Abstract:With the aim of investigating how motor proteins negotiate DNA nanostructures, we produced test circuits based on recombination intermediates in which 1D translocation across a Holliday junction (HJ) could be assessed by subsequent triplex displacement signals on each DNA arm. Using the EcoR124I restriction-modification enzyme, a 3′–5′ double-strand DNA (dsDNA) translocase, we could show that the motor will tend to follow its translocated strand across a junction. Nonetheless, as the frequency of junction bypa… Show more
“…Figure 5.Effect of QxxxY HsdR mutants on the rate of DNA cleavage by EcoR124I. Cleavage of pLKS5 (28) was monitored over a 1 h time course (Materials and methods section). The reaction profiles are shown for ( A ) wild-type HsdR, ( B ) Q179A HsdR, ( C ) Q179K HsdR, ( D ) Y183F HsdR and ( E ) Y183A HsdR.…”
The Type I restriction-modification enzyme EcoR124I is an ATP-dependent endonuclease that uses dsDNA translocation to locate and cleave distant non-specific DNA sites. Bioinformatic analysis of the HsdR subunits of EcoR124I and related Type I enzymes showed that in addition to the principal PD-(E/D)xK Motifs, I, II and III, a QxxxY motif is also present that is characteristic of RecB-family nucleases. The QxxxY motif resides immediately C-terminal to Motif III within a region of predicted α-helix. Using mutagenesis, we examined the role of the Q and Y residues in DNA binding, translocation and cleavage. Roles for the QxxxY motif in coordinating the catalytic residues or in stabilizing the nuclease domain on the DNA are discussed.
“…Figure 5.Effect of QxxxY HsdR mutants on the rate of DNA cleavage by EcoR124I. Cleavage of pLKS5 (28) was monitored over a 1 h time course (Materials and methods section). The reaction profiles are shown for ( A ) wild-type HsdR, ( B ) Q179A HsdR, ( C ) Q179K HsdR, ( D ) Y183F HsdR and ( E ) Y183A HsdR.…”
The Type I restriction-modification enzyme EcoR124I is an ATP-dependent endonuclease that uses dsDNA translocation to locate and cleave distant non-specific DNA sites. Bioinformatic analysis of the HsdR subunits of EcoR124I and related Type I enzymes showed that in addition to the principal PD-(E/D)xK Motifs, I, II and III, a QxxxY motif is also present that is characteristic of RecB-family nucleases. The QxxxY motif resides immediately C-terminal to Motif III within a region of predicted α-helix. Using mutagenesis, we examined the role of the Q and Y residues in DNA binding, translocation and cleavage. Roles for the QxxxY motif in coordinating the catalytic residues or in stabilizing the nuclease domain on the DNA are discussed.
“…QuikChange mutagenesis was also used to mutate an EcoRI site in pLKS5 [a plasmid containing four triplex binding sites (TBSs) (36)] to create plasmid pLKS5dE using the oligonuceotides: 5′-GCGAGTCGACGGCGGCCGC GAACTC CTCGAGCCCGGGTGACTGC-3′ and 5′-GCAGTCACCCGGGCTCGAG GAGTTC GCGGCCGCCGTCGACTCGC-3′. pLKS5dE was digested with AfeI and EcoRV.…”
To cleave DNA, the single polypeptide restriction–modification enzyme LlaGI must communicate between a pair of indirectly repeated recognition sites. We demonstrate that this communication occurs by a 1-dimensional route, namely unidirectional dsDNA loop translocation rightward of the specific recognition sequence 5′-CTnGAyG-3′ as written (where n is either A, G, C or T and y is either C or T). Motion across thousands of base pairs is catalysed by the helicase domain and requires the hydrolysis of 1.5-2 ATP per base pair. DNA loop extrusion is accompanied by changes in DNA twist consistent with the motor following the helical pitch of the polynucleotide track. LlaGI is therefore an example of a polypeptide that is a completely self-contained, multi-functional molecular machine.
“…No activity that promoted branch migration has been detected so far for Type I restriction enzymes (51). Interaction of branch migration and Type I restriction is an interesting subject of future research (see below).…”
Cleavage of a DNA replication fork leads to fork restoration by recombination repair. In prokaryote cells carrying restriction–modification systems, fork passage reduces genome methylation by the modification enzyme and exposes the chromosome to attack by the restriction enzyme. Various observations have suggested a relationship between the fork and Type I restriction enzymes, which cleave DNA at a distance from a recognition sequence. Here, we demonstrate that a Type I restriction enzyme preparation cleaves a model replication fork at its branch. The enzyme probably tracks along the DNA from an unmethylated recognition site on the daughter DNA and cuts the fork upon encountering the branch point. Our finding suggests that these restriction–modification systems contribute to genome maintenance through cell death and indicates that DNA replication fork cleavage represents a critical point in genome maintenance to choose between the restoration pathway and the destruction pathway.
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