Reconstitution of the Very Short Patch Repair Pathway from Escherichia coli

Robertson, Adam B.; Matson, Steven W.

doi:10.1074/jbc.m112.384321

Cited by 16 publications

(15 citation statements)

References 58 publications

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“…(21): briefly, a series of Nt . BbvCI nicking sites were serially incorporated near the middle of the synthetic DNA sequence and after nicking by Nt .…”

Section: Methodsmentioning

confidence: 99%

Atomic force microscopy captures the initiation of methyl-directed DNA mismatch repair

2015

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In E. coli, errors in newly-replicated DNA, such as the incorporation of a mis-paired base or an accidental insertion or deletion of nucleotides, are corrected by a methyl-directed mismatch repair (MMR) pathway. While the enzymology of MMR has long been established, many fundamental aspects of its mechanisms remain elusive, such as the structures, compositions, and orientations of complexes of MutS, MutL, and MutHas they initiate repair. Using atomic force microscopy, we—for the first time—record the structures and locations of individual complexes of MutS, MutL and MutH bound to DNA molecules during the initial stages of mismatch repair. This technique reveals a number of striking and unexpected structures, such as the growth and disassembly of large multimeric complexes at mismatched sites, complexes of MutS and MutL anchoring latent MutH onto hemi-methylated d(GATC) sites or bound themselves at nicks in the DNA, and complexes directly bridging mismatched and hemimethylated d(GATC) sites by looping the DNA. The observations from these single-molecule studies provide new opportunities to resolve some of the long-standing controversies in the field and underscore the dynamic heterogeneity and versatility of MutSLH complexes in the repair process.

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“…(21): briefly, a series of Nt . BbvCI nicking sites were serially incorporated near the middle of the synthetic DNA sequence and after nicking by Nt .…”

Section: Methodsmentioning

confidence: 99%

Atomic force microscopy captures the initiation of methyl-directed DNA mismatch repair

2015

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“…MutL participates in VSP repair, so it was surprising that the rate of G:C > A:T mutations at Dcm sites was increased only threefold in the MutL − strain. However, MutL facilitates but is not required for the relatively inefficient VSP repair (51,52). Because G:C >A:T mutations were increased 100-fold overall in the MutL − strain, other sources of G: C > A:T mutations must occur at much higher frequencies than deamination of 5meC.…”

Section: Discussionmentioning

confidence: 99%

Rate and molecular spectrum of spontaneous mutations in the bacteriumEscherichia colias determined by whole-genome sequencing

Lee¹,

Popodi

Tang³

et al. 2012

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

638

131

859

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Knowledge of the rate and nature of spontaneous mutation is fundamental to understanding evolutionary and molecular processes. In this report, we analyze spontaneous mutations accumulated over thousands of generations by wild-type Escherichia coli and a derivative defective in mismatch repair (MMR), the primary pathway for correcting replication errors. The major conclusions are (i) the mutation rate of a wild-type E. coli strain is ∼1 × 10 −3 per genome per generation; (ii) mutations in the wild-type strain have the expected mutational bias for G:C > A:T mutations, but the bias changes to A:T > G:C mutations in the absence of MMR; (iii) during replication, A:T > G:C transitions preferentially occur with A templating the lagging strand and T templating the leading strand, whereas G:C > A:T transitions preferentially occur with C templating the lagging strand and G templating the leading strand; (iv) there is a strong bias for transition mutations to occur at 5′ApC3′/3′TpG5′ sites (where bases 5′A and 3′T are mutated) and, to a lesser extent, at 5′GpC3′/3′CpG5′ sites (where bases 5′G and 3′C are mutated); (v) although the rate of small (≤4 nt) insertions and deletions is high at repeat sequences, these events occur at only 1/10th the genomic rate of base-pair substitutions. MMR activity is genetically regulated, and bacteria isolated from nature often lack MMR capacity, suggesting that modulation of MMR can be adaptive. Thus, comparing results from the wild-type and MMR-defective strains may lead to a deeper understanding of factors that determine mutation rates and spectra, how these factors may differ among organisms, and how they may be shaped by environmental conditions. evolution | mutation accumulation | neutral mutation | mutational hotspots | indels M utations are the source of variation upon which natural selection acts; thus, a complete understanding of evolutionary processes must include an accurate assessment of mutation rates and of the molecular spectrum of mutational events. In addition, we need to know whether, and how, intrinsic and extrinsic factors influence mutational processes. This understanding must be founded on baseline parameters established by analyzing mutations that accumulate in a neutral fashion, unbiased by selective pressures. Much of our knowledge of spontaneous mutation is based on mutations that occur in nonessential reporter genes during short-term laboratory culture of microorganisms (1). An alternative approach is to compare presumably neutral mutations that have accumulated over evolutionary time periods in diverged species (2). Both methods have substantial uncertainties. The experimental approach may use reporter loci that are not representative of the whole genome and necessarily incorporates assumptions about the expression and neutrality of the mutant phenotypes. The historical approach relies on estimated divergence times and the absence of selective pressure on synonymous sequence changes. High-throughput whole-genome sequencing allows some of these limitations to be...

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“…By engineering two or more nick sites in close proximity to each other, a gap can be formed, and an oligonucleotide that spans the nick sites containing a modified base, lesion, or extra bases can be introduced to anneal this gap, which can then be ligated to leave DNA that has a specific lesion at a known location. This method has been optimized by the Hsieh and Matson labs (Geng et al, 2011; Robertson & Matson, 2012), and the description below is adapted from their methods.…”

Section: Methods To Study Dna Repair Complexes With Afmmentioning

confidence: 99%

“…Plasmid/PCR preparation: A plasmid containing four Nt.BBvCI nickase sites across a 31 base span (pUC19-VSR) (Robertson & Matson, 2012) is purified from E. coli using a miniprep kit. Plasmid DNA obtained from mini/midi preps is suitable for longer substrates.…”

Section: Methods To Study Dna Repair Complexes With Afmmentioning

confidence: 99%

Using Atomic Force Microscopy to Characterize the Conformational Properties of Proteins and Protein–DNA Complexes That Carry Out DNA Repair

LeBlanc

Wilkins

et al. 2017

Methods in Enzymology

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Atomic force microscopy (AFM) is a scanning probe technique that allows visualization of single biomolecules and complexes deposited on a surface with nanometer resolution. AFM is a powerful tool for characterizing protein–protein and protein–DNA interactions. It can be used to capture snapshots of protein–DNA solution dynamics, which in turn, enables the characterization of the conformational properties of transient protein–protein and protein–DNA interactions. With AFM, it is possible to determine the stoichiometries and binding affinities of protein–protein and protein–DNA associations, the specificity of proteins binding to specific sites on DNA, and the conformations of the complexes. We describe methods to prepare and deposit samples, including surface treatments for optimal depositions, and how to quantitatively analyze images. We also discuss a new electrostatic force imaging technique called DREEM, which allows the visualization of the path of DNA within proteins in protein–DNA complexes. Collectively, these methods facilitate the development of comprehensive models of DNA repair and provide a broader understanding of all protein–protein and protein–nucleic acid interactions. The structural details gleaned from analysis of AFM images coupled with biochemistry provide vital information toward establishing the structure–function relationships that govern DNA repair processes.

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Reconstitution of the Very Short Patch Repair Pathway from Escherichia coli

Cited by 16 publications

References 58 publications

Atomic force microscopy captures the initiation of methyl-directed DNA mismatch repair

Atomic force microscopy captures the initiation of methyl-directed DNA mismatch repair

Rate and molecular spectrum of spontaneous mutations in the bacteriumEscherichia colias determined by whole-genome sequencing

Using Atomic Force Microscopy to Characterize the Conformational Properties of Proteins and Protein–DNA Complexes That Carry Out DNA Repair

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