scholarly journals Bulk and single-molecule analysis of a novel DNA2-like helicase-nuclease reveals a single-stranded DNA looping motor

2019 ◽  
Author(s):  
O.J. Wilkinson ◽  
C. Carrasco ◽  
C. Aicart-Ramos ◽  
F. Moreno-Herrero ◽  
M.S. Dillingham
Keyword(s):  
Single Molecule ◽  
Nuclease Activity ◽  
Biochemical Properties ◽  
Dna Helicase ◽  
Dna Looping ◽  
Dna Unwinding ◽  
Ssdna Binding ◽  
Dna Replication And Repair ◽  
Essential Enzyme ◽  
Single Molecule Analysis

ABSTRACTDNA2 is an essential enzyme involved in DNA replication and repair in eukaryotes. In a search for homologues of this protein, we identified and characterised Geobacillus stearothermophilus Bad, a novel bacterial DNA helicase-nuclease with similarity to human DNA2. We show that Bad contains an Fe-S cluster and identify four cysteine residues that are likely to co-ordinate the cluster by analogy to DNA2. The purified enzyme specifically recognises ss-dsDNA junctions and possesses ssDNA-dependent ATPase, ssDNA binding, ssDNA endonuclease, 5’ to 3’ ssDNA translocase and 5’ to 3’ helicase activity. Single molecule analysis reveals that Bad is a highly processive DNA motor capable of moving along DNA for distances of more than 4 kbp at a rate of ∼200 base pairs per second at room temperature. Interestingly, as reported for the homologous human and yeast DNA2 proteins, the DNA unwinding activity of Bad is cryptic and can be unmasked by inactivating the intrinsic nuclease activity. Strikingly, our experiments also show that the enzyme loops DNA while translocating, which is an emerging feature of highly processive DNA unwinding enzymes. The bacterial Bad enzymes will provide an excellent model system for understanding the biochemical properties of DNA2-like helicase-nucleases and DNA looping motor proteins in general.

scholarly journals Bulk and single-molecule analysis of a bacterial DNA2-like helicase–nuclease reveals a single-stranded DNA looping motor

2020 ◽  
Vol 48 (14) ◽  
pp. 7991-8005
Author(s):  
Oliver J Wilkinson ◽  
Carolina Carrasco ◽  
Clara Aicart-Ramos ◽  
Fernando Moreno-Herrero ◽  
Mark S Dillingham
Keyword(s):  
Single Molecule ◽  
Nuclease Activity ◽  
Biochemical Properties ◽  
Dna Helicase ◽  
Dna Looping ◽  
Dna Unwinding ◽  
Ssdna Binding ◽  
Dna Replication And Repair ◽  
Essential Enzyme ◽  
Single Molecule Analysis

Abstract DNA2 is an essential enzyme involved in DNA replication and repair in eukaryotes. In a search for homologues of this protein, we identified and characterised Geobacillus stearothermophilus Bad, a bacterial DNA helicase–nuclease with similarity to human DNA2. We show that Bad contains an Fe-S cluster and identify four cysteine residues that are likely to co-ordinate the cluster by analogy to DNA2. The purified enzyme specifically recognises ss-dsDNA junctions and possesses ssDNA-dependent ATPase, ssDNA binding, ssDNA endonuclease, 5′ to 3′ ssDNA translocase and 5′ to 3′ helicase activity. Single molecule analysis reveals that Bad is a processive DNA motor capable of moving along DNA for distances of >4 kb at a rate of ∼200 bp per second at room temperature. Interestingly, as reported for the homologous human and yeast DNA2 proteins, the DNA unwinding activity of Bad is cryptic and can be unmasked by inactivating the intrinsic nuclease activity. Strikingly, our experiments show that the enzyme loops DNA while translocating, which is an emerging feature of processive DNA unwinding enzymes. The bacterial Bad enzymes will provide an excellent model system for understanding the biochemical properties of DNA2-like helicase–nucleases and DNA looping motor proteins in general.

scholarly journals Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Cosimo Pinto ◽  
Kristina Kasaciunaite ◽  
Ralf Seidel ◽  
Petr Cejka
Keyword(s):  
Single Molecule ◽  
Nuclease Activity ◽  
Molecular Machine ◽  
Dna Unwinding ◽  
Metabolic Processes ◽  
Nuclease Domain

Human DNA2 (hDNA2) contains both a helicase and a nuclease domain within the same polypeptide. The nuclease of hDNA2 is involved in a variety of DNA metabolic processes. Little is known about the role of the hDNA2 helicase. Using bulk and single-molecule approaches, we show that hDNA2 is a processive helicase capable of unwinding kilobases of dsDNA in length. The nuclease activity prevents the engagement of the helicase by competing for the same substrate, hence prominent DNA unwinding by hDNA2 alone can only be observed using the nuclease-deficient variant. We show that the helicase of hDNA2 functionally integrates with BLM or WRN helicases to promote dsDNA degradation by forming a heterodimeric molecular machine. This collectively suggests that the hDNA2 motor promotes the enzyme's capacity to degrade dsDNA in conjunction with BLM or WRN and thus promote the repair of broken DNA.

scholarly journals Mechanisms of improved specificity of engineered Cas9s revealed by single molecule analysis

2017 ◽  
Author(s):  
Digvijay Singh ◽  
Yanbo Wang ◽  
John Mallon ◽  
Olivia Yang ◽  
Jingyi Fei ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Unwinding ◽  
Cleavage Reaction ◽  
Cleavage Rate ◽  
Guide Rna ◽  
Slowing Down ◽  
Rna Targets ◽  
Cleavage Reactions ◽  
Additional Base ◽  
Single Molecule Analysis

ABSTRACTIn microbes, CRISPR-Cas systems provide adaptive immunity against invading genetic elements. Cas9 in complex with a guide-RNA targets complementary DNA for cleavage and has been repurposed for wide-ranging biological applications. New Cas9s have been engineered (eCas9 and Cas9-HF1) to improve specificity, but how they help reduce off-target cleavage is not known. Here, we developed single molecule DNA unwinding assay to show that sequence mismatches affect cleavage reactions through rebalancing the internal unwinding/rewinding equilibrium. Increasing PAM-distal mismatches facilitate rewinding, and the associated cleavage impairment shows that cleavage proceeds from the unwound state. Engineered Cas9s depopulate the unwound state more readily upon mismatch detection. Intrinsic cleavage rate is much lower for engineered Cas9s, preventing cleavage from transiently unwound off-targets. DNA interrogation experiments showed that engineered Cas9s require about one additional base pair match for stable binding, freeing them from sites that would otherwise sequester them. Therefore, engineered Cas9s achieve their improved specificity (1) by inhibiting stable DNA binding to partially matching sequences, (2) by making DNA unwinding more sensitive to mismatches, and (3) by slowing down intrinsic cleavage reaction.

scholarly journals Human RPA activates BLM’s bidirectional DNA unwinding from a nick

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Zhenheng Qin ◽  
Lulu Bi ◽  
Xi-Miao Hou ◽  
Siqi Zhang ◽  
Xia Zhang ◽  
...  
Keyword(s):  
Dna Repair ◽  
Dna Replication ◽  
Single Molecule ◽  
Genome Stability ◽  
Protein A ◽  
Replication Protein A ◽  
Replication Protein ◽  
High Abundance ◽  
Dna Unwinding ◽  
Dna Replication And Repair

BLM is a multifunctional helicase that plays critical roles in maintaining genome stability. It processes distinct DNA substrates, but not nicked DNA, during many steps in DNA replication and repair. However, how BLM prepares itself for diverse functions remains elusive. Here, using a combined single-molecule approach, we find that a high abundance of BLMs can indeed unidirectionally unwind dsDNA from a nick when an external destabilizing force is applied. Strikingly, human replication protein A (hRPA) not only ensures that limited quantities of BLMs processively unwind nicked dsDNA under a reduced force but also permits the translocation of BLMs on both intact and nicked ssDNAs, resulting in a bidirectional unwinding mode. This activation necessitates BLM targeting on the nick and the presence of free hRPAs in solution whereas direct interactions between them are dispensable. Our findings present novel DNA unwinding activities of BLM that potentially facilitate its function switching in DNA repair.

scholarly journals Single molecule analysis of effects of non-canonical guide RNAs and specificity-enhancing mutations on Cas9-induced DNA unwinding

2019 ◽  
Author(s):  
Ikenna C Okafor ◽  
Digvijay Singh ◽  
Yanbo Wang ◽  
Minhee Jung ◽  
Haobo Wang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Genome Engineering ◽  
Dna Unwinding ◽  
Dependent Manner ◽  
Guide Rnas ◽  
Single Molecule Fret ◽  
Wide Range ◽  
The Cost ◽  
Single Molecule Analysis ◽  
Target Activity

Abstract Cas9 has made a wide range of genomic manipulation possible. However, its specificity continues to be a challenge. Non-canonical gRNAs and new engineered variants of Cas9 have been developed to improve specificity, but at the cost of the on-target activity. DNA unwinding is a checkpoint before cleavage by Cas9, and was shown to be made more sensitive to sequence mismatches by specificity-enhancing mutations in engineered Cas9s. Here we performed single-molecule FRET-based DNA unwinding experiments using various combinations of non-canonical gRNAs and different Cas9s. All engineered Cas9s were less promiscuous than wild type when canonical gRNA was used, but HypaCas9 had much-reduced on-target unwinding. Cas9-HF1 and eCas9 showed the best balance between low promiscuity and high on-target activity with canonical gRNA. When extended gRNAs with one or two non-matching guanines added to the 5′ end were used, Sniper1-Cas9 showed the lowest promiscuity while maintaining high on-target activity. Truncated gRNA generally reduced unwinding and adding a non-matching guanine to the 5′ end of gRNA influenced unwinding in a sequence-context dependent manner. Our results are consistent with cell-based cleavage data and provide a mechanistic understanding of how various Cas9/gRNA combinations perform in genome engineering.

scholarly journals Single-Molecule Analysis Reveals Differential Effect of ssDNA-Binding Proteins on DNA Translocation by XPD Helicase

2009 ◽  
Vol 35 (5) ◽  
pp. 694-703 ◽  
Author(s):  
Masayoshi Honda ◽  
Jeehae Park ◽  
Robert A. Pugh ◽  
Taekjip Ha ◽  
Maria Spies
Keyword(s):  
Single Molecule ◽  
Differential Effect ◽  
Binding Proteins ◽  
Dna Translocation ◽  
Ssdna Binding ◽  
Single Molecule Analysis ◽  
Ssdna Binding Proteins ◽  
Xpd Helicase

scholarly journals Differences in the Single-stranded DNA Binding Activities of MCM2-7 and MCM467

2007 ◽  
Vol 282 (46) ◽  
pp. 33795-33804 ◽  
Author(s):  
Matthew L. Bochman ◽  
Anthony Schwacha
Keyword(s):  
Dna Binding ◽  
Active Site ◽  
Biochemical Properties ◽  
Dna Helicase ◽  
Binding Activity ◽  
Helicase Activity ◽  
Single Stranded Dna ◽  
Ssdna Binding ◽  
Better Than

The MCM2-7 complex, a hexamer containing six distinct and essential subunits, is postulated to be the eukaryotic replicative DNA helicase. Although all six subunits function at the replication fork, only a specific subcomplex consisting of the MCM4, 6, and 7 subunits (MCM467) and not the MCM2-7 complex exhibits DNA helicase activity in vitro. To understand why MCM2-7 lacks helicase activity and to address the possible function of the MCM2, 3, and 5 subunits, we have compared the biochemical properties of the Saccharomyces cerevisiae MCM2-7 and MCM467 complexes. We demonstrate that both complexes are toroidal and possess a similar ATP-dependent single-stranded DNA (ssDNA) binding activity, indicating that the lack of helicase activity by MCM2-7 is not due to ineffective ssDNA binding. We identify two important differences between them. MCM467 binds dsDNA better than MCM2-7. In addition, we find that the rate of MCM2-7/ssDNA association is slow compared with MCM467; the association rate can be dramatically increased either by preincubation with ATP or by inclusion of mutations that ablate the MCM2/5 active site. We propose that the DNA binding differences between MCM2-7 and MCM467 correspond to a conformational change at the MCM2/5 active site with putative regulatory significance.

scholarly journals Single-Molecule Analysis Reveals the Kinetics and Physiological Relevance of MutL-ssDNA Binding

PLoS ONE ◽  
2010 ◽  
Vol 5 (11) ◽  
pp. e15496 ◽  
Author(s):  
Jonghyun Park ◽  
Yongmoon Jeon ◽  
Daekil In ◽  
Richard Fishel ◽  
Changill Ban ◽  
...  
Keyword(s):  
Single Molecule ◽  
Ssdna Binding ◽  
Physiological Relevance ◽  
Single Molecule Analysis

scholarly journals Single molecule analysis of effects of non-canonical guide RNAs and specificity-enhancing mutations on Cas9-induced DNA unwinding

2019 ◽  
Author(s):  
Ikenna C. Okafor ◽  
Digvijay Singh ◽  
Yanbo Wang ◽  
Minhee Jung ◽  
Haobo Wang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Genome Engineering ◽  
Dna Unwinding ◽  
Dependent Manner ◽  
Guide Rnas ◽  
Single Molecule Fret ◽  
Wide Range ◽  
The Cost ◽  
Single Molecule Analysis ◽  
Target Activity

ABSTRACTCas9 has made a wide range of genome engineering applications possible. However, its specificity continues to be a challenge. Non-canonical gRNAs and new engineered variants of Cas9 have been developed to improve specificity but at the cost of the on-target activity. DNA unwinding is the primary checkpoint before cleavage by Cas9 and was shown to be made more sensitive to sequence mismatches by specificity-enhancing mutations in Cas9. Here we performed single-molecule FRET-based DNA unwinding experiments using various combinations of non-canonical gRNAs and different Cas9s. All engineered Cas9s were less promiscuous than wild type when canonical gRNA was used but HypaCas9 had much-reduced on-target unwinding. Cas9-HF1 and eCas9 showed the best balance between low promiscuity and high on-target activity with canonical gRNA. When extended gRNAs with one or two guanines added were used, Sniper1-Cas9 showed the lowest promiscuity while maintaining high on-target activity. Truncated gRNA generally reduced unwinding and adding a non-matching guanine to the 5’ end of gRNA influenced unwinding in a sequence-context dependent manner. Our results are consistent with cell-based cleavage data and provide a mechanistic understanding of how various Cas9/gRNA combinations perform in genome engineering.

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