Macromolecular Genetics of Life

Cell Biology, BIOL310

Advancement of eukaryotic cell function
and activities governed by three Rs
Ability of cells to maintain a
higher degree of order in a
chaotic universe depends
upon the accurate duplication
of vast quantities of genetic
information
Maintaining this requires:
Replication with efficiency
(speed times accuracy)
Repair of mistakes and
damages that would ultimately
lead to dysfunctional protein
Recombination allows for the
generation of functional
diversity to better adapt,
survive, and thrive in an
evolving, changing world
Genetic stability via maintenance of
DNA sequences is necessary for survival
Germ cells transmit
genetic information from
parent to offspring
Somatic cells are cells
that give rise to
specialized tissue and
cells in the body
each differentiated to carry
out instructed tasks
Division of labor based on
their ability to access the
genetic code
Synthesis of the polymers for protein,
carbohydrates, and nucleotides
harvests the power of water

Base-pairing underlies DNA
replication and repair and highlights
the value of protein-DNA interactions
Process of recognizing a
nucleotide within a
single strand template
DNA and inserting its
complimentary base (or
nucleotide)
This is principally
mediated by the H-bond
interactions between A-
T and G-C

Semi-conservative inheritance highlights
the importance of covalent bonds vs. H-
bond in control of information
As all life follows the rule of semi-
conservative inheritance, all cellular life is
descendent from a predecessor cell
Each of the two daughter
cells inherits a new DNA
double helix containing
one original and one new
strand
semi-conservative
In all rounds of
replication, each of the
two DNA strands are used
as templates to form
complementary DNA
strands
Eukaryotes DNA replication takes part
only during S phase of cell cycle
Replication occurs only
during DNA synthesis (S)
phase of the cell cycle
Typically last about 8
hours in mammalian cells
About 40 minutes for
single celled organisms like
yeast
Directly proportional to
size and number of start
sites

DNA synthesis begins at replication origins,
which are conserved sequences to which
proteins bind in order to identify start site
DNA replication is begun
by initiator proteins that
bind to dsDNA at specific,
conserved sequences
These initiator proteins
bind and pry the two
strands apart, breaking the
H-bonds between bases
The conserved
sequences, or positions,
are called replication
origins

Eukaryotic chromosomes contain
multiple origins of replication
Because of the vast size of
the eukaryotic genome,
multiple origins of
replication must exist to
which DNA replication of
numerous parts can
proceed concomitantly
Speed x Accuracy
Within in a replication unit,
individual origins are
spaced at intervals of
about 30L-250K
nucleotides apart from one
another

Different regions on the same
chromosome replicate at distinct times
Different regions of each
chromosome are replicated in
a reproducible order during S
phase
This can be identified by the
multiple clusters of replication
forks seen at different time
points by radiography labeling
Highly condensed chromatin
replicated late, while genes in
less condensed chromatin is
replicated early
Accessibility is governed by
differential temporal expression
of genes
Synthesis is catalyzed by
DNA polymerase catalysis of nucleotide
addition of nucleotides in a copy-paste
The backbone of DNA is assembled
through simple monomer addition to a
growing chain a theme of Cell Biology
Multi-enzyme complex that
contains DNA polymerase
synthesizes the DNA of both
new daughter strands.
Importantly, DNA is always
synthesized in the 5 3
direction
It is never, ever possible to
synthesize in the 3 5
direction
Thus, a mechanism must be
put into place to add DNA in
the 53 direction while
simultaneously reading 5
3
H + OH
Finding a work around to traveling and
writing in the 5 3 direction, the
cell uses RNA primers-Okazaki Fragm.
DNA synthesis on the lagging
strand must be made initially
as a series of short DNA
molecules
These molecules are called
okazaki fragments
For the lagging strand, the
direction of nucleotide
polymerization is opposite to
the overall direction of DNA
chain growth
Most importantly, it is still made
in the 53 direction while
reading also in the 53
direction in a discontinuous
fashion
Finding a work around to traveling and
writing in the 5 3 direction, the cell
uses RNA primers-Okazaki Fragments
Most importantly, it is
still made in the 53
direction while reading
also in the 35
direction in a
discontinuous fashion
The DNA polymerase
knows where to start
and stop thanks to a
START SEQUENCE called
a RNA primer

DNA primase synthesizes RNA primer
molecules for lagging strand synthesis
A RNA primer is made
downstream
The short RNA primer can be
elongated by DNA polymerase
to make the complimentary
DNA strand
It begins by generating the
okazaki fragment that runs until
it meets up with the 5 end of
the RNA primer of the previous
fragment.
DNA repair machinery remove
the RNA primers and replace
them with DNA nucleotides,
DNA ligase effectively joins the
3 end of one okazaki fragment
with the 5 end of the next
okazaki fragment to seal the
DNA backbone

The drawback to using RNA primers for
lagging strand and working backwards is
that eventually you run out of room
Remember, the lagging
strand needs RNA primers
to place downstream so it
can synthesize back in the
correct 53 fashion
while reading in the 35
A problem then occurs
when you reach the very
end of the DNA in (non-
circular) Eukaryotic
genomes
How do you then add
primers to synthesize back
when there is no more
room?
Telomerase replicates chromosome ends
Eukaryotic cells have evolved
specific sequences at the
ends of their chromosomes
This is generally a rich repeat of
GGGTTA sequences
sequences serve as DNA binding
motif
Secondly, the eukaryotic cells
use telomerase enzymes that
extends the 3 end by adding
an RNA-sequence template at
the ends of chromosomes
Now, DNA polymerase has more
template to work with to
synthesize the end of the DNA
Telomere length is regulated by cells,
deregulation lead to uncontrolled
division/cell replication
The process of telomere
extension and contraction is
variable
thus lengths of telomere ends
can\are different from cell to
cell
In effect, the absence of
telomerase granted RNA
primers means the end of
appropriate DNA replication
and senescence of that cell
I.e. No more daughter cells;
cellular division is lost
Thus the replication extent of
life can be modulated by
telomerase activity

Numerous different proteins
cooperate in a concerted effort to
drive DNA replication
Proteins at a replication fork cooperate
to form a replication machine
Replication requires work, and the cell
readily uses its ATP molecules to drive
unfavorable energetic reactions
DNA replication requires several
proofreading mechanisms to ensure
the integrity of the copy
DNA thus requires the
necessity to ensure that
the copies it makes are not
filled with errors or every
subsequent copy will be
faulty.
DNA is so efficient that
there is only 1 mistake for
every 10
9
nucleotides copied
The mistakes occur at the
level of mismatched base-
pairing

Energetically favorable nucleotides provide
means to ensure correct addition
DNA polymerase performs
the first proofreading step
immediately before a new
nucleotide is added to the
growing chain
The correct nucleotide has a
higher affinity for a moving
polymerase that does the
incorrect nucleotide
Wrong pairing is
energetically unfavorable
Thus the first true
proofreading step is to
ensure energetically
favorable nucleotides
Strand-directed mismatch repair
system removes replication errors
Strand-directed mismatch
repair system detects the
potential for distortion in
the DNA helix from the
misfit between non-
complementary bases
After removing the DNA
from the newly synthesized
strand, the DNA repair
system adds in the
appropriate nucleotides
following the original DNA
template
How does the machinery tell
the template strand vs. the
newly synthesized strand?
The rate of mutation is exactly related
to the number of proteins (i.e. genes)
Mutation rates: rate at
which changes in nucleotide
base pairs occur; often
times equated to
observable changes
(phenotype)
For an organism to
maximize productivity, it
has evolved to a saturated
point balancing rate of
mutations versus the
probability of accruing that
mutation in genes
This has put pressure on the
genome to keep only a
maximum of 50K genes