of (3 -5 ) excision, measured 30 -60 nucleotides 5 to the nick, but blocked subsequent progress of excision to the mismatch; longer-path hairpins blocked the (lower level) 5 -3 excision to the mismatch. Thus, (a) MMR excision protein(s) cannot move past DNA hairpins. Hairpins at both ends of substrate-derived 0.5-kbp DNA fragments did not prevent ATP-induced dissociation of mismatch-bound human MSH2⅐MSH6, so recognition complexes at mismatches might provoke excision at nicks beyond hairpins, or loosely sliding MSH2⅐MSH6 dimers might move to the nicks.Evolutionarily conserved prokaryotic and eukaryotic mismatch-repair (MMR) 1 systems promote genomic stability by correcting DNA replication errors, antagonizing homeologous recombination between diverged DNA sequences, and responding to a variety of DNA lesions (for reviews, see Refs. 1-3). The Escherichia coli pathway, fully reconstituted from purified proteins, provides a mechanistic paradigm. Here, MutS homodimers bind to DNA mismatches and MutH proteins recognize hemi-methylated d(GATC) sequences, transitory consequences of a brief post-replication delay in GATC-adenine methylation of nascent DNA, to thus provide the two requisite specificity elements for correction. Interaction of MutL homodimers with MutS-mismatch complexes activates DNAstrand nicking of nascent DNA at unmethylated d(GATC) sites. DNA helicase II (UvrD protein) loads at the nick if and only if MutS, a DNA mismatch, and MutL are present; one or more steps in this chain of events require ATP hydrolysis. UvrD presumably separates strands to facilitate excision of the nicked strand towar...

“…23) showing that some polymerases contact the DNA for five base pairs upstream of the polymerase active site, such that the presence of an embedded mismatch could still promote excision over polymerization. It is also possible that errors that escape proofreading could be corrected by mismatch repair (29,30), thereby further contributing to the fidelity of the overall bypass process. Finally, our data indicating that pol can compete for 3Ј-OH termini during replication by leading and lagging strand replication proteins suggest that genome instability could result from conditions that promote this competition, such as a change in the ratio of pol relative to other polymerases.…”

Section: Table IIImentioning

confidence: 99%

Proofreading of DNA Polymerase η-dependent Replication Errors

Bębenek¹,

Matsuda²,

Masutani³

et al. 2001

Human DNA polymerase , the product of the skin cancer susceptibility gene XPV, bypasses UV photoproducts in template DNA that block synthesis by other DNA polymerases. Pol lacks an intrinsic proofreading exonuclease and copies DNA with low fidelity, such that pol errors could contribute to mutagenesis unless they are corrected. Here we provide evidence that pol can compete with other human polymerases during replication of duplex DNA, and in so doing it lowers replication fidelity. However, we show that pol has low processivity and extends mismatched primer termini less efficiently than matched termini. These properties could provide an opportunity for extrinsic exonuclease(s) to proofread pol -induced replication errors. When we tested this hypothesis during replication in human cell extracts, pol -induced replication infidelity was found to be modulated by changing the dNTP concentration and to be enhanced by adding dGMP to a replication reaction. Both effects are classical hallmarks of exonucleolytic proofreading. Thus, pol is ideally suited for its role in reducing UV-induced mutagenesis and skin cancer risk, in that its relaxed base selectivity may facilitate efficient bypass of UV photoproducts, while subsequent proofreading by extrinsic exonuclease(s) may reduce its mutagenic potential.The UmuC/DinB superfamily of DNA polymerases includes human DNA polymerase (pol ), 1 the product of the XPV (Rad30A) skin cancer susceptibility gene (1, 2). Human pol has the ability to efficiently copy cis-syn thymine-thymine dimers in template DNA (1). Mutations in XPV that inactivate pol (1, 2) render cells hypermutable by UV radiation (3-7) and defective in replicating DNA containing UV photoproducts (Ref. 8 and references therein). These facts demonstrate an important role for pol in modulating UV-induced mutagenesis and in reducing the risk of human skin cancer. Kinetic analysis reveals that human pol inserts incorrect nucleotides opposite undamaged (9, 10) and damaged (10) template bases more efficiently than most other DNA polymerases. Moreover, human pol lacks an intrinsic proofreading exonuclease activity (9), and its base substitution error rates when copying undamaged DNA are much higher than are those of most other eukaryotic polymerases, whether they have proofreading activity or not (9). We (9) and others (10) have suggested that this generally relaxed discrimination ability during DNA synthesis may be critical to the ability of human pol to bypass certain DNA lesions that impede synthesis by other DNA polymerases (1,8,11,12).Current models suggest that pol competes with other replicative polymerases for 3Ј-OH termini at a replication fork (reviewed in Refs. 13-15). Given the intrinsically low fidelity of pol , mechanisms may exist to prevent errors by pol from reducing the accuracy of chromosomal replication. We previously suggested (9) two obvious error correction mechanisms, exonucleolytic proofreading of pol mistakes by a separate exonuclease(s) and post-replication DNA repair of mismatches generated by p...