6.4 DNA Replication & Repair Flashcards
DNA structure
Due to the work of Watson, Crick, Franklin, and
Wilkins, an accurate model of DNA was determined in
the 1950s
DNA has a double helix structure, with the “sides”
consisting of alternating deoxyribose sugars and
phosphates
The “rungs” consist of nucleotide base pairs (A, T, C,
&G)
The strands run antiparallel to each other
The hydroxyl on the 3’ carbon of deoxyribose is at one end of the strand
The phosphate on the 5’ carbon is at the other end
The strands run in opposite directions
semiconservative replication
In 1958, Meselson and Stahl verified that DNA
replication was semiconservative
Used “heavy” isotopes of nitrogen (15N) to
label E. coli bacteria (lots of N in DNA!)
Then transferred colonies to a growth medium
of normal N, allowed to replicate for one or
two rounds :. New DNA would contain “light”
N and density could be measured
DNA replication
Eukaryotic DNA replication is similar to
prokaryotic, but more complex due to its linear
configuration and sheer volume
Consists of 3 steps:
1) Strand separation
2) Building complementary
strands
3) Dealing with errors
Step 1: strand separation
DNA helicase binds to specific nucleotide
sequences (replication origins)
Unwinds DNA by breaking apart H-bonds
between base pairs
Replication fork and 2 problems
Replication fork: Y-shaped region of separation
2 problems:
Tension on DNA behind fork (topoisomerase)
Separated strands tend to anneal (SSBPs)
Helicase will separate strands in both
directions, forming a replication bubble
There can be many replication bubbles at any
given time on a strand of DNA; they will
extend until they meet and merge
DNA is replicated at a rate of ~50bp per
second at each fork
It takes about an hour to replicate the entire
genome
step 2: building complementary strands
DNA polymerases are enzymes that add
nucleotides to build new DNA strands
Nucleotides are added to the 3’ end of the existing
“template” strand, which is read in the 3’ to 5’
direction
New strand: 5🡪3
:. strands are synthesized 5’ to 3’
DNA polymerases need energy, which comes
from the hydrolysis of 2 Pi from a nucleoside
triphosphate as it is added to the strand
Nucleoside = Sugar + Base
Nucleotide = Sugar + Base + Phosphate
DNA polymerase lll
DNA polymerase III can only add to the 3’ end
of a strand, so RNA primase builds a short (10
– 60 bp) complementary RNA sequence called
an RNA primer
DNA polymerase III begins adding to the
primer in the 5’ to 3’ direction
step 2 cont.
One strand will be able to be synthesized continuously: leading strand
The other side must be made in smaller
fragments, using multiple RNA primers:
lagging strand
These DNA fragments on the lagging strand
are called Okazaki fragments
100-200 bp long in eukaryotes
1000-2000 bp long in prokaryotes
As each fragment extends, it will run into the RNA primer of the previous Okazaki fragment, then…
DNA polymerase I removes the RNA
nucleotides and replaces them with those of
DNA
DNA ligase catalyzes the formation of a
phosphodiester bond between the nucleotides
of the two fragments
step 3: error correction
DNA polymerases also proofreads and corrects the
newly synthesized strands
For example, if there is a base pair mismatch (e.g. A
and C), DNA polymerase III can’t continue
It will back up, replace the nucleotide, and continue
Sometimes, errors will be missed (1 in every
million bp) which distort the shape of DNA
DNA polymerase II has a repair mechanism that
can determine which is the original correct
template strand, and remove the incorrect bases so they can be replaced
semiconservative replication
a mechanism of DNA replication in which each of the two strands of parent DNA is incorporated into a new double-stranded DNA molecule
replication origin
a specific sequence of DNA that acts as a starting point for replication
helicase
a replication enzyme that separates and unwinds the DNA strands
replication fork
the point of separation of the two parent DNA strands during replication
topoisomerases
a class of enzymes that relieve tension caused by the unwinding of parent DNA; they cleave one or two of the DNA strands, allow the strand(s) to untwist, and then rejoin the strand(s)
