Single-molecule analysis reveals three phases of DNA degradation by an exonuclease

Lee, Gwangrog; Yoo, Jungmin; Leslie, Benjamin J.; Ha, Taekjip

doi:10.1038/nchembio.561

Cited by 50 publications

(65 citation statements)

References 49 publications

(49 reference statements)

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“…47 The distributive phase was found to last for about the first ∼20 bp of the DNA, after which the processive phase ensued. The transition is likely to occur when the 3′-overhang of the DNA substrate is long enough for the λexo trimer to remain stably tethered.…”

Section: ■ Discussionmentioning

confidence: 99%

Mutant Poisoning Demonstrates a Nonsequential Mechanism for Digestion of Double-Stranded DNA by λ Exonuclease Trimers

et al. 2015

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λ Exonuclease (λexo) is a highly processive 5'-3' exonuclease that binds double-stranded DNA (dsDNA) ends and digests the 5'-strand into mononucleotides. The enzyme forms a toroidal homotrimer with a central tapered channel for tracking along the DNA. During catalysis, dsDNA enters the open end of the channel, and the 5'-strand is digested at one of the three active sites. It is currently not known if λexo uses a sequential mechanism, in which the DNA moves from one active site to the next around the trimer for each round of catalysis or a nonsequential mechanism, in which the DNA locks onto a single active site for multiple rounds. To understand how λexo uses its three active sites, we used a mutant poisoning approach, in which a 6xHis-tagged K131A inactive mutant of λexo was mixed with untagged wild type (WT) to form hybrid trimers. Nickel-spin pull-down analysis confirmed complete subunit exchange after 1 h at 37 °C. Exonuclease assays revealed an approximately linear decrease in activity with increasing fraction of mutant, as expected for a nonsequential mechanism. By fitting the observed rates of digestion to a simple mathematical model, the individual rates of the two hybrid species of trimer were determined. This analysis showed that trimers containing only one or two WT subunits contribute significantly to the observed activity, in further agreement with a nonsequential mechanism. Finally, purification of hybrid trimer mixtures by Ni-spin chromatography, to remove the contribution from fully WT trimers, also resulted in significant levels of activity, again consistent with a nonsequential mechanism.

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Section: ■ Discussionmentioning

confidence: 99%

Mutant Poisoning Demonstrates a Nonsequential Mechanism for Digestion of Double-Stranded DNA by λ Exonuclease Trimers

et al. 2015

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“…To exclude the possibility that repetitive unwinding results from the specific substrate geometry or the positions of the fluorophores, we also tested a different forked DNA substrate where the FRET value would increase rather than decrease as unwinding progresses (Fig. S2), displaying similar heterogeneous unwinding patterns (27,28).…”

Section: Resultsmentioning

confidence: 99%

Dynamic look at DNA unwinding by a replicative helicase

Lee

Syed

Enemark

et al. 2014

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

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A prerequisite for DNA replication is the unwinding of duplex DNA catalyzed by a replicative hexameric helicase. Despite a growing body of research, key elements of helicase mechanism remain under substantial debate. In particular, the number of DNA strands encircled by the helicase ring during unwinding and the ring orientation at the replication fork completely contrast in contemporary mechanistic models. Here we use single-molecule and ensemble assays to address these questions for the papillomavirus E1 helicase. We find that E1 unwinds DNA with a strand-exclusion mechanism, with the N-terminal side of the helicase ring facing the replication fork. We show that E1 generates strikingly heterogeneous unwinding patterns stemming from varying degrees of repetitive movements, which is modulated by the DNA-binding domain. Together, our studies reveal previously unrecognized dynamic facets of replicative helicase unwinding mechanisms.ATPase | molecular motors D NA replication is the most fundamental of all of life's processes. One of the key requisites in the initiation of replication is the separation of the two strands of the double helix, which is carried out by a hexameric helicase. Despite their prominent roles in biology, some of the basic aspects of these helicases, whether they use a strand-exclusion mechanism or whether they translocate along double-stranded DNA, for example, have been subjects of considerable debate (1, 2). Viral replicative helicases, such as SV40 Large-T antigen (LTag) and papillomavirus E1, have provided the opportunity to study some of these basic features largely owing to their homohexameric architecture. These viral helicases recognize their respective origin of DNA replication (ori) through their dsDNA-binding domains (DBDs) and assemble in a stepwise fashion, ultimately forming double-hexameric (DH) structures on their ori and unwind the DNA bidirectionally.E1 consists of an N-terminal domain, a DBD, an oligomerization domain (OD), a helicase/ATPase domain (HD), and a C-terminal acidic tail (Fig. 1A). Biochemical and structural data have demonstrated that the DBDs bound to the pseudopalindromic E1 binding site are at the center of the double hexamer and that the helicase domains that bind to the flanks of the ori are on either end in a head-to-head arrangement (3, 4). This arrangement is supported by EM studies of LTag that show a dumbbell-shaped structure for the DH (5, 6), with each half of the dumbbell containing two lobes: a larger HD outer lobe and a smaller DBD inner lobe. The assembled DH appears to place the HD of E1 proximal to the dsDNA to be unwound.However, in the structure of the E1 helicase with ssDNA and Mg 2+ -ADP, the ssDNA is oriented such that the N-terminal part of the polypeptide (the OD) is closest to the 5′ end of the DNA (7). The 3′→5′ polarity of E1 helicase indicates that the translocating helicase moves with the N-terminal OD leading and the C-terminal helicase domain trailing, or alternatively, that the DNA is pumped through the helicase from the OD side...

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“…FRET occurs when two molecules are located sufficiently close to each other, generally less than 10 nanometers. FRET has been applied to study protein-protein interactions, DNA-protein interactions, fluorescently tagged DNA oligomers in conjunction with real-time PCR and single molecule analysis and even RNA-FISH to study alternative splicing variants (Kenworthy, 2001; Robertson et al, 2006; Mukhopadhyay et al, 2007; Matsumoto et al, 2009; Blanco and Artero, 2010; Grecco and Verveer, 2011; Lee et al, 2011). However, prior to this study, this technique has not been used to study DNA-DNA interaction in combination with conventional FISH.…”

Section: Discussionmentioning

confidence: 99%

“…Low levels of FRET (mean Eapp – 5%-15%) have been reported before (Fjorback et al, 2009). In fact, findings have been concluded based on fluctuations between low and high FRET efficiencies (Mukhopadhyay et al, 2007; Lee et al, 2011). Another explanation for observing low levels of FRET efficiencies could be the fact that we used the most conservative method of calculating Eapp values (van Rheenen et al, 2004).…”

Section: Discussionmentioning

confidence: 99%

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Frequency of close positioning of chromosomal loci detected by FRET correlates with their participation in carcinogenic rearrangements in human cells

Gandhi

Evdokimova

Nikiforov

2012

Genes Chromosomes & Cancer

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It has been well established that genes participating in oncogenic rearrangements are non-randomly positioned and frequently close to each other in human cell nuclei. However the actual distance between these fusion partners has never been determined. The phenomenon of Fluorescence Resonance Energy Transfer (FRET) is observed when a donor fluorophore is close (<10 nm) to transfer some of it energy to an acceptor fluorophore. The aim of this study was to validate the use of FRET on directly labeled DNA molecules to assess the frequency of positioning at <10 nm distances between genes known to be involved in rearrangement and to correlate it with their probability to undergo rearrangement. In the validation experiments, the frequency of FRET-SE (sensitized emission) was found to be 93-96% between probes for the immediately adjacent chromosomal regions as compared to 0.1-0.2% between probes for the random loci located on large linear separation. Further, we found that the frequency of FRET-SE between four pairs of genes that form rearrangements in thyroid cancer was 5% for RET and CCDC6, 4% for RET and NCOA4, 2% for BRAF and AKAP9, and 2% for NTRK1 and TPR. Moreover, the frequency with which FRET was observed showed strong correlation (r = 0.9871) with the prevalence of respective rearrangements in thyroid cancer. Our findings demonstrate that FRET can be used as a technique to analyze proximity between specific DNA regions and that the frequency of gene positioning at distances allowing FRET correlates with their probability to undergo chromosomal rearrangements.

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Single-molecule analysis reveals three phases of DNA degradation by an exonuclease

Cited by 50 publications

References 49 publications

Mutant Poisoning Demonstrates a Nonsequential Mechanism for Digestion of Double-Stranded DNA by λ Exonuclease Trimers

Mutant Poisoning Demonstrates a Nonsequential Mechanism for Digestion of Double-Stranded DNA by λ Exonuclease Trimers

Dynamic look at DNA unwinding by a replicative helicase

Frequency of close positioning of chromosomal loci detected by FRET correlates with their participation in carcinogenic rearrangements in human cells

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