Chapter VI: DNA Replication:
Year III Pharm.D
Dr.V. Chitra
I. The Molecular Mechanism of
DNA Replication
„ The copying process of DNA is related
to nitrogenous base pairing rules
– Parent DNA molecule consists of two
strands
™Complementary – A pairs with T; G
pairs with C
™The two strands run in antiparallel
directions (DNA has polarity)
– The two DNA strands separate and
serve as templates to direct the
synthesis of “new” complementary
strands
– New nucleotides are inserted along
the template
– A pairs with T; G pairs with C
– Each nucleotide that is added is covalently
attached to the previous one
™ Enzyme = DNA Polymerase
™ Sugar-phosphate backbone of new strand is
formed
„ The linear DNA sequence exists in many states
– Each gene has its own UNIQUE sequence
– Knowing the sequence of one strand, you can
deduce the sequence of the other
(complementarity)
II. Three Models of DNA Replication
„ Conservative model
– Parent molecule remains the same
– Completely new copy of the double helix is made
„ Semiconservative model
– Parent strands separate and serve as templates
for new strand synthesis
– Hybrid molecules are made
„ Dispersive model
– New strands contain a mixture of old molecules
and newly synthesized molecules
„ Messelson and Stahl Experiment
Supports the Semi-Conservative Model
of DNA Replication
III. DNA Replication Involves a
Complex Assembly of Proteins and
Enzymes
„ Human haploid genome = 3 x 109 bp
– ~>1000 X more complex than
Escherichia coli
– The Human Genome Project has
sequenced the entire genome of our
species
™Worldwide effort – International
Collaborations
– The DNA synthesis phase
(interphase) during mitosis lasts only
a few hours despite its huge size
– Replication of the DNA sequence is
very accurate
™Mutation rate ~1/109 errors
The Sequence of Events
1. Beginning of replication occurs at
the origins of replication
– Prokaryotic cells (e.g. E. coli) contain
only one origin
– Eukaryotic cells contain thousands of
orgins on each chromosome
– Proteins bind to origins and pry open
the two strands
– A replication bubble appears at the site of
strand separation and new DNA synthesis
™Replication forks forma at the ends of
the bubbles
– Replication occurs in both directions on the
two strands
™But ALWAYS in the 5’ Æ 3’ direction per
strand
– Replication bubbles fuse
DNA Replication
„ Origins of replication
1. Replication Forks: hundreds of Y-shaped
regions of replicating DNA molecules
where new strands are growing.
3’
5’ Parental DNA Molecule
3’
Replication
Fork
5’
DNA Replication
„ Origins of replication
2. Replication Bubbles:
a.
Hundreds of replicating bubbles
(Eukaryotes).
b.
Single replication fork (bacteria).
Bubbles
Bubbles
2.
DNA polymerases add on new nucleotides
to the growing DNA strand
– One nucleotide is added at a time to the
3’-OH group of the previous nucleotide
– The 3’-OH group of the ribose sugar is
covalently linked to the nucleoside
triphosphate forming a phosphodiester
bond
– Two phosphate groups are liberated –
energy is released (PPi –
pyrophosphate)
3. How to resolve the replication fork
dilemma of antiparallel strands
– DNA strands have polarity
(antiparallel)
– DNA polymerase can only add new
nucleotides to the 3’ end of the
terminal deoxyribose
– Synthesis always progresses in the
5’Æ 3’ direction
– Leading strand is synthesized
continuously
™DNA polymerase progresses as
DNA is unzipped
™One continuous polymer is made
– Lagging strand is synthesized
discontinuously
™Synthesized in opposite direction
™Synthesized AWAY from the replication
fork
™Initiated as a series of short segments
called Okazaki fragments (100-200
bases long)
™Okazaki fragments are joined together
by DNA ligase (covalent phosphodiester
bonds between fragments)
4.
RNA primers are required for initiation of DNA
synthesis
– DNA polymerase can add nucleotides only to 3’OH group of an already existing nucleotide
paired to its complement on the other strand
– Q: How do things get started?
– A: RNA primers are made by an enzyme called
PRIMASE
™
~10 nucleotides long primers Æ H-bonds to
template and provides substitute for DNA
polymerase
™Leading
strand requires only one
RNA primer
™Lagging strand requires one RNA
primer for every Okazaki fragment
– RNA primers are removed by specific
enzymes and replaced with DNA
nucleotides
™Gaps are sealed with DNA ligase
5.
Helicases and single-stranded binding
proteins are important components of DNA
synthesis
– Helicases unwind the double helix and
separate the two templates
™ Smoothing of twists
™ Breaking of H-bonds
– Single-stranded binding proteins stabilize
the DNA for replication
The Telomere Problem
„ Eukaryotic cells have a problem replicating the 5’ ends of
daughter DNA strands
– The ends of chromosomes contain 100-1000 repeating
(TTAGGG)n segments
– Result: Ends of DNA get shorter and shorter in most
dividing somatic cells
™ Older people have shorter telomeres
„ Some cells have a solution:
– Telomerase
™ Enzyme + RNA fragment – catalyzes the extension of
the ends
™ RNA = template for new telomere pieces
– Examples:
™ Germ cells
™ Some cancerous cells
™ Immortalized cultured cells