BCH323
Information flow and
introduction to Bioinformatics
Dr XH Makhoba
LECTURE #2 – DNA Replication
DNA dependent DNA polymerase
DNA dependent RNA polymerase
RNA dependent DNA polymerase
RNA dependent RNA polymerase
Ribosomes; mRNA; tRNA; rRNA
Page 93
Voet Biochemistry 3e
© 2004 John Wiley & Sons, Inc.
Figure 5-22
Gene expression.
Enzymes/Proteins required
1.
2.
3.
4.
5.
6.
7.
8.
Proteins DnaA from dnaA gene and DnaC from the dnaC gene.
DNA gyrase
o Type II topoisomerase, introduces negative supercoils at the expense of ATP hydrolysis, to overcome torsional stress imposed by unwinding.
Helicase (DnaB)
o Synthesized from the dnaB gene
o Separates the strands of DNA by hydrolyzing ATP and disrupts the hydrogen bonds holding the two strands together
Single strand binding proteins
o Prevent reannealing of the single stranded DNA
Primase (DnaG)
o 60 kD monomeric protein synthesized from the dnaG gene
o DNA dependent RNA polymerase
o Synthesizes a 1-60 bp long RNA primer on the lagging strand to initiate the synthesis of a Okazaki fragment (1000 – 2000 nt long in prokaryotes)
o It is NOT inhibited by rifampicin (a RNA polymerase inhibitor)
DNA dependent RNA polymerase
o Together with Primase, mediates the initiation of the leading strand synthesis, which occurs only once in a replication cycle
DNA dependent DNA polymerases
o DNA polymerase I, II and III in E. coli
o DNA polymerase , ,  and  in Eukaryotes
o Involved in de novo synthesis of DNA strands and DNA repair
DNA ligase
o Catalyzes the reaction that seals the single stranded nicks formed between adjacent Okazaki fragments, as well as those formed on circular DNA after
leading strand synthesis
DNA Replication
Replication of duplex DNA is a complex mechanism that involves a whole
group of enzyme activities involved in:
1) Initiation – recognition of an origin by a protein complex (primosome),
which catalyze the separation of the parental strands, stabilize the
single-stranded state and initiate synthesis of daughter strands at the
replication fork
2) Elongation – protein complex (replisome) that is associated with the
particular structure that DNA takes at the replication fork
3) Termination – at the end of replication joining and/or termination
reactions are necessary followed by separation of the duplicate
chromosomes from each other
Prokaryotes
•
•
•
•
•
Initiation
• Regulation of chromosomal replication occurs at the level of initiation.
• Occurs at a unique genetic site, the replicative origin (ori).
Enzymes/Proteins required include:
• DNA gyrase
• Helicase (DnaB)
• Single strand binding proteins
• Primase (DnaG)
• DNA dependent RNA polymerase
• Proteins DnaA (Recognizes and binds the DnaA boxes) and DnaC (an ATPase)
The oriC is a unique 245-bp segment. The DnaA proteins recognize and bind the five DnaA boxes, each
containing a highly conserved 9-bp consensus sequence (5’-TTATCCACA-3’), to form a complex of negatively
supercoiled oriC DNA wrapped around a central core of five DnaA protein monomers. The DnaA proteins
successively melt three tandemly repeated, 13-bp, AT-rich fragments (5’-GATCTNTTNTTTT-3’) located near
the oriC.
Two DnaB6-DnaC6 complexes are recruited to opposite ends of the replication “bubble” to form the
PREPRIMING COMPLEX. Five additional DnaA proteins bind to the DnaA boxes to form five dimers at the
DnaA boxes. The DnaC (an ATPase that facilitates DnaB loading) is subsequently released.
Together with single strand binding proteins and gyrase, the DnaB helicase further unwinds the DNA in
both directions allowing the entry of the primase and RNA polymerase
Prokaryotes - Elongation
Elongation
• DNA Polymerases:
• An enzyme that can synthesis a new DNA strand on a template strand.
• Multiple DNA polymerase activities present – some involved with replication while others are involved in
subsidiary roles or repair
• All prokaryotic and Eukaryotic DNA polymerases share the same fundamental type of synthetic activity
where the DNA strand is extended by adding nucleotides one at a time to a 3’-OH end dictated by base
pairing with the template strand
• DNA polymerases also have a mechanism to ensure fidelity of replication. This can be achieved by either
scrutinizing the incoming base for proper complementarity with the template (presynthetic error control)
OR scrutinize the base pair after the new base has been added (proofreading). All bacterial enzymes
possess a 3’-5’ exonuclease activity that proceeds in the reverse direction from DNA synthesis, thus
providing a proofreading control
• Different DNA polymerases handle the relationship between the polymerizing and proofreading activities in
different ways – sometimes it is part of the same protein subunit, sometimes it is contained in a different
subunit
Prokaryotes - Elongation
• In E. coli three enzymes have been identified:
• DNA polymerase I – involved in repair and in a subsidiary role in DNA
semiconservative replication
• DNA polymerase II – implicated in SOS response in DNA repair
• DNA polymerase III – a multisubunit protein responsible for de novo synthesis
of new DNA strands
DNA Polymerase III
• responsible for replication in E. coli.
• It has several subunits:
• the dnaE locus which codes for the 130kD  subunit with DNA synthetic
activity;
• the dnaQ locus which codes for the 27.5 kD  subunit with 3’-5’ exonuclease
activity. It works in conjunction with the  subunit by providing proofreading
during DNA replication.
• The role of the third 10kD subunit, , is unknown
DNA polymerase errors include:
• Substitutions due to the incorporation of a wrong nucleotide – this is corrected by
the 3’-5’ proofreading activity of each DNA polymerase
• Frameshifts when an extra nucleotide is inserted of left out – fidelity is affected by
processivity (the tendency to remain on a single template rather than dissociate
and reassociate).
DNA polymerase I
• A single polypeptide (103kD)
• one subunit (68kD, Klenow fragment) containing polymerase and 3’-5’ exonuclease
activity in different regions of the protein
• one subunit (35kD) contains a 5’-3’ exonuclease activity which excises small groups
of nucleotides up to ~10 bases at a time.
• This activity is coordinated with the proofreading activity and enables DNA
polymerase I to have the unique ability to start replication in vitro at a nick in DNA.
This process is called nicked translation. The displaced strand is degraded by the 5’3’ exonuclease activity.
• DNA Polymerase I in bacterium, is not responsible for replication. The 5’-3’
synthetic/3’-5’ exonucleolytic action is used:
• In Nick translation, where the 5’-3’ activity removes the preceeding DNA at the
nick and the polymerase activity replaces the nucleotides at the 3’-OH site.
• In the excising of the RNA primers.
Termination and segregation
Termination of replication occurs at a specific site, diametrically opposite
from oriC, named terC.
• ter binding protein blocks the bidirectionally moving replicative forks from
moving clockwise or counter clockwise.
• The ter region contains a number of short DNA sequences containing a
consensus core element 5’-GTGTGTTGT
• A ter sequence element will empede replication fork progression only if
orientated in the proper direction with respect to the approaching replication fork
and only if a specific replication termination protein, Tus protein (a
contahelicase), is bound to it.
• Following termination of DNA replication, the resulting chromosomes must be
segregated into daughter cells.
DNA Polymerase
•
DNA is synthesized 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 semidiscontinuously (Okazaki
fragments) also in the 5’ -> 3’ direction, but away from the replication
fork
• lagging strand fragments are joined by the enzyme DNA ligase
DNA Polymerase Reaction
• The 3’-OH group at the end of the growing DNA
chain acts as a nucleophile.
• The phosphorus adjacent to the sugar is attacked,
and then added to the growing chain.
Function of DNA Polymerase
• DNA polymerase function has the following requirements:
• all four deoxyribonucleoside triphosphates: dTTP, dATP, dGTP, and dCTP
• Mg2+
• an RNA primer - a short strand of RNA to which the growing
polynucleotide chain is covalently bonded in the early stages of replication
• DNA-Pol I: repair and patching of DNA
• DNA-Pol III: responsible for the polymerization of the newly formed
DNA strand
• DNA-Pol II, IV, and V: proofreading and repair enzymes
Supercoiling and Replication
• DNA gyrase (class II
topoisomerase) catalyzes
reaction involving relaxed
circular DNA:
• creates a nick in relaxed
circular DNA
• a slight unwinding at the
point of the nick
introduces supercoiling
• the nick is resealed
• The energy required for this
process is supplied by the
hydrolysis of ATP to ADP
and Pi
Replication with Supercoiled DNA
• Replication of supercoiled circular DNA
• DNA gyrase has different role here. It introduces a nick in supercoiled
DNA
• a swivel point is created at the site of the nick
• the gyrase opens and reseals the swivel point in advance of the replication
fork
• the newly synthesized DNA automatically assumes the supercoiled form
because it does not have the nick at the swivel point
• helicase, a helix-destabilizing protein, promotes unwinding by binding at
the replication fork
• single-stranded binding (SSB) protein stabilizes single-stranded regions
by binding tightly to them
Primase Reaction
• The primase reaction
• RNA serves as a primer in DNA replication
• primer activity first observed in-vivo.
• Primase - catalyzes the copying of a short stretch of the DNA template
strand to produce RNA primer sequence
• Synthesis and linking of new DNA strands
• begun by DNA polymerase III
• the newly formed DNA is linked to the 3’-OH of the RNA primer
• as the replication fork moves away, the RNA primer is removed by DNA
polymerase I
Replication of duplex DNA in E. coli.
https://www.youtube.com/watch?v=-qAr5Ib_6as
https://www.youtube.com/watch?v=FBYeBb4C5Rc
https://www.khanacademy.org/science/high-school-biology/hs-molecular-genetics/hs-discovery-and-structure-of-dna/v/leading-and-lagging-strands-in-dna-replication
Replication Fork General Features
Replacement of RNA primers by DNA in lagging strand
synthesis.
Function of DNA ligase.
The 3 5 exonuclease function of DNA polymerase I
and DNA polymerase III.
Summary of DNA Replication in Prokaryotes
• DNA synthesis is bidirectional
• 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
• Five DNA polymerases have been found to exist in E. coli
• Pol I is involved in synthesis and repair
• Pol II, IV, and V are for repair under unique conditions
• Pol III is primarily responsible for new synthesis
Summary of DNA Replication in Prokaryotes
• Unwinding
• 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 singlestranded DNA
• Primase catalyzes the synthesis of RNA primer
• Synthesis
• catalyzed by Pol III
• primer removed by Pol I
• DNA ligase seals remaining nicks
Proofreading and Repair
• DNA replication takes place only once each generation in each cell
• Errors in replication (mutations) occur spontaneously only once in every 109 to
1010 base pairs
• Can be lethal to organisms
• Proofreading - the removal of incorrect nucleotides immediately after they are
added to the growing DNA during replication
• Errors in hydrogen bonding lead to errors in a growing DNA chain once in every
104 to 105 base pairs
Proofreading Improves Replication Fidelity
• Cut-and-patch catalyzed by Pol I: cutting is removal of the RNA primer
and patching is incorporation of the required deoxynucleotides
• Nick translation: Pol I removes RNA primer or DNA mistakes as it moves
along the DNA and then fills in behind it with its polymerase activity
• Mismatch repair: enzymes recognize that two bases are incorrectly
paired, the area of mismatch is removed, and the area replicated again
• Base excision repair: a damaged base is removed by DNA glycosylase
leaving an AP site; the sugar and phosphate are removed along with
several more bases, and then Pol I fills the gap
The 5  3 exonuclease function of DNA polymerase I.
DNA Polymerase Repair
Mismatch Repair in Prokaryotes
• Mechanisms of mismatch repair encompass: