Anomalous electrophoretic migration of short oligodeoxynucleotides labelled with 5′‐terminal <scp>C</scp>y5 dyes

Killelea, Tom; Saint-Pierre, Christine; Ralec, Céline; Gasparutto, Didier; Henneke, Ghislaine

doi:10.1002/elps.201400018

Cited by 15 publications

(12 citation statements)

References 58 publications

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“…Besides, higher migration products resulting from subsequent exonucleolytic degradation were also detectable (Figure 6B , lanes 3, 5, 9, 12, 14, 16). Although having an altered electrophoretic mobility running at a higher position than the 17-nt oligonucleotide marker these cleavage fragments mostly ranged from 1 to 8-nt in length as previously demonstrated ( 79 ).…”

Section: Resultssupporting

confidence: 67%

Calcium-driven DNA synthesis by a high-fidelity DNA polymerase

Ralec

Henry

Lemor

et al. 2017

Nucleic Acids Research

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Divalent metal ions, usually Mg2+, are required for both DNA synthesis and proofreading functions by DNA polymerases (DNA Pol). Although used as a non-reactive cofactor substitute for binding and crystallographic studies, Ca2+ supports DNA polymerization by only one DNA Pol, Dpo4. Here, we explore whether Ca2+-driven catalysis might apply to high-fidelity (HiFi) family B DNA Pols. The consequences of replacing Mg2+ by Ca2+ on base pairing at the polymerase active site as well as the editing of terminal nucleotides at the exonuclease active site of the archaeal Pyrococcus abyssi DNA Pol (PabPolB) are characterized and compared to other (families B, A, Y, X, D) DNA Pols. Based on primer extension assays, steady-state kinetics and ion-chased experiments, we demonstrate that Ca2+ (and other metal ions) activates DNA synthesis by PabPolB. While showing a slower rate of phosphodiester bond formation, nucleotide selectivity is improved over that of Mg2+. Further mechanistic studies show that the affinities for primer/template are higher in the presence of Ca2+ and reinforced by a correct incoming nucleotide. Conversely, no exonuclease degradation of the terminal nucleotides occurs with Ca2+. Evolutionary and mechanistic insights among DNA Pols are thus discussed.

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Section: Resultssupporting

confidence: 67%

Calcium-driven DNA synthesis by a high-fidelity DNA polymerase

Ralec

Henry

Lemor

et al. 2017

Nucleic Acids Research

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“…Cy5 confers an anomalous migration to the oligomers since, when the products are 6 or less nucleotides in length, they start migrating as products with a higher molecular weight, as previously demonstrated by Henneke et al . [ 39 ], This behavior explains the signal that can be observed in Fig 3B (as indicated by the arrow) which corresponds to a fragment of about 2 nucleotides. This activity might be due to either exo- or endonuclease activity, or both.…”

Section: Resultsmentioning

confidence: 57%

“…According to Henneke et al . [ 39 ] this product corresponds to a 4 nucleotides Cy5-labelled molecule since it migrates as a 21-mer substrate (indicated by the arrow in Fig 7C , lane 12). The experiments were also conducted on a double-stranded DNA, in which ODN2 was previously annealed to its complementary strand.…”

Section: Resultsmentioning

confidence: 99%

NurA Is Endowed with Endo- and Exonuclease Activities that Are Modulated by HerA: New Insight into Their Role in DNA-End Processing

et al. 2015

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The nuclease NurA and the ATPase HerA are present in all known thermophilic archaea and cooperate with the highly conserved MRE11/RAD50 proteins to facilitate efficient DNA double-strand break end processing during homologous recombinational repair. However, contradictory results have been reported on the exact activities and mutual dependence of these two enzymes. To understand the functional relationship between these two enzymes we deeply characterized Sulfolobus solfataricus NurA and HerA proteins. We found that NurA is endowed with exo- and endonuclease activities on various DNA substrates, including linear (single-stranded and double stranded) as well as circular molecules (single stranded and supercoiled double-stranded). All these activities are not strictly dependent on the presence of HerA, require divalent ions (preferably Mn2+), and are inhibited by the presence of ATP. The endo- and exonculease activities have distinct requirements: whereas the exonuclease activity on linear DNA fragments is stimulated by HerA and depends on the catalytic D58 residue, the endonuclease activity on circular double-stranded DNA is HerA-independent and is not affected by the D58A mutation. On the basis of our results we propose a mechanism of action of NurA/HerA complex during DNA end processing.

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“…Incubation of all three substrates with TtCsm6 resulted in the formation of anomalously migrating short oligonucleotide products, including the terminal dinucleotides (Supplemental Fig. 1; Killelea et al 2014). This suggests that the enzymatic activity of TtCsm6 is largely sequence-unspecific and does not appear to be selective for a specific substrate or product length.…”

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confidence: 95%

Structural basis for the endoribonuclease activity of the type III-A CRISPR-associated protein Csm6

Niewoehner

Jinek

2016

RNA

135

134

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Prokaryotic CRISPR-Cas systems provide an RNA-guided mechanism for genome defense against mobile genetic elements such as viruses and plasmids. In type III-A CRISPR-Cas systems, the RNA-guided multisubunit Csm effector complex targets both singlestranded RNAs and double-stranded DNAs. In addition to the Csm complex, efficient anti-plasmid immunity mediated by type III-A systems also requires the CRISPR-associated protein Csm6. Here we report the crystal structure of Csm6 from Thermus thermophilus and show that the protein is a ssRNA-specific endoribonuclease. The structure reveals a dimeric architecture generated by interactions involving the N-terminal CARF and C-terminal HEPN domains. HEPN domain dimerization leads to the formation of a composite ribonuclease active site. Consistently, mutations of invariant active site residues impair catalytic activity in vitro. We further show that the ribonuclease activity of Csm6 is conserved across orthologs, suggesting that it plays an important functional role in CRISPR-Cas systems. The dimer interface of the CARF domains features a conserved electropositive pocket that may function as a ligand-binding site for allosteric control of ribonuclease activity. Altogether, our work suggests that Csm6 proteins provide an auxiliary RNA-targeting interference mechanism in type III-A CRISPR-Cas systems that operates in conjunction with the RNA-and DNA-targeting endonuclease activities of the Csm effector complex.

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Anomalous electrophoretic migration of short oligodeoxynucleotides labelled with 5′‐terminal Cy5 dyes

Cited by 15 publications

References 58 publications

Calcium-driven DNA synthesis by a high-fidelity DNA polymerase

Calcium-driven DNA synthesis by a high-fidelity DNA polymerase

NurA Is Endowed with Endo- and Exonuclease Activities that Are Modulated by HerA: New Insight into Their Role in DNA-End Processing

Structural basis for the endoribonuclease activity of the type III-A CRISPR-associated protein Csm6

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