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Lecture 8

BIOL 3113 Lecture 8: 8.DNA.Replication, Repair and Recombination


Department
BIOL
Course Code
BIOL 3113
Professor
Barbara S
Lecture
8

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8 DNA - Replication
Must occur before cell division
Cell has to copy its genome with great accuracy - Mistakes in replication cause mutations
Duplication rate - ~ 1000 nucleotides/second, bilions of nucleotides during every cell
division
Each strand of a DNA double helix can serve as a template for the replication of the
other (complementary) strand
Complementary base pairing with the template strand determines which new nucleoside
is added to the new (daughter) strand
The daughter strand is antiparallel to the template strand
DNA replication in cells is semiconservative
*both of the two parent strands are conserved, one in each of the two daughter molecules
*daughter strands are complementary to the respective parent strands
*therefore, the two daughter molecules are identical to the original parent molecule
To be used as a template - two strands must be separated
Initiator proteins - bind to the DNA and open double helix
Replication Origin
Site on the DNA double helix where replication is initiated
Site where the double helix first opens - replication bubble
*consist of specific nucleotide sequences recognized by initiator proteins
*A-T rich (easier to separate)
*100 bp (base pairs) in length
Number of replication origins
Procaryotes
*1 replication origin per chromosome
*replication rate ~ 1000 nucleotides/sec.
Eucaryotes
*multiple replication sites on each chromosome
*replication rate ~ 100 nucleotides/sec. (more complex chromatin structure)
In eucaryotes replication origins are activated in clusters of 20 to 80 adjacent origins =
replication units

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Replication Fork
Replication of DNA occurs at replication forks
Each replication origin generates 2 replication forks
Y-shaped structure resulting from the separation of the DNA double helix into two
strands during replication
Move away from origin in both directions
DNA Polymerase - Enzyme responsible for DNA synthesis
Reaction:
Attaches a 5'-nucleoside triphosphate (energy-rich, provides energy for polymerization)
to the 3'-end of existing DNA strand
Catalyzes the formation of a phosphodiester bonds
Moves along template strand stepwise for many cycles of polymerization reaction
DNA Polymerase - can catalyze DNA synthesis in only one direction: 5’-to-3’
Problem:
DNA strands in a helix have opposite polarity - provides two templates: one 5’-to-3’ and
one 3’-to-5’
Solution:
on 3’-to-5’ template new DNA strand made continuously in 5’-to-3’ direction - Leading
strand - synthesis in the same direction that the replication fork is opening, allows
continuous DNA synthesis
on 5’-to-3’ template new strand made discontinuously in pieces that are “stitched”
together after synthesis - Lagging strand - synthesis in the opposite direction in which
the replication fork is opening (Okazaki fragments)
Proofreading Ability of DNA Polymerase
DNA polymerase is very accurate (~1 error in 107 copied nucleotides), but if the wrong
nucleotide is inserted, the polymerase can correct its mistakes
Contains both polymerizing and editing sites
Proofreading
1. Before adding a new nucleotide polymerase checks if the previous nucleotide is
correctly base-paired to the template strand
2. If the complementary base pairing is inaccurate, DNA polymerase recognizes the
mismatch and hydrolyzes the phosphodiester bond (exonuclease activity), removing the
nucleotide
3. DNA polymerase inserts a new nucleotide and moves forward
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Initiation of DNA Replication
At the replication origin there is only single-stranded DNA
DNA polymerase requires double-stranded DNA to attach
Solution:
~10 nucleotides long RNA primer strand is made on DNA template- forms DNA-RNA
double strand
Enzyme: Primase
*Catalyzes the formation of an RNA strand, complementary and antiparallel to a single
DNA strand
*RNA strand grows 5’ 3’ complementary to the DNA read 3’ 5’
DNA polymerase attaches to the RNA-DNA and starts synthesis of a new DNA strand,
starting at the 3’ end of the RNA strand
On the leading strand only one RNA primer is needed for one replication origin
On the lagging strand new primers are needed for every Okazaki fragment
Removal of RNA primers and Closing of the DNA-DNA gaps
1. Nuclease beaks down and removes the RNA primer
2. Repair polymerase replaces primer with a DNA using adjacent Okazaki fragment as a
primer
3. DNA ligase joins 5’-phospate end of one new DNA fragment to 3’-hydroxyl end of the
next fragment (there is no high energy phosphate bond to supply energy, DNA ligase
uses energy from ATP to catalyze the formation of a phosphodiester bond)
Creates a continuous DNA strand from separately synthesized fragments
Meeting of replication bubbles
When adjacent replication bubbles meet, the daughter DNA strands separate and DNA
replication ceases
DNA - Replication Machine
Several proteins working together with DNA polymerase:
DNA helicase - separates the bases of the DNA double helix at the replication fork
Single-strand binding protein - monomers bind to single stranded DNA in the replication
bubble to stabilize it
Sliding clamp protein - keeps the DNA polymerase firmly attached to the DNA template,
allows it to slide along DNA
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