RESTRICTION ENDONUCLEASES

• The Mechanisms of Different Types of Endonuclease

  1.S1 nuclease

  • cleaves only single-stranded DNA

  2. DNase I

  • cleaves both single- and double-stranded DNA

  3. Restriction Endonuclease

  • cleaves double-stranded DNA
• Restriction Enzymes

  • Endonucleases that recognize specific base sequences and break or restrict the DNA polymer at the sugar-phosphate backbone
  • Originally isolated from bacteria
  • Restriction enzymes are named for the organism from which they were isolated:

  e.g BamHI = Bacillus amyloliquefaciens H

  HindIII = Haemophilus influenzae Rd

  SmaI = Serratia marcescens Sbb
• Derivation of EcoRI name

  • E = Escherichia = genus
  • co = coli = specific species
  • R = RY13 = strain
  • I = fist identified = order of identification in the bacterium
• Four General Types of Restriction Endonucleases

  TYPE I

  • Have both nuclease and methylase activity in a single enzyme
  • They bind to host-specific DNA sites of 4 to 6 bp separated by 6 to 8 bp and containing methylated adenines
  • The site of cleavage of the DNA substrate can be over 1,000 bp from this binding site

  e.g. EcoK from E. coli K
• Four General Types of Restriction Endonucleases

  TYPE II

  • These enzymes do not have inherent methylation activity
  • Type II restriction enzymes cleave the DNA directly at the binding site, producing fragments of predictable size
  • They bind as simple dimers to symmetrical 4- to 8-bp DNA recognition sites
  • Sites are palindromic in nature = bilateral symmetry
• TYPE II (Part 2)

  • Cutting DNA at specific sequences is the basis of many procedures in molecular technology, including mapping, cloning, genetic engineering, and mutation analysis
  • Mode of cutting

  1. Some enzymes cut the duplex with a staggered separation at the recognition site, leaving 2- to 4-base single-strand overhangs at the ends of the DNA = STICKY END
  2. Separates the DNA duplex at the same place on both strands = FLUSH/BLUNT END
• TYPE II (Part 3)

  • Blunt ends = can be joined together regardless of the recognition site
  • Sticky ends = must have matching overhangs to be joined together

  DNA Polymerase

  • Used to convert sticky ends to blunt ends
  • Nucleotides of the overhang as a template

  Adaptors

  • Used to convert blunt ends to specific sticky ends
  • Synthetic short DNA fragments with one blunt end and one sticky end
• Different Sources of Type II Restriction Endonucleases

  1.Isoschizomers

  • Restriction enzymes isolated from different bacteria may recognize and cut DNA at the same site

  e.g. BspEI from Bacillus species

  AccIII from Acinetobacter calcoaceticus

  2. Neoschizomers

  • Restriction enzymes recognize and bind to the same sequence of DNA but cleave at different positions, producing different single-stranded extensions

  e.g. NarI from Nocardia argentinensis

  SfoI from Serratia fonticola
• Different Sources of Type II Restriction Endonucleases

  3. Isocaudomers

  • Restriction endonucleases that produce the same nucleotide extensions but have different recognition sites

  e.g. NcoI from Nocardia corallina

  PagI from Pseudomonas alcaligenes
• Four General Types of Restriction Endonucleases

  TYPE III

  • Resembles as type I enzymes in their ability to both methylate and restrict DNA
  • Recognition sites for these enzymes are asymmetrical
  • the cleavage of the substrate DNA occurs 24 to 26 bp from the site to the 3ʹ side

  e.g. PstIIII from P. stuartii
• Four General Types of Restriction Endonucleases

  TYPE IV

  • Type IV enzymes have cutting and methyltransferase functions

  e.g. BseMII from Bacillus stearothermophilus

  - BseMII also has a methylation function, adding methyl groups to both of the adenine residues in the target sequence
• Factors that affect Restriction Enzyme Activity

  The digestion activity of restriction enzymes depends on the following factors:

  1.Temperature

  • Most endonucleases digest the target DNA at 37°C

  2. Cofactors

  • All enzymes require Mg2+ as a cofactor for the endonuclease activity

  3. Ionic conditions

  • Some enzymes also require ions such as Na+ and K+

  4. Buffer Systems

  • Most restriction enzymes are active in the pH range of 7.0–8.0

  5. Methylation status of DNA

  • Methylation of adenine or cytidine residues affects the digestion of DNA
• Restriction-Modification System

  • Both restriction endonucleases and methylases, are collectively called restriction-modification system (R-M) system
  • Methylation à DNA sites are protected from the cleavage by the restriction endonucleases

  Hemimethylation

  • During the replication, one strand of the daughter duplex is a newly made strand and is unmethylated
• RESTRICTION ENZYME MAPPING OF DNA

  Restriction site mapping determining where in the DNA sequence a particular restriction enzyme recognition site is located

  • Restriction enzymes commonly used in the laboratory (type II restriction enzymes) have 4 to 6 base-pair (bp) recognition sites, or binding/cutting sites, on the DNA
  • Preliminary mapping is usually done to locate the cutting site and to examine the sizes of fragments
  • The location of restriction sites will differ among DNA molecules with different sequences
• Restriction Map

  • DNA is exposed to several restriction enzymes separately and then in different combinations
  • After incubation with the enzyme, the resulting fragments are separated by gel electrophoresis
  • The gel image reveals fragments, produced by the restriction enzyme cutting
• Restriction mapping of a linear DNA fragment

  • A linear fragment of DNA digested with the enzyme PstI
  • Gel electrophoresis

  - The gel image reveals four fragments, labeled A, B, C, and D, produced by PstI

  • Although PstI analysis of this fragment yields a characteristic four-band restriction pattern, it does not indicate the order of the four restriction products in the original fragment
• Restriction mapping of a linear DNA fragment

  • To determine the order of the restriction fragments

  - Another enzyme is used

  e.g. BamHI

  • When the fragment is cut simultaneously with PstI and BamHI, five products are produced

  - Because the BamH1 site is known to be close to one end of the fragment, PstI fragment A is on one end of the DNA fragment
• Restriction mapping of a Circular Plasmid

  • No free ends
  • The size of the fragment is the size of the plasmid

  e.g. 4-kb-pair circular plasmid with one BamHI site and two XhoI sites

  • As with linear mapping, cutting the plasmid with XhoI and BamHI at the same time will start to order the sites with respect to one another on the plasmid.
• Star Activity of Restriction Enzymes

  • Defined as the alteration in the digestion specificity that occurs under sub-optimal enzyme conditions
  • Star activity results in cleavage of DNA at non-specific sites
  • Some of the sub- optimal conditions that result in star activity are as follows:

  - pH >8.0

  - Glycerol concentration of >5%

  - Enzyme concentration >100 units/mg of DNA

  - Increased incubation time with the enzyme

  - Presence of organic solvents in the reaction mixture

  - Incorrect cofactor or buffer
• CRISPR ENZYME SYSTEMS

  • Clustered regularly interspaced short palindromic repeats (CRISPRs) = found in prokaryotic and archaebacterial genomes
  • They are repeated sequences interrupted by spacer sequences matching the genome regions of plasmids or bacteriophages that had previously infected the bacterium.
  • DNA from new invaders is incorporated into the CRISPR locus within a series of short (~20 bp) repeats

  CRISPR Locus = encodes an endonuclease = CRISPR-associated protein (Cas)
• Restriction Fragment Length Polymorphisms

  • RFLP is a difference in homologous DNA sequences that can be detected by the presence of fragments of different lengths after digestion of the DNA samples
  • A genetic variant that can be examined by cleaving the DNA into fragments (restriction fragments) with a restriction enzyme
  • An inherited difference due to genomic variations in the pattern of restriction enzyme digestion is known as a RFLP
  • It is the resulting differences in the size or number of restriction fragments
• Steps in RFLP Typing

  1. Construct a restriction enzyme map of the DNA region under investigation
  2. The number and sizes of the restriction fragments of a test DNA region are compared with the number and sizes of fragments expected based on the restriction map
  3. Polymorphisms are detected by observing fragment numbers and sizes different from those expected from the reference restriction map
• RFLP Typing in Humans

  1. In the first step fragmentation of a sample of DNA is done by a restriction enzyme, which can recognize and cut DNA wherever a specific short sequence occurs, in a process known as a restriction digest
  2. The resulting DNA fragments are then separated by length through a process known as agarose gel electrophoresis
  3. Then transferred to a membrane via the Southern blot procedure
  4. Hybridization of the membrane to a labeled DNA probe will done and then determines the length of the fragments which are complementary to the probe
  5. Then, observe the fragments of different length
• RFLP Typing in humans

  • An RFLP occurs when the length of a detected fragment varies between individuals.
  • Each fragment length is considered an allele, and can be used in genetic analysis.
  • More than 2,000 RFLP loci have been described in human DNA
  • The uniqueness of the collection of polymorphisms in each individual is the basis for human identification at the DNA level
• Recombination and Random Assortment

  • DNA is inherited as one haploid chromosome complement from each parent
  • Each chromosome carries its polymorphisms so that the offspring inherits a combination of the parental polymorphisms
  • When visualized as fragments that hybridize to a probe of a polymorphic region, the band patterns represent the combination of RFLPs inherited from each parent
• Genetic Mapping With RFLPs

  • Polymorphisms can be used as landmarks, or markers, in the genome to determine the location of other genes
  • Clear family history or direct identification of a genetic factor, one can confirm that a disease has a genetic component by demonstrating a close genetic association or linkage to a known marker
  • The more frequently a particular polymorphism is present in persons with a disease phenotype, the more likely an affected gene is located close to the polymorphism
• RFLP and Parentage Testing

  • There is a unique combination of RFLP in each individual, one can infer a parent’s contribution of alleles to a child from the combination of alleles in the child and those of the other parent
  • The fragment sizes of an individual are a combination of those from each parent
• Paternity Test

  • The alleles or fragment sizes of the offspring and the mother are analyzed
  • The remaining fragments have to come from the father
  • Alleged fathers are identified based on the ability to provide the remaining alleles (inclusion)
• Human Identification Using RFLPs

  • RFLP can arise from a number of genetic events:

  - Point mutations in the restriction site

  - Mutations that create a new restriction site

  - Insertion or deletion of repeated sequences (tandem repeats)

  • The insertion or deletion of nucleotides occurs frequently in repeated sequences in DNA
  • Tandem repeats of sequences of all sizes are present in genomic DNA
• DNA Fingerprinting With RFLP

  • Sir Alec Jeffreys’s Southern blot multiple-locus probe (MLP)-RFLP system (1985)

  - This method utilized three to five probes to analyze three to five loci on the same blot

  • Single-locus Probe (SLP) systems (1990)

  - Analysis of one locus at a time yielded simpler patterns, which were much easier to interpret