Review
Chapter 2 and
Figure 3.35 on
nucleotides.
Complementary base pairs
DNA double helix
Anti-parallel
complementary
Sequence of nucleotides in
human beta-globin gene
The DNA sequences
highlighted in color show
the three regions of the
gene that specify the
amino acid sequence.
The complete set of
information in an
organisms’ DNA is its
genome
Not all of the
DNA in genes is
used to encode the
proteins that they
specify; much of
the rest is
concerned with
determining when,
and in what
amounts, the
protein encoded is
made – regulatory
regions of genes
Complementary base
pairing allows each
strand to act as a
template, or mold for
the synthesis of a new
complementary
strand.
DNA Replication
Anti-parallel
Copying must be carried out with speed and accuracy (8 hours
in a dividing animal cell). Is carried out by a cluster of proteins
that together form a “replication machine”
DNA replication is
“semiconservative” because
each daughter DNA double
helix is composed of one
conserved (old) strand and
one newly synthesized strand
DNA double helix is very stable. Many hydrogen bonds, weak
on their own, are very strong together.
The process of replication is begun by initiator proteins that
bind to the DNA and pry the two strands apart
Replication origins are marked by a particular sequence of
nucleotides that 1. bind to initiator proteins and 2. have stretches
that are A-T rich. Why?
Bidirectional and move very fast –
1000 nucleotide pairs /sec
Pyrophosphate
is then
hydrolyzed –
making
polymerization
irreversible
Why?
DNA polymerase adds nucleotides that are
complementary to the template to the 3’ end
by forming phosphodiester bonds between
this end and the 5’-phosphate group of the
incoming nucleotide.
DNA polymerase stays associated with the DNA, with the
help of other molecules that are part of the complex,.and
moves along it stepwise. DNA polymerase can only add
nucleotides in one direction, at the 3’ end.
Replication forks
of all cells,
procaryotic and
eucaryotic have
leading and
lagging strands
DNA polymerase is selfcorrecting. It makes only about
one error in ever 107 nucleotide
pairs replicated, but this is too
many for survival of an
organism.
Error-correcting activity is
called proofreading. Before
adding the next nucleotide,
DNA polymerase checks the one
it just added. If there is a
mispaired nucleotide, it is
removed using the nuclease
activity.
DNA polymerase has both 5’ –
3’ polymerization activity and 3’
– 5’ nuclease activity
DNA
polymerase can
not start a new
strand of DNA,
it only
elongates an
existing strand.
If DNA polymerase could
polymerize 3’ to 5’ it would
lose its ability to proofread.
Why?
DNA polymerase can not polymerize 3’ to
5’. It can not add nucleotides to the 5’ end
and there is no DNA polymerase that can.
DNA polymerase can not start a new
strand of DNA. It can only elongate an
existing strand. Therefore primers are
required These are RNA not DNA since
they are only temporary. This is done by a
primase. Primerase can not proofread and
leaves many mistakes.
Primers are removed by nucleases that
recognize RNA/DNA helices. This leaves
a gap filled in by a DNA repair
polymerase. And DNA ligase joins the
nucleotides at the nick..
One primer is required for the leading
strand. Multiple primers are needed for
the lagging strand.
Okazaki fragments
• DNA helicase uses energy from ATP to
speed along DNA, breaking the many
hydrogen bonds and opening the double
helix as it goes.
• Single-stranded binding proteins keep the
helix open and protect the single strand of
DNA.
• Sliding clamp keeps DNA polymerase
firmly attached to DNA template by
forming a ring around the DNA helix and
binding DNA polymerase.
Theoretical multienzyme complex moves as a unit along
the DNA. However, there is much we do not understand.
DNA Repair
Rare beneficial DNA mutations
(mistakes or damage) allow for
evolution of organisms in the face
of ever changing environments.
However, in the short term, DNA
mutations are almost always
detrimental to an organism.
Genetic stability is the result of
the accuracy of DNA polymerase
and the many mechanisms for
proof-reading and DNA repair.
When these processes fail, a
permanent change in the DNA
sequence occurs - mutation.
Sickle-cell anemia
Glutomic acid to
valine at 6th
position of 146 aa
Mutations in germ cells
(reproductive cells) lead to
genetic diseases like sicklecell anemia.
Somatic cells must also be
protected from damage to the
DNA. Gradual accumulation
of mutations in the DNA of
somatic cells can eventually
lead to a lack of replication
control and cancer - one cell
grows uncontrollably.
These mutations accumulate
and cancer is more likely as
we age.
DNA Mismatach Repair removes replication errors that escape from
the replication machine. Corrects 99% of the replication errors.
Accuracy is one in 107 base pairs. Mismatch repair proteins must
excise (remove) the mismatched nucleotide from the new strand.
How does the enzyme complex know which is which? The new
strand may remain nicked for a short time, also old strands may have
chemical modifications, such as methylation.
DNA mismatch repair
proteins are thought to
recognize (bind to) the
distortion in the geometry
of the DNA double helix
resulting from an incorrect
base pairing. They may
recognize the correct (old)
strand by occasional nicks
(which would only be
present for a short time
after replication is
complete.
Even though DNA is one of the most stable molecules,
thermal collisions do sometimes cause changes - including
depurination and deamination.
Ultraviolet radiation in sunlight damages DNA and
promotes covalent linkage between two adjacent
pyrimidine bases - forming thymine dimers.
Many different repair enzymes exist to correct and repair
the damage. When someone inherits a mutation in the
gene responsible for producing one of the mismatch
repair proteins, they may be predisposed to certain
cancers.
If uncorrected, the result is a permanent mutation.
1. There are a
variety of enzymes
that recognize and
exise different types
of DNA damage
Step 2 and 3 are
nearly the same for
most types of DNA
repair.
Since the damaged
strand usually has a
distinct structure,
different from normal
DNA structure, it can
be “recognized” by the
DNA repair enzyme.
And, because each
strand is
complementary to each
other, there is a
template to copy.
• Single-celled organisms like yeast have
more than 50 different proteins that function
in DNA repair. Probably much more
complex in humans.
• Xeroderma pigmentosum is a genetic
disease in which the gene for one of these
DNA repair enzymes is mutated. Severe
skin lesions, including cancer, are the result
of an accumulation of thymine dimers in
cells exposed to sunlight.
DNA is extremely stable. Changes accumulate slowly
in the course of evolution, as evidenced by the degree of
homology between many genes even in unrelated
organisms like fruit flies and humans.