DNA replication in prokaryotes

Part 1

Dr. Kara van Aelst

School of Biochemistry
 Biochemistry: Cellular Composition BIOC1003

Lecture 20
DNA replication and repair in Prokaryotes

• DNA replication
• DNA polymerases
• DNA synthesis
• Introduction to DNA repair pathways
 Central Dogma
DNA
Replication

DNA RNA protein

Transcription Translation
(RNA synthesis) (protein synthesis)
In Watson and Crick’s understated turn-of-
phrase from their 1953 paper:
“It has not escaped our notice that the
specific pairing we have postulated
immediately suggests a possible copying
mechanism for the genetic material.”

Two strands of DNA, each acting as a

template for the other
 DNA replication is semi-conservative

semi-conservative; one strand is the old strand and one strand is new

Meselson-Stahl experiment – e.g. Voet & Voet, p88-90, Figure 5-13

DNA is synthesis

DNA is synthesized from dNTP building

blocks using a DNA template
 Genome replication in Escherichia coli

Replication
origin
(oriC)
Escherichia coli K12
Circular genome
4,639,221 bp
4485 genes
(4288 protein coding)

Terminator
sequences
(Ter)

this DNA is replicated about once every 20 minutes

 E. coli polymerases

DNA polymerase I - DNA replication & repair

(1957)

DNA polymerase II - DNA repair

DNA polymerase III - Main DNA replicating enzyme

(1972)

DNA polymerases IV & V - DNA Repair

DNA polymerases core enzyme fold

Template
DNA

Newly-synthesized DNA
 DNA polymerase III

• DNA polymerase III is the main replicating enzyme in E. coli

• 9 protein subunits

• DNA synthesis activity

• Independent of sequence

• 250-1000 nucleotides (nt) per second

• Capable of synthesising 1000s of nt before dissociating

• 3′ - 5′ exonuclease activity

• Proofreading to minimise copying mistakes

 DNA polymerases requirements

• DNA polymerase uses all 4 dNTPs (i.e. dATP, dGTP, dCTP, dTTP) as the
immediate precursors of DNA synthesis.

• DNA polymerase synthesises a complementary copy of the DNA template

• DNA polymerase can only add a dNTP to the 3' end of a pre-existing DNA
or RNA molecule, so it needs a primer with a free 3'-hydroxyl group.

• DNA polymerase requires Mg2+ to function.

 Replication starts at the origin

Specific binding sequences in oriC

e.g. DnaA in E. coli

Allow two replication

forks to establish
 DNA Helicase
• DNA helicase unwinds dsDNA to produce ssDNA for replication

e.g. DnaB in E. coli

 Single stranded binding (SSB) proteins
• Single stranded binding protein binds ssDNA non-specifically and
cooperatively, removing secondary structure

e.g. SSB in E. coli

 Primase
• Primase synthesizes RNA primers de novo

NTPs not dNTPs e.g. DnaG in E. coli

• Primase is a specialised RNA

polymerase
• Makes short RNA primers (5-10 nt)
• No proofreading
• No specific sequences for initiation
• Frequency of priming different on
leading/lagging strands
 Replication fork

Helicase
= RNA primer
Single-stranded
binding proteins
Bi-directional replication fork
oriC

Initiation

Bidirectional replication established

 DNA replication in prokaryotes
Part 2

Dr. Kara van Aelst

School of Biochemistry
 DNA polymerisation
• DNA polymerase adds deoxynucleotide triphosphates (dNTPs) to the 3' end of
the growing chain.

• DNA polymerase makes DNA in the 5' to 3' direction.

• Each addition creates a new 3’ OH for the next addition.

Nucleophilic attack

DNA chain grows in 5’ to 3’ direction

 Catalytic site DNA polymerase III

Mg2+ play a role in: TEMPLATE

• stabilising these phosphates

• activating the OH to make it a
better nucleophile
NEW
NUCLEOTIDE

PRIMER

Catalytic site requires:

• Aspartates (acidic residues)
• Mg2+
• Triphosphate (high energy
bond)
 DNA proofreading
Incorrect base!

3′ - 5 ′ exonuclease activity of Pol III

DNA Proofreading – 3′ - 5 ′ exonuclease removes incorrect nucleotide

Nuclease Enzyme that cleaves phosphodiester bonds

Exonuclease Cleaves nucleotides one-at-a-time from ends of polynucleotides
Endonuclease Cleaves within polynucleotides
 Discontinuous DNA replication
5' 3' 5' 3'

Replication Fork

3'
5' 3' 5' 3'
5' 3'
5'

The “leading strand” section is made continuously but the “lagging strand”
section is made discontinuously.

As the DNA is unwound at each fork primase repeatedly makes new primers on
the lagging strands.
 Discontinuous DNA replication
Lagging strand Leading strand

Leading strand Lagging strand

DNA
 Connecting Okazaki fragments

DNA Pol I
• DNA Pol I removes RNA primers 5´- 3´ exonuclease activity
(5´-3´ exonuclease)

• DNA Pol I replaces with DNA (5´-3´

polymerase) DNA Pol I
5´- 3´polymerase activity

• 3´-5´ exonuclease proofreading

• DNA ligase catalyses phosphate DNA ligase

linkage
 Replication fork

Helicase
= RNA primer
Single-stranded
binding proteins
https://youtu.be/TEQMeP9GG6M

https://youtu.be/49fmm2WoWBs
Replication termination
oriC

Initiation
 Replication termination

Terminator Ter DNA sequences, TerA, TerB, ..., TerJ.

sequences
(Ter)

e.g. Tus binds Ter sites in E. coli

Replication fork

Physical block to replication fork

 DNA repair

• Multiple pathways for DNA repair in E. coli

Direct Repair
Mismatch Repair (MMR)
Base Excision Repair (BER)
Nucleotide Excision Repair (NER)
Type of damage
Specific base damage
Incorrect base pairs introduced during
replication
Range of damaged bases
Wide range of bulky DNA damage

Homologous Recombination dsDNA breaks

More detail in GER in year 2

 DNA repair

Direct Repair

Damage directly
removed by enzyme

MMR, BER and NER - Gap repair

Damage recognised Patch repair by DNA

and a section of DNA polymerase (II,IV,V)
removed to give a gap and DNA ligase
 Thank you!

Dr. Kara van Aelst

School of Biochemistry