Interactions of Human O6-Alkylguanine-DNA Alkyltransferase (AGT) with Short Single-stranded DNAs

Rasimas, Joseph J.; Kar, S.; Pegg, Anthony E.; Fried, Mike

doi:10.1074/jbc.m608876200

Cited by 42 publications

(91 citation statements)

References 54 publications

(92 reference statements)

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“…Comparison of the rates for sliding with those for flipping suggests that the kinetic gate-keeping mechanism of lesion discrimination would be operative and would allow the protein to scan the sequence rapidly compared with one requiring full flipping. At higher concentrations and in physiological contexts, it is likely that the DNA will be crowded with proteins (18,19,21,22). Then, there will be more chance that undamaged bases enter the active site, but this possibility is not dangerous because the protein directly removes alkyl groups rather than excising bases.…”

Section: Discussionmentioning

confidence: 99%

“…Because of its medical relevance (9), AGT has been the focus of many biochemical (16)(17)(18)(19) and structural (17,(20)(21)(22) studies, making it an attractive system for computational investigation. For the calculations, we constructed a complex between the wild type (wt) and a DNA duplex containing mGua from the structure of the catalytically inactive C145S mutant bound to that ligand (21).…”

Section: Path Sampling Simulations Reveal a Two-mentioning

confidence: 99%

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A two-step nucleotide-flipping mechanism enables kinetic discrimination of DNA lesions by AGT

Dinner

2008

Proc. Natl. Acad. Sci. U.S.A.

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O 6 -alkylguanine-DNA alkyltransferase (AGT) repairs damage to the human genome by flipping guanine and thymine bases into its active site for irreversible transfer of alkyl lesions to Cys-145, but how the protein identifies its targets has remained unknown. Understanding molecular recognition in this system, which can serve as a paradigm for the many nucleotide-flipping proteins that regulate genes and repair DNA in all kingdoms of life, is particularly important given that inhibitors are in clinical trials as anticancer therapeutics. Computational approaches introduced recently for harvesting and statistically characterizing trajectories of molecularly rare events now enable us to elucidate a pathway for nucleotide flipping by AGT and the forces that promote it. In contrast to previously proposed flipping mechanisms, we observe a two-step process that promotes a kinetic, rather than a thermodynamic, gate-keeping strategy for lesion discrimination. Connection is made to recent single-molecule studies of DNA-repair proteins sliding on DNA to understand how they sense subtle chemical differences between bases efficiently. commitment probabilities ͉ DNA repair ͉ reaction coordinates ͉ transition path sampling

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Section: Discussionmentioning

confidence: 99%

Section: Path Sampling Simulations Reveal a Two-mentioning

confidence: 99%

A two-step nucleotide-flipping mechanism enables kinetic discrimination of DNA lesions by AGT

Dinner

2008

Proc. Natl. Acad. Sci. U.S.A.

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“…When such high sensitivity is not needed, variants or the assay using fluorescence, chemiluminescence and immunohistochemical detection are also available [13][14][15][16][17] . A wide range of nucleic acid sizes (lengths from short oligonucleotides to several thousand nt/bp 18,19 ) and structures (single-stranded, duplex, triplex 20 and quadruplex 21 nucleic acids as well as small circular DNAs 22 ) are compatible with the assay. Under favorable conditions, the distribution of proteins between several nucleic acid molecules can be monitored within a single solution 18,23 , as can the presence of complexes differing in protein stoichiometry and/or binding site distribution 7,24 .…”

Section: Advantages and Limitations Of Emsamentioning

confidence: 99%

Electrophoretic mobility shift assay (EMSA) for detecting protein–nucleic acid interactions

2007

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The gel electrophoresis mobility shift assay (EMSA) is used to detect protein complexes with nucleic acids. It is the core technology underlying a wide range of qualitative and quantitative analyses for the characterization of interacting systems. In the classical assay, solutions of protein and nucleic acid are combined and the resulting mixtures are subjected to electrophoresis under native conditions through polyacrylamide or agarose gel. After electrophoresis, the distribution of species containing nucleic acid is determined, usually by autoradiography of 32 P-labeled nucleic acid. In general, protein-nucleic acid complexes migrate more slowly than the corresponding free nucleic acid. In this article, we identify the most important factors that determine the stabilities and electrophoretic mobilities of complexes under assay conditions. A representative protocol is provided and commonly used variants are discussed. Expected outcomes are briefly described. References to extensions of the method and a troubleshooting guide are provided.

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“…Results from computational approaches have suggested a two-step base-flipping mechanism of hAGT, in which the existence of an extra-helical intermediate was postulated (Hu et al 2008). It has also been revealed that while searching along a duplex DNA, hAGT shows 5 0 to 3 0 preference and binds DNA cooperatively (Daniels et al 2004;Rasimas et al 2007;Adams et al 2009). Last, single-molecule spatial tracking measurements of C-Ada, the E. coli equivalence of hAGT, showed that sliding, with essentially no hopping, is the mechanism of C-Ada motion along stretched DNA (Lin et al 2009).…”

Section: Direct Reversal Of Alkylation Damage By Alkyltransferasesmentioning

confidence: 99%

DNA Repair by Reversal of DNA Damage

2013

Cold Spring Harbor Perspectives in Biology

137

115

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Endogenous and exogenous factors constantly challenge cellular DNA, generating cytotoxic and/or mutagenic DNA adducts. As a result, organisms have evolved different mechanisms to defend against the deleterious effects of DNA damage. Among these diverse repair pathways, direct DNA-repair systems provide cells with simple yet efficient solutions to reverse covalent DNA adducts. In this review, we focus on recent advances in the field of direct DNA repair, namely, photolyase-, alkyltransferase-, and dioxygenase-mediated repair processes. We present specific examples to describe new findings of known enzymes and appealing discoveries of new proteins. At the end of this article, we also briefly discuss the influence of direct DNA repair on other fields of biology and its implication on the discovery of new biology. E ndogenous and environmental agents continuously threaten the genomic integrity of all living organisms. Replication of damaged DNA can lead to mutations that are tumorigenic, whereas DNA lesions that block replication or transcription can result in senescence and cell death. Therefore, cellular DNA must be promptly repaired. Well-known mechanisms include base-excision repair, nucleotide excision repair, mismatch repair, homologous recombination, and nonhomologous end joining. In addition, nature has also evolved several mechanisms in which the damage is directly reversed most often by a single repair protein without the incision of DNA backbone. Although such "direct repair" processes mediate the reversal of a relatively small set of DNA lesions, the simplicity and essentially error-free property of the direct reversal processes make them particularly attractive for a cell. Three major mechanisms of direct DNA repair have been identified to date: (i) photolyases reverse UV light-induced photolesions; (ii) O 6 -alkylguanine-DNA alkyltransferases (AGTs) reverse a set of O-alkylated DNA damage; and (iii) the AlkB family dioxygenases reverse N-alkylated base adducts (Fig. 1). This concise article intends to update knowledge since the publication of the second edition of DNA Repair and Mutagenesis (ASM) (Friedberg et al. 2006).

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Interactions of Human O6-Alkylguanine-DNA Alkyltransferase (AGT) with Short Single-stranded DNAs

Cited by 42 publications

References 54 publications

A two-step nucleotide-flipping mechanism enables kinetic discrimination of DNA lesions by AGT

A two-step nucleotide-flipping mechanism enables kinetic discrimination of DNA lesions by AGT

Electrophoretic mobility shift assay (EMSA) for detecting protein–nucleic acid interactions

DNA Repair by Reversal of DNA Damage

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