CHAPTER 14
DNA REPLICATION –MOLECULAR ASPECTS
DNA REPAIR AND TELOMERES
SUMMARY: DNA REPLICATION
• The two strands of the DNA molecule unwind
• DNA polymerase adds nucleotides to an existing chain
• Direction of new synthesis is in the 5′→3′ direction, which is antiparallel to the template
strand
• Nucleotides enter a newly synthesized chain according to the A-T and G-C
complementary base-pairing rules
PROKARYOTIC REPLICATION
• E. coli model
• Single circular molecule of DNA
• Replication begins at one origin of replication
• Proceeds in both directions around the chromosome
• Replicon – DNA controlled by an origin
EUKARYOTIC DNA REPLICATION
• Eukaryotes usually have multiple, large
chromosomes.
• multiple origins of replication
6 EUKARYOTIC REPLICATION
• Complicated by
• Larger amount of DNA in multiple chromosomes
• Linear structure
• Basic enzymology is similar
• Requires new enzymatic activity for dealing with ends only
MULTIPLE ORIGINS OF REPLICATION
Origin DNA double
helix
Replication
forks
Replication
direction
REPLISOME
• Enzymes involved in DNA replication form a macromolecular assembly
• 2 main components
• Primosome
• Primase, helicase, SSB, Topoisomerase
• Complex of 2 DNA pol III
• One for each strand
ENZYMES IN DNA REPLICATION
• Many enzymes coordinate to replicate DNA.
• DNA helicase unwinds DNA.
• Primase initiates all new strands.
• DNA polymerase III is the main polymerase.
• DNA polymerase I forms the lagging strand.
• DNA ligase binds Okazaki fragments together.
• E. coli has 3 DNA polymerases
• DNA polymerase I (pol I)
• Acts on lagging strand to remove primers and
replace them with DNA
• DNA polymerase II (pol II)
• Involved in DNA repair processes
• DNA polymerase III (pol III)
• Main replication enzyme
• All 3 have 3′-to-5′ exonuclease activity – proofreading
• DNA pol I has 5′-to-3′ exonuclase activity
MOLECULAR MODEL OF DNA REPLICATION
MOLECULAR MODEL OF DNA REPLICATION
MOLECULAR MODEL OF DNA REPLICATION
19 TELOMERES
• Specialized structures found on the ends of eukaryotic chromosomes
• Protect ends of chromosomes from nucleases and maintain the integrity of linear
chromosomes
• Gradual shortening of chromosomes with each round of cell division
• Unable to replicate last section of lagging strand
TELOMERES
• The RNA primer in DNA replication causes a problem for the replicating linear
chromosomes of eukaryotes.
• When the primer is removed, it leaves a gap at the 5′ end of the new DNA strand that
DNA polymerase can’t fill, – causing the chromosome to shorten with each replication.
• The ends of most eukaryotic chromosomes are protected by a buffer of noncoding DNA
(the telomere), consisting of short, repeating sequences (the telomere repeat).
21
TELOMERASE
• With each replication, some telomere repeats are lost, but the genes are unaffected.
• Buffering fails when the entire telomere is lost.
• Telomerase stops the shortening of telomeres by adding telomere repeats to
chromosome ends.
• An RNA section binds to DNA and is the template for addition of telomere repeats.
• Active only in rapidly dividing embryonic cells, in germ cells, and in cancerous somatic cells.
TELOMERE REPEAT
24
• Telomeres composed of short repeated sequences of
DNA
• Telomerase – enzyme makes telomere of lagging
strand using and internal RNA template (not the
DNA itself)
• Leading strand can be replicated to the end
• Telomerase developmentally regulated
• Relationship between senescence and telomere length
• Cancer cells generally show activation of telomerase
REVIEW
1.
What is the importance of complementary base pairing to
DNA replication?
2.
Why is a primer needed for DNA replication? How is the
primer made?
3.
DNA polymerase III and DNA polymerase I are used in DNA
replication in E. coli. What are their roles?
4.
Why are telomeres important?
26 DNA REPAIR
• Mutagens – any agent that increases the number of mutations above background level
• Radiation and chemicals
• Importance of DNA repair is indicated by the multiplicity of repair systems that have
been discovered
REPAIR OF ERRORS IN DNA
• Errors made during replication (base-pair mismatches) are corrected in a proofreading
mechanism by DNA polymerases
• After replication is complete, remaining base-pair mismatches are corrected by a DNA
repair mechanism
PROOFREADING MECHANISM
• The proofreading mechanism allows DNA polymerases to back up and remove
mispaired nucleotides
• If a newly added nucleotide is mismatched, DNA polymerase reverses, using 3′→5′
exonuclease activity to remove the newly added incorrect nucleotide
• DNA polymerase then resumes forward synthesis and inserts the correct nucleotide
Template
strand
PROOFREADING
DNA polymerase
Polymerization activity of DNA
polymerase adds DNA nucleotides to
the new chain in the 5'
3'
direction using complementary base
pairing rules.
New strand
Rarely, DNA polymerase
adds a mispaired
nucleotide.
New
strand
DNA polymerase recognizes
the mismatched base pair.The
enzyme reverses, using its 3'
to 5' exonuclease to remove
the mispaired nucleotide from
the strand.
DNA polymerase resumes
its polymerization activity
in the forward direction,
extending the
new chain in the 5' ⟶ 3'
direction.
DNA REPAIR MECHANISMS
• DNA repair mechanisms correct base-pair mismatches that remain after proofreading
(mismatch repair)
• Distortions in DNA structure caused by mispaired bases provide recognition sites for
mismatch repair enzymes
• Repair enzymes cut the new DNA strand on each side of the mismatch, and remove a
portion of the chain
• DNA polymerase fills the gap with new DNA, and DNA ligase seals the nucleotide chain
MISMATCH REPAIR
Base-pair mismatch
Template strand
Mismatch repair proteins scan
DNA
for a mispaired base that was missed in
proofreading and cleave the backbone of
the new strand on
each side of the
mismatch.
New strand
The repair proteins
remove several to many
bases, including the
mismatched base, leaving a
gap in the DNA.
A repair DNA polymerase fills in
the gap with its 5’ to 3'
polymerizing activity, using the
template strand as a guide.
Nick left after
filled in
gap
DNA ligase seals the nick left
after gap filling to complete the
repair.
EXCISION REPAIR
• DNA damage occurs constantly in cells.
• Base-excision repair mechanisms repair nonbulky damage by removing the erroneous
base and replacing it with the correct one based on complementary pairing rules.
• Nucleotide-excision repair repairs bulky distortions in DNA (such as in thymine
dimers) by removing an entire segment of DNA.
THYMINE DIMER
MUTATION AND EVOLUTIONARY PROCESSES
• Errors that remain in DNA after proofreading and DNA repair are a primary source of
mutations (changes in DNA sequence that are passed on in replicated copies).
• Mutation in a gene can alter the protein encoded by the gene, which may alter how the
organism functions – mutations are the ultimate source of variability acted on by natural
selection.
DRAW A GRADIENT AT THE END OF SIX ROUNDS
OF REPLICATION IN SEMI-CONSERVATIVE MODE
• Bacteria start out in N14 and then get switched to N 15. Draw the gradient at the end of six
rounds of replication in N15.