共查询到9条相似文献,搜索用时 15 毫秒
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Mismatch repair (MMR) is essential for eliminating biosynthetic errors generated during replication or genetic recombination in virtually all organisms. The critical first step in Escherichia coli MMR is the specific recognition and binding of MutS to a heteroduplex, containing either a mismatch or an insertion/deletion loop of up to four nucleotides. All known MutS homologs recognize a similar broad spectrum of substrates. Binding and hydrolysis of nucleotide cofactors by the MutS-heteroduplex complex are required for downstream MMR activity, although the exact role of the nucleotide cofactors is less clear. Here, we showed that MutS bound to a 30-bp heteroduplex containing an unpaired T with a binding affinity ≈ 400-fold stronger than to a 30-bp homoduplex, a much higher specificity than previously reported. The binding of nucleotide cofactors decreased both MutS specific and nonspecific binding affinity, with the latter marked by a larger drop, further increasing MutS specificity by ≈ 3-fold. Kinetic studies showed that the difference in MutS Kd for various heteroduplexes was attributable to the difference in intrinsic dissociation rate of a particular MutS-heteroduplex complex. Furthermore, the kinetic association event of MutS binding to heteroduplexes was marked by positive cooperativity. Our studies showed that the positive cooperativity in MutS binding was modulated by the binding of nucleotide cofactors. The binding of nucleotide cofactors transformed E. coli MutS tetramers, the functional unit in E. coli MMR, from a cooperative to a noncooperative binding form. Finally, we found that E. coli MutS bound to single-strand DNA with significant affinity, which could have important implication for strand discrimination in eukaryotic MMR mechanism. 相似文献
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Sharma S Gong P Temple B Bhattacharyya D Dokholyan NV Chaney SG 《Journal of molecular biology》2007,373(5):1123-1140
Mismatch repair proteins, DNA damage-recognition proteins and translesion DNA polymerases discriminate between Pt-GG adducts containing cis-diammine ligands (formed by cisplatin (CP) and carboplatin) and trans-RR-diaminocyclohexane ligands (formed by oxaliplatin (OX)) and this discrimination is thought to be important in determining differences in the efficacy, toxicity and mutagenicity of these platinum anticancer agents. We have postulated that these proteins recognize differences in conformation and/or conformational dynamics of the DNA containing the adducts. We have previously determined the NMR solution structure of OX-DNA, CP-DNA and undamaged duplex DNA in the 5'-d(CCTCAGGCCTCC)-3' sequence context and have shown the existence of several conformational differences in the vicinity of the Pt-GG adduct. Here we have used molecular dynamics simulations to explore differences in the conformational dynamics between OX-DNA, CP-DNA and undamaged DNA in the same sequence context. Twenty-five 10 ns unrestrained fully solvated molecular dynamics simulations were performed starting from two different DNA conformations using AMBER v8.0. All 25 simulations reached equilibrium within 4 ns, were independent of the starting structure and were in close agreement with previous crystal and NMR structures. Our data show that the cis-diammine (CP) ligand preferentially forms hydrogen bonds on the 5' side of the Pt-GG adduct, while the trans-RR-diaminocyclohexane (OX) ligand preferentially forms hydrogen bonds on the 3' side of the adduct. In addition, our data show that these differences in hydrogen bond formation are strongly correlated with differences in conformational dynamics, specifically the fraction of time spent in different DNA conformations in the vicinity of the adduct, for CP- and OX-DNA adducts. We postulate that differential recognition of CP- and OX-GG adducts by mismatch repair proteins, DNA damage-recognition proteins and DNA polymerases may be due, in part, to differences in the fraction of time that the adducts spend in a conformation favorable for protein binding. 相似文献
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Jarmila Mlcouskova Jaroslav Malina Vojtech Novohradsky Jana Kasparkova Seiji Komeda Viktor Brabec 《Biochimica et Biophysica Acta (BBA)/General Subjects》2012
Background
The design of anticancer metallodrugs is currently focused on platinum complexes which form on DNA major adducts that cannot readily be removed by DNA repair systems. Hence, antitumor azolato-bridged dinuclear PtII complexes, such as [{cis-Pt(NH3)2}2(μ‐OH)(μ-pyrazolate)]2+ (AMPZ), have been designed and synthesized. These complexes exhibit markedly higher toxic effects in tumor cell lines than mononuclear conventional cisplatin.Methods
Biophysical and biochemical aspects of the alterations induced in short DNA duplexes uniquely and site-specifically modified by the major DNA adduct of AMPZ, namely 1,2-GG intrastrand cross-links, were examined. Attention was also paid to conformational distortions induced in DNA by the adducts of AMPZ and cisplatin, associated alterations in the thermodynamic stability of the duplexes, and recognition of these adducts by high-mobility-group (HMG) domain proteins.Results
Chemical probing of DNA conformation, DNA bending studies and translesion synthesis by DNA polymerase across the platinum adduct revealed that the distortion induced in DNA by the major adduct of AMPZ was significantly less pronounced than that induced by similar cross-links from cisplatin. Concomitantly, the cross-link from AMPZ reduced the thermodynamic stability of the modified duplex considerably less. In addition, HMGB1 protein recognizes major DNA adducts of AMPZ markedly less than those of cisplatin.General significance
The experimental evidence demonstrates why the major DNA adducts of the new anticancer azolato-bridged dinuclear PtII complexes are poor substrates for DNA repair observed in a previously published report. The relative resistance to DNA repair explains why these platinum complexes show major pharmacological advantages over cisplatin in tumor cells. 相似文献5.
Ruchika Sharma 《Journal of molecular biology》2009,385(5):1375-6732
RecJ exonuclease plays crucial roles in several DNA repair and recombination pathways, and its ubiquity in bacterial species points to its ancient origin and vital cellular function. RecJ exonuclease from Haemophilus influenzae is a 575-amino-acid protein that harbors the characteristic motifs conserved among RecJ homologs. The purified protein exhibits a processive 5′-3′ single-stranded-DNA-specific exonuclease activity. The exonuclease activity of H. influenzae RecJ (HiRecJ) was supported by Mg2 + or Mn2 + and inhibited by Cd2 +, suggesting a different mode of metal binding in HiRecJ as compared to Escherichia coli RecJ (EcoRecJ). Site-directed mutagenesis of highly conserved residues in HiRecJ abolished enzymatic activity. Interestingly, substitution of alanine for aspartate 77 resulted in a catalytically inactive enzyme that bound to DNA with a significantly higher affinity as compared to the wild-type enzyme. Noticeably, steady-state kinetic studies showed that H. influenzae single-stranded DNA-binding protein (HiSSB) increased the affinity of HiRecJ for single-stranded DNA and stimulated its exonuclease activity. HiSSB, whose C-terminal tail had been deleted, failed to enhance RecJ exonuclease activity. More importantly, HiRecJ was found to directly associate with its cognate single-stranded DNA-binding protein (SSB), as demonstrated by various in vitro assays. Interaction studies carried out with the truncated variants of HiRecJ and HiSSB revealed that the two proteins interact via the C-terminus of SSB protein and the core-catalytic domain of RecJ. Taken together, these results emphasize direct interaction between RecJ and SSB, which confers functional cooperativity to these two proteins. In addition, these results implicate SSB as being involved in the recruitment of RecJ to DNA and provide insights into the interplay between these proteins in repair and recombination pathways. 相似文献
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