Chapter 3 Structures and Functions of Nucleic Acids

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Chapter 3Structures and Functions of Nucleic
Acids
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Nucleic acid

• A biopolymer composed of nucleotides linked in
  a linear sequential order through 3,5
  phosphodiester bonds

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Classification of nucleic acid

• Ribonucleic acid (RNA) is composed of
  ribonucleotides.
• in nuclei and cytoplasm
• participate in the gene expression
• Deoxyribonucleic acid (DNA) is composed of
  deoxyribonucleotides.
• 90 in nuclei and the rest in mitochondria
• store and carry genetic information determine
  the genotype of cells

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Interesting history

• 1944 proved DNA is genetic materials (Avery et
  al.)
• 1953 discovered DNA double helix (Watson and
  Crick)
• 1968 decoded the genetic codes (Nirenberg)
• 1975 discovered reverse transcriptase (Temin and
  Baltimore)
• 1981 invented DNA sequencing method (Gilbert and
  Sanger)
• 1985 invented PCR technique (Mullis)
• 1987 launched the human genome project
• 1994 HGP in China
• 2001 accomplished the draft map of human genome

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Section 1Chemical Components of Nucleic Acids
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1.1 Molecular Constituents
Nucleic acid can be hydrolyzed into nucleotides
by nucleases. The hydrolyzed nucleic acid has
equal quantity of base, pentose and phosphate.
phosphate
pentose
nucleic acid
nucleotides
nucleosides
bases
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Base Purine
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Base Pyrimidine
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Pentose
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Ribonucleoside
glycosidic bond
Purine N-9 or pyrimidine N-1 is connected to
pentose (or deoxypentose) C-1 through a
glycosidic bond.
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Ribonucleotide
phosphoester bond
A nucleoside (or deoxynucleoside) and a
phosphoric acid are linked together through the
5-phosphoester bond.
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Nomenclature
 
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Nomenclature
 
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Composition of DNA and RNA
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Nucleic acid derivatives

• Multiple phosphate nucleotides
• adenosine monophosphate (AMP)
• adenosine diphosphate (ADP)
• adenosine triphosphate (ATP)

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Nucleic acid derivatives
Cyclic ribonucleotide 3,5-cAMP, 3,5-cGMP,
used in signal transduction
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Nucleic acid derivatives

• Biologically active systems containing
  ribonucleotide NAD, NADP, CoA-SH

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Phosphoester bond formation
The ?-P atom of the triphosphate group of a dNTP
attacks the C-3 OH group of a nucleotide or an
existing DNA chain, and forms a 3-phosphoester
bond.
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Nucleic acid chain extension
A nucleic acid chain, having a phosphate group at
5 end and a -OH group at 3 end, can only be
extended from the 3 end.
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Phosphodiester bonds
Alternative phosphodiester bonds and pentoses
constitute the 5-3 backbone of nucleic acids.
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Section 2 Structures and Functions of Nucleic
Acids
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2.1 Primary Structure

• The primary structure of DNA and RNA is defined
  as the nucleotide sequence in the 5 3
  direction.
• Since the difference among nucleotides is the
  bases, the primary structure of DNA and RNA is
  actually the base sequence.
• The nucleotide chain can be as long as thousands
  and even more, so that the base sequence
  variations create phenomenal genetic information.
  

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2.2 Secondary structure

• The secondary structure is defined as the
  relative spatial position of all the atoms of
  nucleotide residues.

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2.2.a Chargaffs rules

• The base composition of DNA generally varies from
  one species to another.
• DNA isolated from different tissues of the same
  species have the same base composition.
• The base composition of DNA in a given species
  does not change with its age, nutritional state,
  and environmental variations.
• The molarity of A equals to that of T, and the
  molarity of G is equal to that of C.

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Molarity of bases
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Historic X-ray diffraction picture
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Building a milestone of life
James Watson and Francis Crick proposed a double
helix model of DNA in 1953. It symbolized the
new era of modern biology.
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2.2.b Double helix of DNA

• Two DNA strands coil together around the same
  axis to form a right-handed double helix (also
  called duplex).
• The two strands run in opposite directions, i.e.,
  antiparallel.
• There are 10 base pairs or 3.4nm per turn and the
  diameter of the helix is 2.0nm.

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Antiparallel
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Backbone and bases

• The hydrophilic backbone is on the outside of the
  duplex, and the bases lie in the inner portion of
  the duplex.

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Base interactions

• The two strands of DNA are stabilized by the base
  interactions.
• The bases on one strand are paired with the
  complementary bases on another strand through
  H-bonds, namely GC and AT.
• The paired bases are nearly planar and
  perpendicular to helical axis.
• Two adjacent base pairs have base-stacking
  interactions to further enhance the stability of
  the duplex.

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Watson-Crick base pair
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Watson-Crick base pair
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Base-stacking interaction
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Major and minor grooves
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Groove binding
Small molecules like drugs bind in the minor
groove, whereas particular protein motifs can
interact with the major grooves.
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2.2.c Polymorphisms of DNA

• DNA can resume different forms depending upon
  their chemical microenvironment, such as ionic
  strength and relative humidity.
• B-form DNA is the predominant structure in the
  aqueous environment of the cells.
• A-form and Z-form are also native structures
  found in biological systems.

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Structural features of DNAs
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Triplet DNA
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Hoogsteen base pair
The third strand is using Hoogsteen H-bonds to
pair with bases on the first strand.
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G-quartet DNA

• The telomere of DNA is a G-righ sequence, such as
  
• 5 (TTGGGG)n 3
• 4 G residues constitute a plane which is
  stabilized by Hoogsteen H-bonds.

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G-quartet of DNA
Four strands are arranged in either parallel or
antiparallel manner.
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2.3 Supercoil Structure
2.3.a Supercoil structure

• The two termini of a linear DNA could be joined
  to form a circular DNA.
• The circular DNA is supercoiled, and supercoil
  can be either positive or negative.
• Only the supercoiled DNA demonstrate biological
  activities.

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EM image of supercoiled DNA
Circular DNAs in nature, in general, are
negatively supercoiled.
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2.3.b Prokaryotic DNA

• Most prokaryotic DNAs are supercoiled.
• Different regions have different degrees of
  supercoiled structures.
• About 200 nts will have a supercoil on average.

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2.3.c Eukaryotic DNA

• DNA in eukaryotic cells is highly packed.
• DNA appears in a highly ordered form called
  chromosomes during metaphase, whereas shows a
  relatively loose form of chromatin in other
  phases.
• The basic unit of chromatin is nucleosome.
• Nucleosomes are composed of DNA and histone
  proteins.

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Nucleosome

• DNA 200 bps
• Histone basic proteins with many Lys and Arg
  residues
• H2A (x2),
• H2B (x2),
• H3 (x2),
• H4 (x2)

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Beads on a string

• 146 bp of negatively supercoiled DNA winds 1 ¾
  turns around a histone octomer.
• H1 histone binds to the DNA spacer.

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The total length of 46 human chromosomes is about
1.7 m, and becomes 200 nm long after 5 times
condensation.
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2.4 Functions of DNA

• DNA is fundamental to individual life in terms
  of
• They are the material basis of life inheritance,
  providing the template for RNA synthesis.
• They are the information basis for biological
  actions, carrying the genetic information.

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• DNA is able to replicate itself in a high
  fidelity to ensure the genetic information
  transfer from one generation to the next.
• DNA can be used as a template to synthesize RNA
  (transcription), and RNA is further used as the
  template to synthesize proteins (translation).
• DNA posses the inherent and the mutant properties
  to create the diversity and the uniformity of the
  biological world.

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Gene and genome

• A gene is defined as a DNA segment that encodes
  the genetic information required to produce
  functional biological products.
• A gene includes coding regions as well as
  non-coding regions.
• Genome is a complete set of genes of a given
  species.

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Section 3 Structures and Functions of RNA
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Classification

• mRNA (messenger RNA) template for protein
  synthesis
• tRNA (transfer RNA) AA carrier
• rRNA (ribosomal RNA) a component of ribosome for
  protein synthesis
• hnRNA (heterogeneous nuclear RNA) precursor of
  mRNA
• snRNA (small nuclei RNA) small RNAs for
  processing and transporting hnRNA

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Classes of eukaryotic RNAs
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Unique features

• RNA is single stranded, in general.
• RNA has self-complementary intrachain base
  paring.
• The double helical regions of RNA are of the
  A-form.
• RNA is susceptible to hydrolysis.

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3.1 Messenger RNA
mRNA is the template for protein synthesis, that
is, to translate each genetic codon on mRNA into
each AA in proteins. Each genetic codon is a set
of three continuous nucleotides on mRNA.

• mRNAs constitute 5 of total RNAs.
• mRNAs vary significantly in life spans.
• hnRNA is the precursor of mRNA.

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mRNA structure
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mRNA maturation

• hnRNA contains introns and exons.
• Exons are the sequences encoding proteins, and
  introns are non-coding portions.
• Splicing process of hnRNA removes introns and
  makes mRNA become matured.
• The matured mRNA has special structure features,
  including 5-cap and 3-poly A tail.

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5-cap
mRNA chain
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5-cap addition
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5-cap addition

• Methylation can occur at different sites on G or
  A.
• 5-cap can be bound with CBP, benefiting
  transporting from nucleus to cytoplasm.
• 5-cap can be recognized by translation
  initiation factor.
• It protects the 5-end from exonucleases.

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Poly A tail

• 20-200 adenine nucleotides at 3 end
• a un-translated sequence.
• Related with mRNA degradation that begins with
  poly A tail shortening.
• Associate with poly A tail binding proteins for
  protection

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Poly A tailing
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hnRNA splicing
intron
exon
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Matured mRNA of eukaryote
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3.2 Transfer RNA
tRNA serves as an amino acid carrier to
transport AA for protein synthesis.

• tRNA is about 15 of total RNA.
• tRNA is 65-100 nucleotides long.
• There are at least 20 types of tRNA in one cell.

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Structure of tRNA

• The overall structure is a cloveleaf, reversed
  L-shape structure.
• There are three loops (DHU loop, anticodon loop,
  T?C loop), and four stems.
• The 3-D structure is stabilized by hydrogen bonds
  of local intrachain base pairs on these stems.

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Reversed L-shape structure
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Two key sites of tRNA

• A tRNA molecule has an amino acid attachment site
  and a template-recognition site, bridging DNA and
  protein.
• The template-recognition site is a sequence of
  three bases called the anticodon complementary to
  the mRNA codon.

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Codon and anticodon
The anticodon on tRNA pairs with the codon on
mRNA.
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Amino acid attachment

• The OH group at the 3' end of tRNA links
  covalently to an amino acid.
• Only the attached AA becomes activated and
  capable of being transported.

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Rare Bases
tRNA contains a high portion of unusual bases.
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3.3 Ribosomal RNA
rRNA provides a proper place for protein
synthesis.

• rRNA is the most abundant RNA in cells (gt80).
• rRNA assembles with numerous ribosomal proteins
  to form ribosomes.

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Ribosomes

• Ribosomes associate with mRNA to form a place for
  protein synthesis.
• Ribosomes of eukaryotes and prokaryotes are
  similar in shapes and functions.

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Components of ribosomes
Prokaryote Eukaryote (E.coli) (Liver of
mouse) Smaller subunit 30s 40s rRNA
16s 1542 nucleotides 18s 1874
nucleotides proteins 21 40 of
total weight 33 50 of total weight
Larger subunit 50s 60s rRNA
23s 2940 nucleotides 28s 4718
nucleotides 5s 120 nucleotides
5.85s 160nucleotides 5s
120nucleotides proteins 31 30 of
total weight 49 35 of total weight
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Ribosome of E. coli
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Secondary structure of 18S rRNA
The secondary structure of rRNA has many loops
and stems, which can bind ribosomal proteins to
form an assembly for protein synthesis.
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Ribosomal complex
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Polysomes
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EM of polysomes
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Section 4 Physical and Chemical Properties of
Nucleic Acids
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General properties

• Acidity
• Negative backbone
• Viscosity
• Concentration and aggregation effects
• Optical absorption
• UV absorption due to aromatic groups
• Thermal stability
• Disassociation of dsDNA (double-stranded DNA)
  into two ssDNAs (single-stranded DNA)

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4.1 UV Absorption
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Application of OD260
Quantify DNAs or RNAs OD2601.0 equals to
50µg/ml dsDNA 40µg/ml ssDNA (or RNA) 20µg/ml
oligonucleotide Determine the purity of nucleic
acid samples pure DNA OD260/OD280 1.8 pure
RNA OD260/OD280 2.0
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Transition of dsDNA to ssDNA
The absorbance at 260nm of a DNA solution
increases when a dsDNA is melted into two single
strands. The change is called hyperchromicity.
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Melting curve of dsDNA
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DNA melting

• Melting curve a graphic presentation of the
  absorbance of dsDNA at 260nm versus the
  temperature.
• Melting temperature (Tm) the temperature at
  which the UV adsorption reaches the half of the
  maximum value, also means that about 50 of the
  dsDNA is disassociated into the single-stranded
  DNA.

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Melting curve shift
Tm of dsDNA depends on its average GC content.
The higher the GC content, the higher the Tm.
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4.2 Thermal stability

• Dissociation of dsDNA into two ssDNAs is referred
  to as denaturation.
• Denaturation can be partially and completely.
• The nature of the denaturation is the breakage of
  H-bonds.
• Denaturation is a common and important process in
  nature.

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Denaturation of DNA
Extremes in pH or high temperature
Cooperative unwinding of DNA strands
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EM image of denatured DNA
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Renaturation of DNA

• Two separated complementary DNA strands can
  rejoin together to form a double helical form
  spontaneously when the temperature or pH returns
  to the biological range. This process is called
  renaturation or annealing.

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4.3 Hybridization

• The ability of DNA to melt and anneal reversibly
  is extremely important.
• An association between two different
  polynucleotide chains whose base sequences are
  complementary is referred to as hybridization.
• The stability of the hybridized strand depends on
  the complementary degree.

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Two dsDNA molecules from different species are
completely denutured by heating. When mixed and
slowly cooled, complementary DNA strands of each
species will associate and anneal to form normal
duplexes.
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• Two ssDNAs, two ssRNAs, as well as one ssDNA and
  one ssRNA can also be hybridized.
• Ionic strength, degree of complementary,
  temperature, as well as base composition,
  fragment length of nucleic acids will affect the
  hybridization.
• It is a common phenomenon in biology, and has
  been used as a convenient techniques in medicine
  and biology.

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complementary hybridization
Target DNA detection
probe . TAGCTGAG target .
ATCGACTC

• mismatched hybridization

probe . TAGCTGAG non-target .
ATCAGCTC
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Applications

• Gene structure and expression
• Microarray or gene chip
• mRNA separation
• Gene diagnosis and therapy
• PCR technique

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Section 5Nuclease
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Nucleases are enzymes that are able to hydrolyze
phosphoester bonds and cleave DNA or RNA into
fragments.
Definition and classification

• Deoxyribonuclease (DNase) - specially cleave
  DNARibonuclease (RNase) - specially cleave
  RNA

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ExonucleasesThey can cleave terminal nucleotides
either from 5-end or from 3-end, such as
enzymes used in the DNA replication.
Endonucleases They can cleave internally at
either 3 or 5 side of a phosphate group, such
as the restriction endonucleases used to
construct the recombinant DNA.
Classification
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5
3
Exonuclease
Endonuclease
Endonuclease
3
Exonuclease
5
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Applications

• Participate in DNA synthesis and repair, as well
  as RNA post-translational modification
• Digest nucleic acids of food for better
  absorption
• Degrade the invaded nucleic acids
• Construct the recombinant DNA