Molecular and
Cell Biology
Dr. LAWAL A.Z. MB;BS, Ph.D.
Department of Department of
Medical Biochemistry, College of
Health Sciences, LAUTECH,
Ogbomoso
Overview
DNA
• Linear polymer of deoxyribonucleotides
• Double stranded
RNA
• Linear polymer of ribonucleotides
• Single stranded
DNA vs. RNA
DNA RNA
Sugar = deoxyribose Sugar = ribose
Double-stranded Single-stranded
molecule molecule
Thymine bonds with Uracil instead of thymine
adenine
DNA vs. RNA
DNA RNA
Nuclear DNA mRNA = messenger
Mitochondrial DNA tRNA = transfer
Chloroplast DNA rRNA = ribosomal
Nuclear DNA never Assembled in nucleus,
leaves the nucleus moves to cytoplasm
(leaves the nucleus)
DNA vs. RNA
?’s 7-12
Gene
• Stretch of DNA that codes for a type of protein that has a
function in the organism
• Unit of heredity in a living organism.
• All living things depend on genes
• Hold the information to build and maintain an organism’s
cells and pass genetic traits to offspring
Genome
• Sum total of genes in an organism
Genetic Code
• Triplet sequence of nucleotides on the mRNA
represent codon for each amino acid
• Four different bases can generate 64 (43)
different codons or code words by permutations
and combinations
• 31 tRNA species, carrying 20 amino acids, which
translate 61 codons
DNA Replication
• The process of producing two identical copies from one
original DNA
molecule
• Facilitates the transfer of genetic information from one
generation to another
• Each new cell must get a complete set of the DNA
molecules
• Before cell division DNA must be replicated so that every
daughter cell receive a complete copy of the genome
Types of DNA
Replication
Semiconservative model
• Daughter DNA molecules contain one parental strand and
one newly-replicated strand
Conservative model
• The parent helix is conserved, the daughter helix is
completely new
Dispersive model
• Parent helix is broken into fragments, dispersed, copied
then assembled into two new helices.
• New and old DNA are completely dispersed
DNA Replication
Process
Process involve 3 stages :
Initiation
• The replication of both prokaryotic and eukaryotic DNAs
starts at a unique sequence called the origin of
replication, which serves as a specific binding site for
proteins that initiate the replication process.
• The proteins binds specifically to the AT-rich replicator
sequence oriC to form a specific DNA-oriC complex.
• An enzyme called helicase unwinds the DNA by breaking
the hydrogen bonds between the nitrogenous base pairs.
• ATP hydrolysis is required for this process.
• Eukaryotic DNA replication is similar to that of prokaryotic
but more complex.
• There are multiple origins of replication
• special sequences known as Autonomously Replicating
Sequences (ARS) are found on the chromosomes.
• Single-strand binding proteins (SSBPs) bind to the
single strands of DNA near the replication fork to prevent
the ssDNA strands from winding back into a double
helix,thus maintaining the strand separation.
• DNA polymerase add nucleotides only in the 5' to 3'
direction (a new DNA strand can be only extended in this
direction).
• It also requires a free 3'-OH group to which it can add
nucleotides by forming a phosphodister bond between the
3'-OH end and the 5' phosphate of the next nucleotide
• RNA primase, synthesizes an RNA primer that is about
five to ten nucleotides long and complementary to the
DNA template
• DNA polymerase extend this RNA primer by adding nucleotides
one by one that are complementary to the template strand.
Elongation step
• A new DNA strand is always synthesized in a 5’ to 3’
direction
• Thus, the replication of both the strands goes in two
different ways
• DNA polymerase III adds nucleotides in this direction
• This continuously synthesized strand is known as the
leading strand
• In prokaryote, DNA polymerase III begins the synthesis
of the leading strand by using the RNA primer formed by
primase ( from 5’-3’direction or the same direction as the
replication fork movement) and add the nucleotides
according to complement base paring to the
template( A=T,T=A; G≅ C; C≅G)
• As synthesis proceeds, the RNA primers are replaced by
DNA
• The primers are removed by the exonuclease activity
of DNA polymerase I in prokaryotes, and the gaps are
filled in by deoxyribonucleotides
• The nicks that remain between the newly synthesized DNA
(that replaced the RNA primer) and the previously
synthesized DNA are sealed by the enzyme DNA ligase
• That catalyzes the formation of phosphodister linkage
between the 3’-OH end of one nucleotide and the 5'
phosphate end of the other fragment.
• This is the reason why the synthesis of the lagging strand is
more complicated than the leading strand.
• A protein called the sliding clamp holds the DNA
polymerase in place as it continues to add nucleotides
• In eukaryote, The enzyme ribonuclease H (RNase H),
instead of a DNA polymerase I, removes the RNA primer,
which is then replaced with DNA nucleotides
• The gaps that remain are sealed by DNA Ligase.
Termination
1. Progress of the DNA replication fork must stop or be blocked
• Termination at a specific locus involves the interaction between two
components:
1. a termination site sequence in the DNA,
2. a protein which binds to this sequence to physically stop DNA
replication
• In various bacterial species, this is named the DNA replication
terminus site-binding protein, or Ter protein
2. Bacteria have circular chromosomes, termination of replication
occurs when the
two replication forks meet each other on the opposite end of the
parental
chromosome
• Thus, replication forks are constrained to always meet within the
termination region of the chromosome.
3. Removes the primer (RNA fragments), by 5'-3' exonuclease
activity
of polymerase I, and replaces the RNA nucleotides with DNA
nucleotides. and fill the gaps
4. Ligase works to fill nicks completing the newly replicated
DNA
molecule
5. Topoisomerase IV separate the two complete daughter
chromosome
in to two chromosome
•To form a continuous lagging strand of DNA, the RNA primers
must
eventually be removed from the Okazaki fragments and
replaced with
DNA.
In Eukaryotic cell
• Eukaryote cell initiate DNA replication at multiple points
in the chromosome, so replication forks meet and
terminate at many points in the chromosome
• Primer removal at the end of the chromosome leaves a
gap that can’t be filled
Enzymes involved in DNA replication
• Primase: RNA polymerase thus the formed primer is
RNA rather than DNA and it will removed
latter by DNA
polymerase I
• Topoisomerase I: break the 3́ 5́ phosphodiester bond
converting super coiled to relax form which opposite to
ligase
• DNA Helicase Also known as helix destabilizing
enzyme cases formation of Replication Fork due to
broken hydrogen bonds
• DNA Gyrase (and Topoisomerase IV) ; this is a specific
type of topisomerase II convert relaxed form to super
coiled
• DNA Ligase Re-anneals the semi-conservative strands
and joins Okazak’i
Fragments of the lagging strand
• Telomerase Lengthens telomeric DNA by adding
repetitive nucleotide
sequences to the ends of eukaryotic chromosomes
• DNA Polymerase Builds a new duplex DNA strand by
adding nucleotides in the 5' to 3' direction. performs
proof-reading and error correction
• DNA clamp: A protein (unit from polymerase which prevents DNA
polymerase III from dissociating from the DNA parent strand
• Single-Strand Binding (SSB) Proteins Bind to ssDNA and prevent
the DNA double helix from re-annealing after DNA helicase
unwinds it thus maintaining the strand separation