Professional Documents
Culture Documents
Biochemistry Ch. 6.1 DNA AND Biotechnology
Biochemistry Ch. 6.1 DNA AND Biotechnology
DNA Structure
• Two chemically distinct forms of nucleic acids within eukaryotic cells: Deoxyribonucleic
acid (DNA) and Ribonucleic acid (RNA). DNA and RNA are polymers of one another
• Bulk of DNA is found in chromosomes in the nucleus
Nucleosides and Nucleotides
• DNA is a polydeoxyribonucleotide which is composed of many
monodeoxyribonucleotides linked together.
• Nucleosides: composed of five-carbon sugar (pentose) bonded to a nitrogenous base
and are formed by covalently linking the base C-1’ of the sugar
o Carbon atoms in sugar are labeled with a prime symbol to distinguish them from
the nitrogenous base
• Nucleotides: formed when one or more phosphate groups are attached to C-5’ of a
nucleoside.
o Often named according to the number of phosphates present
▪ E.g. – adenosine di-/triphosphate
o High energy compounds since there is repulsion between the closely associated
negative phosphate groups.
• Nucleic acids are classified according to the pentose they contain
o If pentose is ribose, then the nucleic acid is RNA
o If pentose is deoxyribose (2’- OH groups replaced by –H), then it is DNA
Sugar-Phosphate Backbone
• Backbone of DNA is composed of alternating sugar and phosphate groups
• This determines the directionality of the DNA and is always read from 5’ to 3’
• Backbone is formed as the nucleotides are joined by 3’-5’ phosphodiester bonds
o Phosphate group link the 3’ carbon of one sugar to the 5’ phosphate group of the
next incoming sugar in the chain
• Since phosphates carry a negative charge, DNA and RNA strands have an overall
negative charge.
• Each strand of DNA has a distinct 5’ and 3’ end, which creates polarity within the
backbone
o The 5’ end of DNA will have an –OH or phosphate group bonded to C-5’ of the
sugar
o The 3’ end of DNA will have a free –OH on the C-3’ of the sugar.
• Base sequence of nucleic acid strand will always be written and read in the 5’ to 3’
direction
o If written backwards, the ends must be labeled as “3’” and “5’” (e.g. – 3’–GTA-5’)
o Position of phosphates may be shown (e.g. – pApTpG)
o “d” may be used as shorthand for deoxyribose (e.g. – dAdTdG)
• DNA is generally double-stranded (dsDNA) and RNA is generally single-stranded (ssRNA)
Purines and Pyrimidines
• Two families of nitrogen-containing bases found in nucleotides: purines and pyrimidines
• Overall, there are five common bases, but there may be exceptions in tRNA or in some
prokaryotes and viruses
• Purines: Contain two rings in their structure.
o Adenine (A) and Guanine (G).
o Both are found in DNA and RNA
• Pyrimidines: contain one ring in their structure
o Cytosine (C), Thymine (T), & Uracil (U)
o Cytosine is found in both DNA and RNA, thymine is only in DNA and uracil is only
in RNA
• These are examples of biological aromatic heterocycles. Aromatic describes an
unusually stable ring system that adheres to the following rules
o Compound is cyclic
o Compound is planar
o Compound is conjugated (has alternating single and multiple bonds, or lone
pairs, creating at least one unhybridized p-orbital for each atom in the ring)
o Huckel’s Rule: compound has 4n+2 electrons.
• Stability from aromatic compounds results from delocalized pi-electrons which are able
to travel throughout the entire compound using available molecular orbitals
• Heterocycles are ring structures that contain at least two different elements in the ring
o Both purines and pyrimidines contain nitrogen in their aromatic rings
o Thus nucleic acids have exceptional stability
Watson-Crick Model
• Proposed the three-dimensional structure of DNA in which the double-helical nature of
it was deduced and also proposed specific base-pairings that would be the basis of a
copying mechanism.
• In the double helix, two linear polynucleotide chains of DNA are wound together in a
spiral orientation along a common axis.
• Key features:
o Two strands of DNA are antiparallel: strands oriented in opposite directions
▪ One strand has polarity 5’ to 3’ down the page while the other strand has
5’ to 3’ polarity up the page
o Sugar-phosphate backbone is on the outside of the helix with the nitrogenous
bases on the inside
Chargaff’s o Complementary base-pairing: Adenine (A) is always base-paired with a thymine
Rules (T) via two hydrogen bonds. A guanine (G) always pairs with a cytosine (C) via
three hydrogen bonds.
▪ 3 hydrogen bonds make the base pair interaction stronger
▪ hydrogen bonds and the hydrophobic interactions between bases
provides DNA stability
o Amount of purines will be equal to total pyrimidines due to complementary
base-pairing
• Double helix of DNA is a right-handed helix forms what is called B-DNA
o Contains 10 bases per every turn (every turn is 3.4 nm)
o Has major and minor grooves, which are usually the site of protein binding
• Another form of DNA is called Z-DNA, which is a left-handed helix
o Contains 12 bases with each turn (every turn is 4.6 nm)
o Usually formed when there is a high GC-content or a high salt concentration
o No biological activity since it is unstable and difficult to research
Denaturation and Reannealing
• For replication and transcription, it is necessary to gain access to the DNA
• Double helical nature of DNA can be denatured by disrupting the hydrogen bonding and
base-pairing which results in the “melting” of the double helix into two single strands
o No covalent link between the nucleotide in the backbone of the DNA break
o Heat, alkaline pH, and chemicals like formaldehyde and urea are commonly used
to denature DNA
• Denatured DNA can be reannealed if the denaturing conditions are slowly removed
DNA Replication
Strand Separation and Origins of Replication
• Replisome or Replication Complex: set of specialized proteins that assist the DNA
polymerases
• Origins of Replication: Points where DNA begins to unwind. Beginning of replication
process
• Replication Forks: generation of new DNA proceeds in both direction, which creates
forks on both sides of the origin.
• Bacterial chromosome is a closed, double stranded circular DNA with a single origin of
replication.
• Eukaryotic replication must copy many more bases and is a slower process
o Each eukaryotic chromosome contains one linear molecule of double-stranded
DNA that has multiple origins of replication.
▪ This allows the chromosomes to duplicate efficiently
o As replication forks move forwards towards each other, sister chromatids are
created. Chromatids remain connected at the centromere.
• Helicase: enzyme responsible for unwinding the DNA, generating two single-stranded
template strands ahead of polymerase.
• Once opened, the unpaired strands want to hydrogen bond with other molecules (there
are free purines and pyrimidines)
o Proteins are required to hold the strands apart. Single-stranded DNA-binding
proteins bind to the unraveled strand which prevents the reassociation of the
DNA strands and prevents the degradation of DNA by nucleases
• Supercoiling: wrapping of DNA on itself as its helical structure is pushed further toward
the telomeres during replication.
o Occurs as helicase unwraps DNA.
o This strains the DNA helix and increases the chances of strand breakage.
• DNA topoisomerases: introduces negative supercoils which alleviates the torsional stress
o Work ahead of helicase and “nick (cut)” one or both strands. This allows
relaxation and the cut strands are then resealed.
• Parental strands will serve as templates for the generation of new daughter strands.
o Process is termed semiconservative since one parental strand is retained in each
of the two resulting identical double-stranded DNA molecules.
Synthesis of Daughter Strands
• DNA polymerase: responsible for reading the DNA template and synthesizing the new
daughter strand
o Can read the template strand in a 3’ to 5’ direction while synthesizing the
complementary strand in the 5’ to 3’ direction
▪ The separated parental strands of the helix are antiparallel to each other,
so one strand is oriented in the right direction for polymerase while the
other is not.
• Leading Strand: the strand in the replication fork that is copied continuously (parallel to
the polymerase direction)
• Lagging Strand: strand that is copied in a direction opposite to the direction of the
replication fork.
o Has 5’ to 3’ polarity but polymerase only reads 3’ to 5’
o Solution for lagging strand is to use Okazaki fragments
• First step in DNA replication Is to lay down an RNA primer since DNA needs another
molecule to “hook on”
o Primase synthesizes a short primer in the 5’ to 3’ direction to start replication on
each strand
▪ These primers are constantly being added to the lagging strand because
each Okazaki fragment starts with a new primer
• Leading strand only requires one in theory
o DNA polymerase III (prokaryotes) or DNA polymerases ,, & (eukaryotes) will
then synthesize the daughter strands of DNA in the 5’ to 3’ direction
▪ Incoming nucleotides are 5’ deoxyribonucleotide triphosphates: dATP,
dCTP, dGTP, and dTTP
▪ As new phosphodiester bonds are made, a free pyrophosphate (PPi) is
releases.
o DNA polymerase I (prokaryotes)/RNase H (eukaryotes) enzymes remove RNA
from the sequence
o DNA polymerase I (prokaryotes)/DNA polymerase (eukaryotes) add DNA
nucleotides where RNA primer used to be
o DNA ligase seals the end of the DNA molecule which creates one continuous
strand of DNA.
• Eukaryotic synthesis is considered similar to prokaryotic DNA synthesis. However, the
five DNA polymerases in eukaryotic cells should be noted:
o DNA polymerases ,, & work together to synthesize both the leading and
lagging strands. Delta also fills in the gaps left behind when RNA primers are
removed
o DNA polymerase replicates mitochondrial DNA
o DNA polymerase & are used in the process of DNA repair
o DNA polymerase & are assisted by PCNA protein. This assembles into a trimer
to form a sliding clamp, that helps to strengthen the interaction between these
DNA polymerases and the template strand
Replicating the Ends of Chromosomes
• DNA polymerase cannot complete the synthesis of the 5’ end of the strand
o Thus each time the DNA synthesis is carried out, the chromosome becomes
shorter
• Telomeres are used to lengthen the time that cells can replicate and synthesize DNA
before necessary genes are damaged.
o Repetitive sequence with high GC-content, and located at tips of the
chromosome.
DNA Repair
• DNA can be damaged from exposure to chemicals or radiation
• If not repaired, the damaged DNA will be passed on to daughter cell
• And damage causes an increased risk of cancer
Oncogenes and Tumor Suppressor Genes
• Cancer can result from mutated genes. These cells can proliferate excessively since they
are able to divide without stimulation from other cells
o Able to migrate by local invasion or metastasis. Allows migration to distant
tissue by the bloodstream or lymphatic system
• Oncogenes: mutated genes that cause cancer
o Primarily encode cell cycle-related proteins
• Proto-oncogenes: before oncogene genes are mutated
o Abnormal alleles encode proteins that are more active than normal proteins
which promotes rapid cell cycle advancement
• Antioncogenes: tumor suppressor genes (like p53 or Rb) that encode proteins that
inhibit the cell cycle or participate in DNA repair processes.
o These function to stop tumor progression
o Mutation of these genes result in the loss of tumor suppression activity which
then promotes cancer
o Generally, both alleles need to be inactivated since even one copy of the normal
protein is usually enough to inhibit tumor formation.
Proofreading and Mismatch Repair
• DNA polymerase is usually 100% accurate, but it does occasionally make errors
Proofreading
• During synthesis, two double-stranded DNA molecules will pass through a part of the
DNA polymerase enzyme for proofreading
• If the wrong bases are paired, the hydrogen bonds between the bases are unstable. This
instability can be detected as the DNA passes through the specified part of polymerase.
o Incorrect base is removed and replaced with correct one.
o Enzyme determines which is the template strand by analyzing level of
methylation. The template strand has existed in the cell for a longer period of
time so it will therefore be more methylated.
• DNA ligase does not have proofreading ability as it closes the gaps between Okazaki
fragments. Thus, it much more likely to have a mutation in the lagging strand as
compared to the leading strand.
Mismatch Repair
• The G2 phase of the cell cycle is able to perform mismatch repair
• Enzymes are encoded by genes MSH2 and MLH1, which detect and remove errors
introduced in replication that were missed during the S phase of the cell
• In prokaryotes, the enzymes that do a similar function are MutS and MutL
Nucleotide and Base Excision Repair
• Cell machinery is able to recognize two specific types of DNA damage in the G1 and G2
cell cycle phases and fixes them through nucleotide or base excision repair
Nucleotide Excision Repair
• UV light induces the formation of dimers between adjacent thymine residues in DNA.
o These dimers interfere with DNA replication and normal gene expression and
distort the shape of the double helix.
• Thymine dimers are eliminated from DNA by a nucleotide excision repair (NER)
o This is a cut and patch process.
o Specific proteins scan the DNA molecule and recognize the lesion because of a
bulge in the strand
o An excision endonuclease then makes cuts in the phosphodiester backbone of
the damaged strand on both sides of the thymine dimer and removes the
defective oligonucleotide.
o DNA polymerase then fills in the by synthesizing DNA in the 5’ to 3’ direction
(uses the undamaged strand as a template)
o The cut in the strand is sealed by DNA ligase
Base Excision Repair
• Cytosine deamination is the loss of an amino group from cytosine and results in its
conversion to uracil. This is usually caused by thermal energy being absorbed by DNA
o This is easily detected since uracil should not be found in the DNA molecule
• Alteration to other bases can occur through other mechanisms as well, and detection
systems exist for many of these bases as well. Base excision repair is used to repair
these base abnormalities.
• Base Excision Repair:
o Affected base is recognized and removed by a glycosylase enzyme. This leaves
behind an apurinic/apyrimidinic (AP) site also known as an abasic site.
o AP site is recognized by AP endonuclease which removes the damaged sequence
from DNA
o DNA polymerase and DNA ligase can then fill in the gap and seal the strand.