Flow of Genetic Information

Mechanisms for the transfer of information in the cell

DNA RNA
replication replication

DNA Trans cription RNA Trans lation Protein

Reverse
transcription
How do we get these
guys?
From what and how are
they made?
DNA Replication

Challenges to ds-DNA rep

DNA template antiparallel strands
has one 5 3 strand and one 3 5 strand
Enzymes (polymerases) only work in one direction!
How and why?
Enzymes (polymerases) have proofreading function
single-stranded DNA more susceptible to damage
ss-DNAases (nucleases)
Radiation and chemical attack
Errors more likely during replication (or in just ss-DNA)
continuous unwinding and separation of the two DNA strands?
Two strand intertwined! Not two circles!!!
DNA Replication

Advantage to ds-DNA, even though only one strand is used in some

(many) cases
Protection of DNA
back-up copy of information
Replication leads to two new copies
Not needed just better evolutionarily
DNA Replication

requires separation of two original strands which are templates for

synthesis of two new daughter strands
Semiconservative replication: each daughter double helix contains
one template strand and one newly synthesized strand
was established in the late 1950s by experiments of Meselson and Stahl
a key observation is that 15N-DNA has a higher density than 14N-DNA, and the
two can be separated by density-gradient ultracentrifugation
Semiconservative
replication
DNA Replication

DNA double helix unwinds at a specific point called an origin of replication

polynucleotide chains are synthesized in both directions ON BOTH STRANDS
from the origin of replication at replication forks
there is one origin of replication and two replication forks in the circular DNA
of prokaryotes
in replication of a eukaryotic chromosome, there are several origins of
replication and two replication forks at each origin
Why the difference?
DNA Polymerase Reaction

DNA is synthesized semidiscontinuously

from its 5 3 end (from the 3 5 direction of the template)
the leading strand is synthesized continuously in the 5 3 direction toward
the replication fork
the lagging strand is synthesized discontinuously as a series of Okazaki
fragments, also in the 5 3 direction, but away from the replication fork
Okazaki fragments of the lagging strand are joined by the enzyme DNA ligase
Why the difference?
DNA Polymerase Reaction
DNA Replication

DNA double helix unwinds at a specific point called an origin of

replication
polynucleotide chains are synthesized in both directions ON BOTH
STRANDS from the origin of replication at replication forks

there is one origin of replication and two replication forks in the

circular DNA of prokaryotes
in replication of a eukaryotic chromosome, there are several origins
of replication and two replication forks at each origin
Why the difference?
BUT WAIT! DNA IS ONLY MADE IN
5 3 DIRECTION
DNA Replication

DNA polymerase reaction requires

DNA template! (RNA template for RT)
all four deoxyribonucleoside triphosphates: dTTP, dATP, dGTP, and dCTP
only mono in product phosphate hydrolysis provides energy
Mg2+
an RNA primer - a short strand of RNA to which the growing polynucleotide
chain is covalently bonded in the early stages of replication

dTTP DNA-dependent
dATP DNA polymerase DNA + PP i
dGTP
dCTP
DNA Replication

Figure 9.10 General features of a replication fork

Eukaryotic Replication

Figure 9.21 Basics of a eukaryotic replication fork

Summary

1. DNA synthesis is bidirectional

Though polymerases only read in 3 5 direction and synthesize in 5 3
direction
Strand (fork) opens in both directions!
2. DNA synthesis is in the 5 3 direction
the leading strand is formed continuously
the lagging strand is formed as a series of Okazaki fragments which are later
joined
3. Primer is RNA
For proofreading
Summary

4. Other topographical and protection proteins needed!

DNA gyrase introduces a swivel point in advance of the replication fork
a helicase binds at the replication fork and promotes unwinding
single-stranded binding (SSB) protein protects exposed regions of single-
stranded DNA
5. Primer removed and DNA ligase seals remaining nicks
RNA removal by exonuclease
Created by discontinuous synthesis
Why How
double-stranded
is double helix used? DNA?
ONLY ONE STRAND IS USED!! How do you know?
The other strand is not a back up, per se.
Disadvantages?
SS-DNA found in nature
Cost? Both of antisense strand and of DNA
Advantages?
Limited reactivity
Repair
Of mismatches
Strand breaks!!!!
 Normal
A-T and G-C
Basepairing
differences between these pairs
purine and pyrimidine match
Are these the only possibilities?
Abnormal basepairing

Why are these considered abnormal?

Why are they seldom observed/tolerated in normal DNA?
 Why have Uracil and Thymine?
Disadvantages?
Costs more (time and energy)
New pool to maintain
Advantages???
Difference between uracil and cytosine
Increased mutations at 5-mCyt
Why?
Telomerase and Cancer