Molecular genetics lecture on replication and polymerase

1. MIRPUR UNIVERSITY OF SCIENCE AND TECHNOLOGY (MUST), MIRPUR
DEPARMENT OF BIOTECHNOLOGY

2. INTRODUCTION TO MOLECULAR GENETICS
HND-2302
Lecture: DNA Replication and Polymerases
Faculty of Natural and applied sciences
Dr Zeeshan Shamim
(Assistant Professor)

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DNA REPLICATION IS SEMICONSERVATIVE
That’s all for
DNA
replication
 DNA replication is the process of copying cell’s DNA.
Three billion base pairs are accurately copied during
division of one of your trillions cells.
 The basic mechanism of DNA replication in organisms is
similar.
 DNA replication if semiconservative, each strand acts as
template for the synthesis of new complementary strand.
 Thus, two daughter molecules are formed from one
starting molecule.
INTRODUCTION TO MOLECULAR GENETICS HND-2302. DR. Zeeshan Shamim

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 In a cell, DNA needs to be copied accurately so to avoid defective DNA in next cell.
 To perform the task smoothly, cell uses variety of proteins and enzymes to accomplish the process of
replication.
 In prokaryotes, three types of main DNA polymerases are known:
• DNA polymerse I
• DNA polymerase II
• DNA polymerase III
 DNA polymerases are responsible for synthesizing DNA by adding nucleotides one by one to the
growing chain of DNA.
DNA POLYMERASES
INTRODUCTION TO MOLECULAR GENETICS HND-2302. DR. Zeeshan Shamim

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KEY FEATURES OF ESCHERICHIA COLI DNA
POLYMERASES
 A template is always required.
 DNA polymerases add nucleotides to the 3’ of a DNA
strand.
 A short sequence of oligonucleotide called primer is
required to initiate the replication process.
 They have “proof reading” abilities which are to
remove wrong nucleotides which are incorporated
accidently during the replication process.
 DNA polymerase I and III are the major polymerases in
E. coli.
INTRODUCTION TO MOLECULAR GENETICS HND-2302. DR. Zeeshan Shamim

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DNA POLYMERASE I
 This is not the primary enzyme of replication, instead it performs clean up
functions during replication, repair and recombination.
Pol I (109 kDa) possesses four enzymatic activities:
 A 5'→3' (forward) DNA-dependent DNA polymerase activity, requiring a 3'
primer site and a template strand
 A 3'→5' (reverse) exonuclease activity that mediates proofreading (deletion of
this domain leaves a large Klenow fragment which has polymerization activity)
 A 5'→3' (forward) exonuclease activity mediating nick translation during DNA
repair.
 A 5'→3' (forward) RNA-dependent DNA polymerase activity. Pol I operates on
RNA templates with considerably lower efficiency (0.1–0.4%) than it does DNA
templates, and this activity is probably of only limited biological significance.
INTRODUCTION TO MOLECULAR GENETICS HND-2302. DR. Zeeshan Shamim

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INTRODUCTION TO MOLECULAR GENETICS HND-2302. DR. Zeeshan Shamim
DNA POLYMERASE I 3’-5’ EXONUCLEASE ACTIVITY

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KLENOW FRAGMENT
INTRODUCTION TO MOLECULAR GENETICS HND-2302. DR. Zeeshan Shamim

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DNA POLYMERASE I 5’-3’ EXONUCLEASE ACTIVITY
INTRODUCTION TO MOLECULAR GENETICS HND-2302. DR. Zeeshan Shamim

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DNA POLYMERASE III
Thomas Kornberg
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DNA POLYMERASE III
The replisome (multiprotein complex) is composed of the following:
•2 DNA Pol III enzymes, each comprising α, ε and θ subunits. (It has been proven that there is a third
copy of Pol III at the replisome.)
• the α subunit (encoded by the dnaE gene) has the polymerase activity.
• the ε subunit (dnaQ) has 3'→5' exonuclease activity.
• the θ subunit (holE) stimulates the ε subunit's proofreading.
•2 β units (dnaN) which act as sliding DNA clamps, they keep the polymerase bound to the DNA.
•2 τ units (dnaX) which act to dimerize two of the core enzymes (α, ε, and θ subunits).
•1 γ unit (also dnaX) which acts as a clamp loader for the lagging strand Okazaki fragments, helping the
two β subunits to form a unit and bind to DNA. The γ unit is made up of 5 γ subunits which include 3 γ
subunits, 1 δ subunit (holA), and 1 δ' subunit (holB). The δ is involved in copying of the lagging strand.
• (holC) and Ψ (holD) which form a 1:1 complex and bind to γ or τ. X can also mediate the switch from
RNA primer to DNA
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DNA POLYMERASE III ACTIVITY
DNA polymerase III synthesizes base pairs at a rate of around 1000
nucleotides per second. DNA Pol III activity begins after strand
separation at the origin of replication. Because DNA synthesis
cannot start de novo, an RNA primer, complementary to part of the
single-stranded DNA, is synthesized by primase (an RNA
polymerase)
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ORIGIN OF REPLICATION (OriC)
HOW DO DNA
POLYMERASES THINK
WHERE TO BEGIN
REPLICATION
DNA replication starts from a very specific region on DNA which is
called Origin of replication which is recognized by its specific
nucleotides.
Most bacteria including E. coli have single A/T rich OriC which is
245 bp in case of E. coli.
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ORIGIN OF REPLICATION (OriC)
• The origin of replication in E. coli is
termed oriC
– origin of Chromosomal replication
• Three types of DNA sequences in
oriC are functionally significant
– AT-rich region (DnaB box)
– DnaA boxes
– GATC methylation sites
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OriC OF E. COLI CHROMOSOME
 DnaA is a protein that activates initiation of DNA replication in
bacteria. It is a replication initiation factor which promotes the
unwinding of DNA at oriC.
 DnaB helicase is an enzyme in bacteria which opens the
replication fork during DNA replication.
 dnaC is a loading factor that complexes with the C-terminus of
helicase dnaB and inhibits it from unwinding the dsDNA at a
replication fork.
 Single-stranded DNA-binding protein (SSB) binds to single-
stranded regions of DNA. It prevents the strands from hardening
too early during replication, it protects the single-stranded DNA
from being broken down by nucleases during repair, and it
removes the secondary structure of the strands so that other
enzymes are able to access them and act effectively upon the
strands
INTRODUCTION TO MOLECULAR GENETICS HND-2302. DR. Zeeshan Shamim

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PRIMERS AND PRIMASE
 DNA polymerases can not initiate chain extension by binding on
single stranded chain (with some exceptions).
 They always need DNA-RNA duplex to initiate nucleotide addition
process on template strand.
 This problem is solved by an enzyme primase (encoded by dnaG
gene).
 The enzyme DnaG, and any other DNA primase, synthesizes short
strands of RNA known as oligonucleotides during DNA replication.
These oligonucleotides are known as primers because they act as
a starting point for DNA synthesis. DnaG catalyzes the synthesis
of oligonucleotides that are 10 to 60 nucleotides (the fundamental
unit of DNA and RNA) long, however most of the oligonucleotides
synthesized are 11 nucleotides
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LEADING AND LAGGING STRANDS
 In E. coli, DNA polymerase III is the main polymerase
involved in the replication process.
 There are two DNA molecules hard on work on each of the
replication forks to synthesized new daughter strand.
 DNA polymerization can only proceed form 5’-3’ direction
only.
 DNA double helix is antiparallel, one strand runs from 5’-3’
while other runs from 3’-5’ direction.
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LEADING AND LAGGING STRANDS
 The helicase unzips the double-stranded DNA for replication, making a
forked structure.
 The primase generates short strands of RNA that bind to the single-
stranded DNA to initiate DNA synthesis by the DNA polymerase.
 This enzyme can work only in the 5' to 3' direction, so it replicates the
leading strand continuously.
 Lagging-strand replication is discontinuous, with short Okazaki fragments
being formed and later linked together.
 The leading strand can be extended from one primer alone, whereas the
lagging strand needs a new primer for each of the short Okazaki fragments.
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MAINTENANCE AND CLEAN UP CREW
There are few more enzymes other than discussed previously which are needed for smooth
replication process.
 A DNA clamp, also known as a sliding clamp or β-clamp, is a protein complex that protein
binds DNA polymerase and prevents this enzyme from dissociating from the template DNA
strand.
 Topoisomerases also plays an important maintenance role in DNA replication. They
participate in the overwinding or underwinding of DNA. The winding problem of DNA arises
due to the intertwined nature of its double-helical structure. During DNA replication and
transcription, DNA becomes overwound ahead of a replication fork.
 Finally, the RNA primers are removed by DNA polymerase I.
 The nicks that remain after the primers are replaced get sealed by the enzyme DNA ligase.
INTRODUCTION TO MOLECULAR GENETICS HND-2302. DR. Zeeshan Shamim

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DNA REPLICATION PROCESS
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DNA REPLICATION TERMINATION
• At the last stage of termination, two replication
fork meets at terminator recognizing sequences,
called as a Ter.
• It contains ten sequences, TerA to TerJ, which are
binding sites for ''tus'', terminator utilization
substance.
• The Ter sequences are ~ 20 bp long and contain
the conserved sequence
5'-GTGTGTTGT-3'.
• Ter sequences with TUS protein create a complex
which arrests the replication fork and prevent
them to escape.
• At this complex, the process of replication is
completed and all other proteins and enzymes
leave this site.
• Only DNA topoisomerase II remains in action, it
cuts both strands, dissociates Ter-TUS complex
and two different circular DNA is generated.
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SUMMARY
 Helicase unzips two DNA strands by disrupting hydrogen bonds between nitrogenous bases
 Single strand binding proteins bind to single strands to prevent rewinding of the DNA strands.
 Topoisomerase move ahead of the replication fork to ease supercoiling.
 Primase synthesizes RNA primers complementary to the DNA template strand.
 DNA polymerase III strands DNA chain extension process by adding nucleotides to 3’-end.
 DNA polymerase I removes RNA primers from lagging and leading strands.
 DNA ligase seals nicks between DNA fragments.
 Tus protein binds to ter sequences, hence stops helicase activity and the replication process is
terminated.
INTRODUCTION TO MOLECULAR GENETICS HND-2302. DR. Zeeshan Shamim