Restriction Endonucleases: Mechanisms

• Restriction endonucleases play a key role in molecular biology and diagnostics.
• The goal is to equip students with knowledge of restriction endonucleases.

Introduction to Restriction Endonucleases

• Very important for metabolizing DNA.
• Able to recognize short nucleotide sequences within DNA.
• Once recognized, they cleave DNA in their sugar-phosphate backbone.
• Has different mechanisms for cleaving DNA.

Mechanisms of Endonucleases

• S1 nuclease cleaves only single-stranded DNA and is utilized to induce nick formation and remove hairpin loops in RT-PCR for cDNA formation.
• S1 nuclease from Aspergillus oryzae hydrolyzes single-stranded DNA or RNA into 5' mononucleotides and hydrolyzes single-stranded regions in duplex DNA.
• DNase I cleaves both single- and double-stranded DNA nonspecifically, regardless of the sequence.
• Deoxyribonuclease I from bovine pancreas digests single-and double-stranded DNA at pyrimidines to oligodeoxyribonucleotides.
• DNase I is used in research and clinical labs to remove DNA from RNA preparations and also to detect exposed regions of DNA in DNA protein binding experiments.
• Restriction endonucleases cleave double-stranded DNA at specific nucleotide sequences and are synthesized by many bacteria, they provide immunity against invading organisms.

Restriction Enzymes

• Discovered in 1950, are enzymes found in bacteria that inactivate viruses, protecting the bacteria from bacteriophages.

Restriction Enzymes and Bacterial DNA

• Restriction enzymes were originally isolated from bacteria to cleave foreign DNA entering the bacterial cell.
• The ability of the cell to recognize foreign DNA depended on both DNA sequence recognition and methylation.
• Bacterial DNA is methylated to protect it from digestion by its own restriction enzymes.
• Methylation is a modification that makes bacteria resistant to their own restriction enzymes.

Four General Types of Restriction Endonucleases

• Type I enzymes possess both nuclease and methylase activity within a single enzyme, binding to specific DNA sites and cleaving the DNA substrate over 1,000 bp away.
• Function is to have restriction (nuclease) and modification (methylase) and can add a methyl group to the DNA.
• Type II restriction enzymes, commonly used in the lab, recognize unmethylated sequences and cleave DNA directly at the binding site.
• Cleavage produces fragments of predictable size.
• These bind as simple dimers to symmetrical 4- to 8-bp DNA recognition sites, cutting recognition sites in the presence of magnesium.
• Type III enzymes resemble Type I enzymes in their ability to both methylate and restrict DNA, but ONE strand of DNA is methylated.
• Recognition sites are asymmetrical, and cleavage occurs 24-26 bp from the 3' side.
• Type IV enzymes have cutting and methyltransferase functions that are methylation-dependent, preferring modified DNA, such as glycosylated or methylated DNA.

Cutting Modes of Type II Restriction Enzymes

• Some enzymes cut the duplex with a staggered separation at the recognition site, leaving 2- to 4-base single-strand overhangs creating sticky ends.
• Others separate the DNA duplex at the same place on both strands, creating flush/blunt ends.

Sticky vs Blunt Ends

• Blunt ends can be joined together regardless of the recognition site.
• Enzymes used to bind two blunt ends is Ligase enzyme.
• Sticky ends must have matching overhangs to be joined.

Sticky Ends

• Some enzymes cut the duplex with a staggered separation at the recognition site, leaving 2–4 base single-strand overhangs at the ends of the DNA.
• Single-strand ends can hybridize with complementary ends on other DNA fragments, directing the efficient joining of cut ends.
• Hability to form hydrogen bonds with complementary overhangs, these cuts are said to produce “sticky ends” at the cut site, always near the end, either 5' or 3'.

Blunt Ends

• DNA duplex at the same place on both strands, leaving flush, or blunt, ends.
• Termini can be rejoined, although not as efficiently as sticky ends.
• The cut is always in the middle.

Enzymes

• DNA polymerase used to convert sticky ends to blunt ends.
• Nucleotides of the overhang become a template.

Adaptors

• Short, synthetic DNA fragments with one blunt end and one sticky end are used to convert blunt ends to sticky ends.

Most Used Restriction Enzymes

• Restriction enzymes in sticky ends include BAMHI, EcoRI, HindIII, PstIII, EcoRI.
• They are produced by sticky ends.
• EcoRV produced by Blunt ends.

Sources of Type II Restriction Endonucleases

• Isoschizomers are restriction enzymes isolated from different bacteria that may recognize and cut DNA at the same site.
• Neoschizomers recognize and bind to the same sequence of DNA but cleave at different positions
• Isocaudomers produce the same nucleotide extensions but have different recognition sites.

Factors Affecting Restriction Enzyme Activity

• The digestion activity of restriction enzymes depends on temperature, cofactors, ionic conditions, buffer systems and methylation status.
• Most endonucleases digest at 37°C, require Mg2+ as a cofactor, are active in the pH range of 7.0–8.0 and function to counteract the negative charge of DNA.
• Methylation of adenine or cytidine residues affects DNA digestion, with methylated DNA being digestion-resistant and unmethylated DNA being digestion-prone.

Restriction-Modification (R-M) Systems

• Restriction endonucleases and methylases are collectively called R-M systems.
• Methylation protects DNA sites from cleavage by restriction endonucleases.
• Hemimethylation: During replication, one strand of the daughter duplex is newly made and is unmethylated.

Hemimethylation

• Source of imprinting of DNA, a system the provides a predetermined program of gene expression during development indicating a mechanism for keeping methylation patterns in the genome.

Restriction Site Mapping

• Involves determining where in the DNA sequence a particular restriction enzyme recognition site is located.
• Commonly used type II restriction enzymes have 4 to 6 base-pair recognition sites.
• Preliminary mapping locates the cutting site and examines the sizes of fragments.
• Varying DNA molecules differ in locations of restriction sites.

Restriction Maps

• Involve exposing DNA to several restriction enzymes separately and then in different combinations, generating different fragments based on the enzymes used.
• They are then separated by gel electrophoresis and resolved using PFGE.

Mapping Linear DNA Fragments

• Since location isn't known, restriction mapping is used.
• Requires 3 restriction sites to form 4 fragments of DNA that is digested with enzyme Pstl forming four fragments labeled A, B, C, and D through Gel electrophoresis
• You can know fragment size by use of molecular weight fragments.

Star Activity of Restriction Enzymes

• Defined as digestion specificity alteration that occurs under sub-optimal enzyme conditions.
• Binding to intended sequence and cutting also results in cleavage of DNA at non-specific sites.
• The conditions that can alter normal activity include pH >8.0, high glycerol concentration, high ratio of enzyme concentration, increased incubation time, presence of organic solvents, or incorrect cofactor/buffer.

CRISPR Enzyme Systems:

• Clustered regularly interspaced short palindromic repeats (CRISPRs) found in prokaryotic and archaebacterial genomes which are sequences matching genome regions of plasmids or bacteriophages used against new invaders.
• CRISPR Locus consists an endonuclease = CRISPR-associated protein (Cas) and serves as the immunity of the bacteria.

Restriction Fragment Length Polymorphisms (RFLPs)

• Arise from inherited or somatic differences in nucleotide sequences in human DNA.
• A difference in homologous DNA sequences detected by fragments of varying lengths after DNA digestion
• A genetic variant examined by cleaving DNA using restriction enzymes
• Analyzed using polymorphism.

RFLP Typing in Humans

• Involves fragmenting DNA with a restriction enzyme, separating the fragments by length through gel electrophoresis, transferring them to a membrane via Southern blotting, and hybridizing to a labeled DNA probe.
• An RFLP occurs when a detected fragment’s length varies between individuals, with each fragment length considered an allele used in genetic analysis.

Recombination and Random Assortment

• Each person has a unique set of RFLPs, with genetic diversity increasing over generations due to mutations, recombination, and other genetic events.
• Human beings are diploid and have two copies of every locus.

Genetic Mapping with RFLPs

• They are used as landmarks to determine the location of other genes.
• A polymorphism's presence in persons with a disease phenotype, an affected gene is likely located close to the polymorphism.

RFLP and Parentage Testing

• Based on the parental contribution of alleles of a child, given their unique combination of RFLPs to identify a parent.
• The alleles or fragment sizes of the offspring and the mother are analyzed.

Paternity Testing

• The remaining fragments have to come from the father.
• Alleged fathers are identified based on the ability to provide the remaining alleles (inclusion)

DNA Fingerprinting

• Utilizes Sir Alec Jeffreys’s Southern blot multiple-locus probe (MLP)-RFLP system: three to five probes to analyze three to five loci on the same blot
• Single-locus Probe (SLP) systems (1990) involve analysis of one locus at a time, yielding simpler patterns that are more specific and analyzed one locus at a time using an observed fragment.

Human Identification

• Done through RFLP can arise from point mutations in the restriction site, mutations that create new restriction sites, or insertion/deletion of repeated sequences.
• The number and location of the restriction site or the given restriction enzyme are not the same in all individuals
• The resulting differences in the size or number of restriction fragments.
• Insertion or deletion of nucleotides occurs frequently in repeated genomic sequences.
• Blood grouping was the 1st genetic tool in human identification and analyzed the polymorphic HLA loci to add a higher level of discrimination.
• It requires the use of a restriction enzyme digest to resolve tandem repeats.