(L

2, 3) DNA Replication in Prokaryotes and Eukaryotes. (Dr. Carmen Cadilla)

In eukaryotic cells, DNA is found associated with various types of proteins (known collectively as
nucleoprotein) present in the nucleus.
o Each chromosome in the nucleus of a eukaryote contains one long, linear molecule of dsDNA,
which is bound to a complex mixture of proteins (histone and non-histone) to form chromatin
Chromatin = complex of DNA + protein
o Eukaryotes have closed, circular DNA molecules in their mitochondria.
o In prokaryotes, the proteinDNA complex is present in a nonmembrane-bound region known as
the nucleoid.
Prokaryotic cells: lack nuclei, have a single chromosome, and may also contain
nonchromosomal DNA in the form of plasmids.
Plasmids may carry genes that convey antibiotic resistance to the host bacterium, and
may facilitate the transfer of genetic information from one bacterium to another.
The plasmid contains an origin of replication and associated DNA regulatory sequences
that are together called a replicon
A prokaryotic organism typically contains a single, double-stranded, supercoiled, circular
chromosome.
DNA exists as a double-stranded (ds) molecule, in which the two strands wind around each other,
forming a double helix. *Con la excepcion de unos cuantos viruses q tienen ss-DNA, el DNA es por lo
general ds-DNA.
o DNA double helix:
Each DNA molecule is composed of two polynucleotide chains joined by hydrogen bonds
between the bases
The chains are antiparallel: one chain runs in a 5 to 3 direction and the other chain runs 3
to 5
Base pairing:
The base pairs are held together by hydrogen bonds: 2 between A and T and 3 between
G and C
These hydrogen bonds, plus the hydrophobic interactions between the stacked bases,
stabilize the structure of the double helix.
Purines & Pyrimidines:
o Pyrimidines are C U T
o Purines G, A
o Phosphodiester linkages between nucleotides (in DNA or RNA) can be cleaved hydrolytically by
chemicals, or hydrolyzed enzymatically by a family of nucleases: deoxyribonucleases for DNA
and ribonucleases for RNA
o Nucleosides can be phosphorylated by specific kinases in the cell on the sugar's primary alcohol
group (-CH2-OH) to produce nucleotides.
Nucleotides are the molecular building-blocks of DNA and RNA.
Semiconservative Model = The double-stranded DNA contains one parental and one daughter
strand following replication
DNA is thought to consist primarily of B-DNA, which is right-handed and contains10 base pairs per
turn.
o Other forms of DNA include the A form, which is similar to the B form but more compact, and
the Z form, which is left-handed and has its bases positioned more toward the periphery of the
helix.

Central Dogma of Molecular Biology:

o DNA RNA proteins
Replication, transcription, translation
Replication in prokaryotes = DNA replication begins at a single, unique nucleotide sequencea site
called the origin of replication.
Replication in eukaryotes = begins at multiple sites along the DNA helix
DNA replication relies on the complementarity of DNA strands = AT/GC rule (Chargaffs rule)
The enzymes involved in the DNA replication process are template-directed polymerases that can
synthesize the complementary sequence of each strand with extraordinary fidelity.

Prokaryotic Replication:
As the two DNA strands unwind and separate, they form a V where active synthesis occurs, a
region called the replication fork.
As the two strands of the double helix are separated, positive supercoils are produced in the region
of DNA ahead of the replication fork.
DNA Supercoiling:
o Positive supercoiling of DNA occurs when the right-handed, double-helical conformation of DNA
is twisted even tighter (twisted in a right-handed fashion) until the helix begins to distort and
"knot."
o Negative supercoiling, on the other hand, involves twisting against the helical conformation
(twisting in a left-handed fashion), which preferentially underwinds and "straightens" the helix
at low twisting stress, and knots the DNA into negative supercoils at high twisting stress.
Linking number in relaxed DNA: # of base pairs divided by the # of base pairs per helical turn.
o Example: Relaxed circular dsDNA of 2,100 base pairs in the B form (10.5 base pairs per turn) has
linking number of 200.
Replication of dsDNA is bidirectionalthat is, the replication forks move in opposite directions from
the origin, generating a replication bubble.
3 types of DNA sequences in oriC are functionally significant:
o AT-rich region
o DnaA boxes
o GATC methylation sites
Initiation of DNA replication requires the recognition of the origin of replication by a group of
proteins that form the prepriming complex: **Example used is E. coli, with oriC (origin of
Chromosomal replication)
o DnaA protein: binds to specific nucleotide sequences at the origin of replication, causing short,
tandemly arranged (one after the other) AT-rich regions in the origin to melt.
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.
The binding of DnaA protein to its DNA binding sitesDnaA boxesin the chromosomal
oriC region is essential for initiation of chromosome replication.
In E. coli, oriC is bound by an initiator protein called DnaA. DnaC then associates and acts
like a matchmaker to allow DnaB, the helicase, to bind and begin separating the parental
strands to create a replication fork. A replication bubble is formed at each origin and a pair
of replication forks is established that move away from the origin, one in each direction.
Thus, replication is bidirectional.
o GATC methylation sites: Before replication, GATC sites are methylated on both strands. This full
methylation facilitates initiation of DNA replication.

Following replication, GATC sites are not methylated on the daughter strands
(hemimethylated).
This half-methylation does not efficiently initiate replication. Several minutes will pass
before Dam methylase will methylate the GATC sites in the daughter strands
DNA helicases: bind to ssDNA near the replication fork, and then move into the neighboring double-
stranded region, forcing the strands apartin effect, unwinding the double helix by breaking the
hydrogen bonds between the 2 strands, creating 2 replication forks (bidirectional replication).
o Composed of 6 subunits
o Travels along the DNA in the 5 to 3 direction
o Uses energy from ATP
Single-stranded DNA-binding (SSB) proteins: bind to the ssDNA generated by helicases.
o keep the 2 strands of DNA separated in the area of the replication origin, thus providing the
single-stranded template required by polymerases, and also protect the DNA from nucleases
that degrade ssDNA.
DNA topoisomerases: responsible for removing supercoils in the helix.
o 2 major types of topoisomerases:
Type I topoisomerases: work by making a transient cut in one DNA strand
Type IA topoisomerases:
o break one strand
o connect broken ends to the enzyme
o pass the intact one through the gap
o join the ends of the broken strand
o change linking number strictly in increments of one
Type IB topoisomerases:
o break one strand
o allows the free cleaved strand to rotate around the other
o join the strands
o change linking number by more than one
Type II topoisomerases: work by making a transient cut in both DNA strands
o break both strands
o pass a DNA segment through the break
o join the strands
o change linking number in increments of 2
o DNA gyrase, often referred to simply as gyrase, is an enzyme that relieves strain while double-
strand DNA is being unwound by helicase. It is also known as DNA topoisomerase II
o Topoisomerase I = mammalian nicking-closing enzyme
Direction of DNA Replication:
o The 2 new daughter strands are synthesized in different ways:
Leading strand:
One RNA primer is made at the origin
DNA pol III attaches nucleotides in a 5 to 3 direction as it slides toward the opening of
the replication fork
Lagging strand:
Synthesis is also in the 5 to 3 direction
However it occurs away from the replication fork
Many RNA primers are required

DNA pol III uses the RNA primers to synthesize small DNA fragments (1000 to 2000
nucleotides each)
These are termed Okazaki fragments
RNA primer:
o DNA polymerases cannot initiate synthesis of a complementary strand of DNA on a totally
single-stranded template. Rather, they require an RNA primerthat is, a short, double-stranded
region consisting of RNA base-paired to the DNA template, with a free hydroxyl group (-OH) on
the 3'-end of the RNA strand.
o DNA primase (DnaG) synthesizes the short stretches of RNA (~10 nucleotides long) which serves
as primer
o Primosome: makes the RNA primer required for leading strand synthesis, and initiates Okazaki
fragment formation in lagging strand synthesis.
o DNA helicase + DNA primase = primosome complex
o primosome is physically associated with the DNA polymerase holoenzyme forming the
replisome
Chain elongation:
o Prokaryotic (and eukaryotic) DNA polymerases elongate a new DNA strand by adding
deoxyribonucleotides, one at a time, to the 3'- end of the growing chain
o DNA polymerases catalyzes a phosphodiester bond between the innermost phosphate group of
the incoming deoxynucleoside triphosphate & 3-OH of the sugar of the previous
deoxynucleotide
o In the process, the last 2 phosphates of the incoming nucleotide are released in the form of
pyrophosphate (PPi)
DNA pol I & DNA pol III = normal replication
o DNA pol I: removes the RNA primers and fills the resulting gap with DNA
1. Uses 5 3 exonuclease activity to digest the RNA
2. Uses 5 3 polymerase activity to synthetize DNA
3. Uses 3 5 exonuclease activity for proofreading
o DNA pol III: remains attached to the template as it is synthesizing the daughter strand
(processive feature)
Processive means it remains bound to the template strand as it moves along, and does not
diffuse away and then rebind before adding each new nucleotide.
This processive feature is due to several different subunits in the DNA pol III holoenzyme
subunit is in the shape of a ring. It is termed the clamp protein
subunit is needed for to initially clamp onto the DNA. It is termed the clamp-loader
protein
DNA pol III is composed of 10 subunits, which all together are known as DNA pol III
holoenzyme
The alpha subunit synthesizes DNA
DNA pol III continues to synthesize DNA on the lagging strand until it is blocked by proximity
to an RNA primer. When this occurs, the RNA is excised and the gap filled by DNA pol I.
In the absence of the subunit DNA pol III falls off the DNA template after a few dozen
nucleotides have been polymerized
In the presence of the subunit DNA pol III stays on the DNA template long enough to
polymerize up to 50,000 nucleotides.
DNA pol II, DNA pol IV, DNA pol V = DNA repair & replication of damaged DNA

Termination of Replication: Opposite to oriC is a pair of termination sequences called ter

sequences, designated T1 and T2
o The protein tus (termination utilization substance) binds to these sequences and it can then
stop the movement of the replication forks
o DNA replication ends when oppositely advancing forks meet (usually at T1 or T2)
o Finally DNA ligase covalently links all 4 DNA strands
o DNA replication often results in 2 intertwined molecules
Intertwined circular molecules are termed catenanes
These are separated by the action of topoisomerases
Proofreading mechanisms:
Mistakes during the process are extremely rare
3 main reasons why fidelity is high:
o Instability of mismatched pairs
o Configuration of the DNA polymerase active site
o Proofreading function of DNA polymerase 3 5 exonuclease activity.

Eukaryotic DNA Replication

Multiple origins of replication
o Tandemly arrayed along the chromosomes (50k-100k bp intervals)
o Proceeds bidirectionally
o Origin Recognition Complex (ORC)
A six subunit complex that acts as the initiator of replication (ATP required)
ORC binds to the origin.
MCM complex also binds to this, with the help of cdc6 protein.
The initiator complex is activated and the helicase activity opens the parental
strands, forming a small bubble.
SSB binds to exposed single strands, helicases are loaded onto the DNA, and the
bubble is enlarged.
Pol /primase synthetizes the first RNA primer containing 10 nt., the switches to elongating
with deoxyribonucleotides, and adds about 15-30 nt. (RNA-DNA hybrid is formed). Pol/primase
leaves and
The chain is then elongated by polymerase , which incorporates deoxyribonucleotides to the
strand.
Eukaryotic DNA Polymerases

DNA pol/primase creates the RNA-DNA hybrid that is used by DNA pol or for the
processive elongation of the leading and lagging strand (processive polymerases).
This exchange is known as polymerase switch.
Eukaryotic Lagging Strand Synthesis
o Helicase activity separates parental strands
o Single-strand DNA binding protein called RPA binds to exposed strands
o Pol/primase complex initiates the Okazaki fragment by synthetizing the RNA-DNA
hybrid primer.
o When the hybrid is synthesized, pol/primase complex dissociates and RPC binds to this
elongated primer and serves as a clamp loader to assemble the PCNA sliding clamp.
o Pol binds the PCNA and completes Okazaki fragment to final length of 130-200bp
Further Okazaki Fragment Processing
o Primer removal is carried is carried out in two steps by RNase H and FEN1
RNase H degrades the RNA primer, leaving a single ribonucleotide attached to
the end of the Okazaki fragment.
FEN1 removes the last ribonucleotide (and some deoxyribonucleotides) and
forms a small flap of a few nucleotides which are cleaved at the angle of the flap
o If pol mismatched the first few nucleotides synthetizing the Okazaki fragment, a larger
flap is created by destabilization and FEN1 excises it. This increases the accuracy of repl.
o The remaining gap is filled by pol .
DNA pol plays a role in Base Excision Repair (BER)
Lesion Replicating Polymerases are involved in replication of damaged DNA.
Nucleosomes and DNA Replication
o More histones are synthesized when DNA is being actively replicated. (S phase)
o They associate with the newly made DNA near the replication fork
o Daughter strands contain mixture of old and new histones
Telomeres and DNA replication
o Telomere = telomeric DNA sequences + bound proteins
o Telomere replication problem there is no place to synthesize a primer that would
allow the daughter strand to be completed, so the daughter strand will be shorter than
the parental strand.
o Telomeric sequences
moderately repetitive tandem arrays of 6 nt sequence (TTAGGG)
3 overhang 12-16 nt. long
o Telomerase a ribonucleoprotein (RNA-containing)
Short RNA strand that partly base-pairs with the telomeric sequences and serves
as the template for the reaction.
Protein component serves as a reverse transcriptase, synthetizing DNA from an
RNA template.
It adds 6-nt repeats at a time; it can also dissociate & bind again to add 6 more

DNA gets progressively shorter with each round of DNA replication; telomerase can
elongate these ends. However, telomerase activity diminishes in the aging cell, which
can cause chromosomal aberrations and eventually cell death.