Sequence-specific and DNA structure-dependent interactions of Escherichia coli MutS and human p53 with DNA (original) (raw)
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Impact of alternative DNA structures on DNA damage, DNA repair, and genetic instability
DNA Repair, 2014
Repetitive genomic sequences can adopt a number of alternative DNA structures that differ from the canonical B-form duplex (i.e. non-B DNA). These non-B DNA-forming sequences have been shown to have many important biological functions related to DNA metabolic processes; for example, they may have regulatory roles in DNA transcription and replication. In addition to these regulatory functions, non-B DNA can stimulate genetic instability in the presence or absence of DNA damage, via replication-dependent and/or replication-independent pathways. This review focuses on the interactions of non-B DNA conformations with DNA repair proteins and how these interactions impact genetic instability.
Role of tumor suppressor p53 domains in selective binding to supercoiled DNA
Nucleic Acids Research, 2002
We showed previously that bacterially expressed full-length human wild-type p53b(1±393) binds selectively to supercoiled (sc)DNA in sc/linear DNA competition experiments, a process we termed supercoil-selective (SCS) binding. Using p53 deletion mutants and pBluescript scDNA (lacking the p53 recognition sequence) at native superhelix density we demonstrate here that the p53 C-terminal domain (amino acids 347±382) and a p53 oligomeric state are important for SCS binding. Monomeric p53(361±393) protein (lacking the p53 tetramerization domain, amino acids 325±356) did not exhibit SCS binding while both dimeric mutant p53(319± 393)L344A and fusion protein GCN4±p53(347±393) were effective in SCS binding. Supershifting of p53(320±393)±scDNA complexes with monoclonal antibodies revealed that the amino acid region 375±378, constituting the epitope of the Bp53-10.1 antibody, plays a role in binding of the p53(320±393) protein to scDNA. Using electron microscopy we observed p53±scDNA nucleoprotein ®laments produced by all the C-terminal proteins that displayed SCS binding in the gel electrophoresis experiments; no ®laments formed with the monomeric p53(361± 393) protein. We propose a model according to which two DNA duplexes are compacted into p53±scDNA ®laments and discuss a role for ®lament formation in recombination.
The formation of adjacent triplex-duplex domains in DNA
Nucleic Acids Research, 1999
The ability of single-stranded DNA oligomers to form adjacent triplex and duplex domains with two DNA structural motifs was examined. Helix-coil transition curves and a gel mobility shift assay were used to characterize the interaction of single-stranded oligomers 12-20 nt in length with a DNA hairpin and with a DNA duplex that has a dangling end. The 12 nt on the 5′-ends of the oligomers could form a triplex structure with the 12 bp stem of the hairpin or the duplex portion of the DNA with a dangling end. The 3′-ends of the 17-20 nt strands could form Watson-Crick pairs to the five base loop of the hairpin or the dangling end of the duplex. Complexes of the hairpin DNA with the singlestranded oligomers showed two step transitions consistent with unwinding of the triplex strand followed by hairpin denaturation. Melting curve and gel competition results indicated that the complex of the hairpin and the 12 nt oligomer was more stable than the complexes involving the extended single strands. In contrast, results indicated that the extended single-stranded oligomers formed Watson-Crick base pairs with the dangling end of the duplex DNA and enhanced the stability of the adjacent triplex region.
Interactions between branched DNAs and peptide inhibitors of DNA repair
Nucleic Acids Research, 2008
The RecG helicase of Escherichia coli unwinds both Holliday junction (HJ) and replication fork DNA substrates. Our lab previously identified and characterized peptides (WRWYCR and KWWCRW) that block the activity of RecG on these substrates. We determined that the peptides bind HJ DNA and prevent the binding of RecG. Herein, we present further evidence that the peptides are competitive inhibitors of RecG binding to its substrates. We have generated structural models of interactions between WRWYCR and a junction substrate. Using the fluorescent probe 2-aminopurine, we show that inhibitors interact with highest affinity with HJs (K d = 14 nM) and 4-to 9-fold more weakly with replication fork substrates. The fluorescence assay results agree with the structural model, and predict the molecular basis for interactions between HJ-trapping peptides and branched DNA molecules. Specifically, aromatic amino acids in the peptides stack with bases at the center of the DNA substrates. These interactions are stabilized by hydrogen bonds to the DNA and by intrapeptide interactions. These peptides inhibit several proteins involved in DNA repair in addition to RecG, have been useful as tools to dissect recombination, and possess antibiotic activity. Greater understanding of the peptides' mechanism of action will further increase their utility.
Bimolecular DNA Triplexes: Duplex Extensions Show Implications for H-Form DNA Stability
Biochemistry, 1997
H-form DNA has recently been shown to be biologically relevant by its involvement in the process of homologous recombination ) Genes DeV. 7, 1766-1778. A bimolecular DNA triple-stranded structure (triplex) is central to the formation of H-form DNA. Understanding the formation and factors governing the stability of such bimolecular triplexes is necessary to fully elucidate the structure/function relationship of H-form DNA. In this study, we extend known information on bimolecular triplexes by examining the effect of a variable CNC base triad (where N ) A, C, T, or G) on a 10 base triad triplex that mimics the triplex motif in H-form DNA. We also examine the effect that a duplex extension of four base pairs has on triplex stability and selectivity for the base N. Results from thermal denaturation experiments indicate that the fully complementary triplex is more stable than its duplex counterpart (∆T m ) 13°C) and is resistant to degradation by bovine spleen phosphodiesterase for at least 24 h at 10°C. A single-base mismatch in the purine strand of the triplex structure is destabilizing (∆T m ) ∼20°C), and all structures containing a mismatch were readily degraded by bovine spleen phosphodiesterase. An extension of four duplex base pairs onto the triplex structure affects the stability of the DNA complex and may have implications relevant to H-form DNA.
A bouquet of DNA structures: Emerging diversity
BB Reports, 2016
Structural polymorphism of DNA has constantly been evolving from the time of illustration of the double helical model of DNA by Watson and Crick. A variety of non-canonical DNA structures have constantly been documented across the globe. DNA attracted worldwide attention as a carrier of genetic information. In addition to the classical Watson-Crick duplex, DNA can actually adopt diverse structures during its active participation in cellular processes like replication, transcription, recombination and repair. Structures like hairpin, cruciform, triplex, G-triplex, quadruplex, i-motif and other alternative non-ca-nonical DNA structures have been studied at length and have also shown their in vivo occurrence. This review mainly focuses on non-canonical structures adopted by DNA oligonucleotides which have certain prerequisites for their formation in terms of sequence, its length, number and orientation of strands along with varied solution conditions. This conformational polymorphism of DNA might be the basis of different functional properties of a specific set of DNA sequences, further giving some insights for various extremely complicated biological phenomena. Many of these structures have already shown their linkages with diseases like cancer and genetic disorders, hence making them an extremely striking target for structure-specific drug designing and therapeutic applications.
Nucleic Acids Research, 2011
Heterocyclic diamidines are compounds with antiparasitic properties that target the minor groove of kinetoplast DNA. The mechanism of action of these compounds is unknown, but topological changes to DNA structures are likely to be involved. In this study, we have developed a polyacrylamide gel electrophoresis-based screening method to determine topological effects of heterocyclic diamidines on four minor groove target sequences: AAAAA, TTT AA, AAATT and ATATA. The AAAAA and AAATT sequences have the largest intrinsic bend, whereas the TTTAA and ATATA sequences are relatively straight. The changes caused by binding of the compounds are sequence dependent, but generally the topological effects on AAAAA and AA ATT are similar as are the effects on TTTAA and ATATA. A total of 13 compounds with a variety of structural differences were evaluated for topological changes to DNA. All compounds decrease the mobility of the ATATA sequence that is consistent with decreased minor groove width and bending of the relatively straight DNA into the minor groove. Similar, but generally smaller, effects are seen with TTTAA. The intrinsically bent AAAAA and AAATT sequences, which have more narrow minor grooves, have smaller mobility changes on binding that are consistent with increased or decreased bending depending on compound structure.
DNA binding and nucleotide flipping by the human DNA repair protein AGT
Nature Structural & Molecular Biology, 2004
DNA repair proteins carry out the essential function of protecting the genomic integrity of cells from both endogenous and exogenous DNA-damaging agents 1. Although in bacterial systems proteins such as photolyase can directly reverse thymine dimers in DNA to restore two adjacent thymine bases, the only known human proteins that directly reverse DNA damage are the AlkB enzymes 2-4 and AGT (or MGMT). AGT directly repairs O 6-alkylguanine lesions in DNA, which result from endogenous sources such as S-adenosylmethionine and environmental toxins. These lesions are mutagenic, causing GC-to-AT transition mutations, and cytotoxic, forming the basis of anticancer chemotherapies that involve DNA methylation or chloroethylation to cause apoptosis 5,6. Hence, increased AGT levels confer alkylation resistance in human tumors 7 and inhibitors of AGT, such as O 6-benzylguanine, are currently in clinical trials as anticancer therapeutics 8. AGT repairs O 6-alkylguanines by irreversibly transferring the alkyl lesion to an active site cysteine (Fig. 1a), and this unique, stoichiometric mechanism makes it particularly amenable to inhibition. Despite many years of intense effort on the structural biology of damage reversal proteins such as AGT, no structures have been published for any direct damage reversal proteins in complex with DNA. To address the major questions regarding how AGT finds, accesses and repairs O 6-alkylguanine lesions in DNA and to aid in inhibitor design, we determined two structures of AGT bound to different DNA substrates by X-ray crystallography (Fig. 1). The first complex, an inactive C145S mutant bound to an O 6-methylguanine-containing oligonucleotide (Fig. 1c), is a pretransfer complex of the predominant biological substrate. In the second complex, active AGT is covalently crosslinked to an oligonucleotide containing the substrate analog N 1 ,O 6-ethanoxanthosine (Fig. 1b,d), a mechanistic, crosslinking inhibitor that is analogous to DNA treated with the chloroethylating therapeutic agents 9. These structures and biochemical data reveal a novel protein-DNA architecture, provide a detailed structural basis for the reaction mechanism and suggest novel mechanisms by which AGT finds and accesses damaged guanines in the context of genomic DNA. RESULTS Overall structure of the complex Crystals of the O 6-methylguanine and ethanoxanthosine complexes diffract, respectively, to 3.2 Å and 3.3 Å (Table 1), and despite crystallizing in different space groups, have essentially identical structures, with a main chain r.m.s. deviation of 0.77 Å. Despite this moderate resolution, 2F oF c and composite omit electron density maps (Figs. 1c,d and 2) show the DNA and protein clearly and define the AGT-DNA binding architecture and DNA conformational change unambiguously. The C-terminal domain of the two-domain α/β-fold houses the cysteine nucleophile in a conserved active site PCHR sequence adjacent to the DNA-binding HTH motif. The N-terminal domain is composed of a three-stranded β-sheet separated from two
Repair and recombination induced by triple helix DNA
Frontiers in Bioscience, 2007
Introduction 3. Biological applications 3.1. Enhanced recombination 3.2. Targeted mutagenesis 4. TFO-induced repair 4.1. Endogenous triple helices as sources of genetic instability 4.2. Repair of complex psoralen-TFO lesions 5. Perspective 6. Acknowledgements 7. References