Restriction of single-stranded M13 DNA using synthetic oligonucleotides: the structural requirement of restriction enzymes

1987 ◽  
Vol 65 (1) ◽  
pp. 50-55
Author(s):  
Guo-Rong Qi ◽  
Pierre Wong ◽  
Robert Cedergren
Keyword(s):  
Dna Cleavage ◽  
Restriction Enzymes ◽  
Recognition Site ◽  
Duplex Dna ◽  
Structural Requirement ◽  
Single Stranded Dna ◽  
Dna Manipulation ◽  
Synthetic Oligonucleotides

A targeted ss (single stranded) DNA cleavage technique is reported which involves the use of synthetic oligomers complementary to the ss M13 DNA polylinker. BamHI, SmaI, and KpnI restriction enzymes were tested with a partial duplex DNA formed from ss M13 DNA and a nested series of fragments derived from a synthetic 21 -mer which were complementary to the polylinker region. These enzymes require up to two flanking nucleotides in addition to the hexameric recognition site for efficient cleavage. This technique could be useful for effecting unique cleavages of DNA with enzymes which generally give a large number of fragments and for strategies of ss DNA manipulation.

Hopping, jumping and looping by restriction enzymes

2001 ◽  
Vol 29 (4) ◽  
pp. 363-373 ◽  
Author(s):  
S. E. Halford
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Dna Cleavage ◽  
Restriction Enzymes ◽  
Test System ◽  
Recognition Site ◽  
Dna Looping ◽  
Type Ii ◽  
Common View ◽  
Recognition Sites

Type II restriction endonucleases recognize specific DNA sequences and cleave both strands of the DNA at fixed locations at or near their recognition sites. Many of these enzymes are dimeric proteins that recognize, in symmetrical fashion, palindromic DNA sequences. They generally catalyse independent reactions at each recognition site on the DNA, although in some cases they act processively; cutting the DNA first at one site, then translocating along the DNA to another site and cutting that before leaving the DNA. The way in which the degree of processivity varies with the length of DNA between the sites can reveal the mechanism of translocation. In contrast with the common view that proteins move along DNA by ‘sliding’, the principal mode of transfer of the EcoRV endonuclease is by ‘hopping’ and ‘jumping’, i.e. the dissociation of the protein from one site followed by its re-association with another site in the same DNA molecule, either close to or distant from the original site. Other type II restriction enzymes require two copies of their recognition sites for their DNA cleavage reactions. Many of these enzymes, such as SfiI, are tetramers with two DNA-binding surfaces. SfiI has no activity when bound to just one recognition site, and instead both DNA-binding surfaces have to be filled before it becomes active. Although the two sites can be on separate DNA molecules, SfiI acts optimally with two sites on the same DNA, where it traps the DNA between the sites in a loop. SfiI thus constitutes a test system for the analysis of DNA looping.

trans Single‐Stranded DNA Cleavage via CRISPR/Cas14a1 Activated by Target RNA without Destruction

2021 ◽  
Author(s):  
Yangdao Wei ◽  
Zhiqing Yang ◽  
Chengli Zong ◽  
Buhua Wang ◽  
Xiaolin Ge ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Single Stranded Dna ◽  
Target Rna

3‘-5‘ Exonuclease of Klenow Fragment:  Role of Amino Acid Residues within the Single-Stranded DNA Binding Region in Exonucleolysis and Duplex DNA Melting†

Biochemistry ◽  
2002 ◽  
Vol 41 (12) ◽  
pp. 3943-3951 ◽  
Author(s):  
Wai-Chung Lam ◽  
Elizabeth H. Z. Thompson ◽  
Olga Potapova ◽  
Xiaojun Chen Sun ◽  
Catherine M. Joyce ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Dna Melting ◽  
Duplex Dna ◽  
Amino Acid Residues ◽  
Klenow Fragment ◽  
Binding Region ◽  
Single Stranded Dna

scholarly journals A comparison of DNA cleavage by the restriction enzymes SalPI and PstI

1980 ◽  
Vol 8 (21) ◽  
pp. 4943-4954 ◽  
Author(s):  
Jacqueline A. Carter ◽  
Keith F. Chater ◽  
Celia J. Bruton ◽  
Nigel L. Brown
Keyword(s):  
Dna Cleavage ◽  
Restriction Enzymes

SSB Protein—Its Interaction with DNA and Use as a Tool in Studying Genome Structure

1981 ◽  
Vol 39 ◽  
pp. 448-451
Author(s):  
Susan Chrysegelos ◽  
Kathi Dunn ◽  
Jack Griffith ◽  
Marcia Manning ◽  
Claire Moore
Keyword(s):  
Genome Structure ◽  
Duplex Dna ◽  
Single Stranded Dna ◽  
Functional Roles ◽  
E Coli ◽  
Gene 5 Protein ◽  
Nucleoprotein Complex ◽  
Infected Cells ◽  
Gene 32

A protein which binds tightly to single stranded DNA but not duplex DNA was first isolated from Escherichia coli (E. coli) by Sigal et. al and is called SSB for single stranded DNA binding protein. Together with SSB the gene 32 protein of T4 infected E. coli cells, and the gene 5 protein of phage M13 infected cells, are the best characterized members of the helix destabilizing family of proteins. They all share the properties (reviewed by Kbrnberg) of binding very tightly and cooperatively to single stranded DNA, of binding somewhat less well to single stranded RNA, and of binding poorly if at all to duplex DNA or RNA. In binding single stranded polynucleotides these proteins disrupt all secondary structure yielding a linear nucleoprotein complex. The details of binding however are very different from one protein to another and must reflect their functional roles in vivo.Physical studies of SSB have showi it to exist as a 75,000 dalton tetramer in solution which is assumed to be the active unit.

A rapid and efficient PCR-based mutagenesis method applicable to cell physiology study

2005 ◽  
Vol 288 (6) ◽  
pp. C1273-C1278 ◽  
Author(s):  
Jae-Kyun Ko ◽  
Jianjie Ma
Keyword(s):  
Molecular Biology ◽  
Protein Engineering ◽  
Restriction Enzyme ◽  
Cost Effective ◽  
Restriction Enzymes ◽  
Recognition Site ◽  
Cell Physiology ◽  
Multiple Site ◽  
Functional Studies ◽  
Engineering Studies

PCR-based mutagenesis is a cornerstone of molecular biology and protein engineering studies. Herein we describe a rapid and highly efficient mutagenesis method using type IIs restriction enzymes. A template gene is amplified into two separate PCR fragments using two pairs of anchor and mutagenic primers. Mutated sequences are located near the recognition site of a type IIs restriction enzyme. After digestion of two fragments with a type IIs enzyme, exposed cohesive ends that are complementary to each other are then ligated together to generate a mutated gene. We applied this method to introduce multiple site-directed mutations in EGFP and Bcl-2 family genes and observed perfect mutagenesis efficiency at the desired sites. This efficient and cost-effective mutagenesis method can be applied to a wide variety of structural and functional studies in cell physiology.

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