TRINITY SCHOOL OF

MEDICINE

GENETICS
MIGUEL MIYARES CALÁS M.D. Ph.D.

PROFESSOR
REPLICATION
 OBJECTIVES
• List the general molecular genetics mechanisms
• List the steps of any molecular genetics mechanism
• Enunciate the definition of replication
• Describe the general mechanism of replication
• List the enzymes and proteins required for replication in prokaryotes and
eukaryotes
• Explain the action of DNA dependent DNA polymerases
• Describe the initiation of replication in prokaryotes
• Describe the elongation of replication in prokaryotes
• Differentiate the leading and lagging strand synthesis mechanisms
• Enunciate the definition and mention the importance of the Okazaki
fragments
• Describe the mechanism of termination of replication in prokaryotes
• Compare the replication in prokaryotes and eukaryotes
 GENERAL MOLECULAR
GENETICS MECHANISMS

Synthesis of DNA (Replication)

Synthesis of RNA (Transcription)

Synthesis of proteins (Translation)

GENERAL CHARACTERISTICS
OF MOLECULAR BIOLOGY
MECHANISMS
All biosynthetic process has been divided in 5 steps:

1.- Activation of monomeric subunits

2- Initiation

3.- Elongation

4.- Termination

5.- Modification (maturation or processing)

Mechanisms are different in prokaryotes than in eukaryotes

 DNA REPLICATION

Arthur Kornberg

It’s the synthesis of DNA

 GENERAL CHARACTERISTICS
OF REPLICATION
1.- Synthesis is semiconservative
2.- The helix must be unwound
3.- It’s highly accurate
4.- It’s very rapid
5.- The enzyme that copy DNA polymerize
nucleotides only in the 5’→3’ direction
6.- One strand is synthesized in a continuous
fashion whereas the opposite strand is
synthesized in fragment (discontinuous fashion)

Leading Lagging
strand strand
GENERAL CHARACTERISTICS OF
PROKARYOTE REPLICATION
1. It has only one origin
2. It proceeds bidirectionally
3. It is semidiscontinuous

PROTEINS REQUIRED
 POLYMERIZING ENZYMES
DNA Polymerase I 3’→5’ and 5’→3’ exonuclease Proofreading
DNA Polymerase II 3’→5’ exonuclease DNA repair
DNA Polymerase III 3’→5’ exonuclease Elongation
DNA Polymerase IV None of them is able to DNA repair
DNA Polymerase V synthesize a dinucleotide DNA repair
 INITIATION
Dam methylase methylated ori (regulated)
Origin of synthesis (Ori)
DnaA binds as a complex to the 9 bp repeats
HU partially denatures the double helix
DnaB binds with the help of DnaC and
acts as a helicase, unwinding DNA
SSB stabilizes the single strands
DNA gyrase relieves the positive
torsional strain from unwinding
ELONGATION
Occurs at the replication fork
A single complex replicates both strands
DNA loops around in order to undergo
bidirectional replication
Helicase unwinds DNA
Topoisomerase relieves stress
SSB stabilizes the single strands
DNA polymerase III synthesizes DNA

LEADING STRAND LAGGING STRAND

• Primase synthesizes an RNA primer • Primase makes an RNA primer
• DNA polymerase begins by adding its • Pol III makes a short stretch of DNA,
first nucleotide to an RNA 3’ hydroxyl until it reaches the previous RNA
primer (Okazaki fragment)
• DNA polymerase synthesizes a new
strand in a continuous fashion by • Pol I remove the RNA primer and fill
reading the template strand and the gap with deoxyribonucleotides
processively creating new strand • DNA ligase close the nick
 TERMINATION
Two replication forks traveling in two
directions would meet and collide
Termination sequences (ter) exist
about 180 degrees from ori
One set of termination sequences acts
on the counterclockwise fork, the
other on the clockwise fork
The termination sequences bind tus
protein (terminus utilization substance)
Topoisomerase IV separates
catenated chromosomes
 EUKARYOTIC REPLICATION
It is similar to Prokaryotic replication but ENZYMES
more complex
Pol a Primase
The enzymes are different
Pol b Repair
There are multiple origins of replication
Pol g Mitochondrial
Eukaryotic chromosomes are large and
multiple origins are necessary to complete Pol d Replication
DNA synthesis in a reasonable amount of time Pol e Unclear
Each origin gives rise to bidirectional Pol  Repair
replication, forming replication bubbles
Pol  Repair
Bubbles arise at regular intervals along a
eukaryotic chromosome Pol i Repair
By inference one would expect ori like
sequences, however the ori sequences are not
well identified in higher eukaryotes

Replication of linear chromosomes

results in the shortened of them
 TELOMERASE
Telomerase extends the lagging strand template by using an RNA
oligonucleotide as a template for DNA synthesis
It is a complex structure resulting from the interaction of a 150 nucleotides
RNA with proteins
It copies just a small fragment of the RNA several times
This is a reverse transcriptase