DNA REPLICATION

INTRODUCTION:

DNA replication, the basis for biological inheritance, is a fundamental process

occurring in all living organisms to copy their DNA. This process is "replication" in that
each strand of the original double-stranded DNA molecule serves as template for the
reproduction of the complementary strand. Hence, following DNA replication, two
identical DNA molecules have been produced from a single double-stranded DNA
molecule. Cellular proofreading and error checking mechanisms ensure near perfect
fidelity for DNA replication.

In a cell, DNA replication begins at specific locations in the genome, called

"origins". Unwinding of DNA at the origin, and synthesis of new strands, forms a
replication fork. In addition to DNA polymerase, the enzyme that synthesizes the new
DNA by adding nucleotides matched to the template strand, a number of other proteins
are associated with the fork and assist in the initiation and continuation of DNA
synthesis.

DNA STRUCTURE:
DNA is a long polymer made from
repeating units called nucleotides.

In living organisms, DNA does not usually exist

as a single molecule, but instead as a pair of
molecules that are held tightly together.

These two long strands represent themselves in

the shape of a double helix. The nucleotide
repeats contain both the segment of the backbone
of the molecule, which holds the chain together,
and a base, which interacts with the other DNA
strand in the helix. A base linked to a sugar is
called a nucleoside and a base linked to a sugar
Fig. Chemical Structure of DNA
and one or more phosphate groups is called a nucleotide. If multiple nucleotides are
linked together, as in DNA, this polymer is called a polynucleotide.

The backbone of the DNA strand is made from alternating phosphate and sugar
residues. The sugar in DNA is 2-deoxyribose, a pentose (five-carbon) sugar. The sugars
are joined together by phosphate groups that form phosphodiester bonds between the
third and fifth carbon atoms of adjacent sugar rings. These asymmetric bonds mean a
strand of DNA has a direction. In a double helix the direction of the nucleotides in one
strand is opposite to their direction in the other strand: the strands are antiparallel. The
asymmetric ends of DNA strands are called the 5′ (five prime) and 3′ (three prime) ends,
with the 5' end having a terminal phosphate group and the 3' end a terminal hydroxyl
group. This directionality has consequences in DNA synthesis, because DNA polymerase
can only synthesize DNA in one direction by adding nucleotides to the 3' end of a DNA
strand.
One major difference between DNA and RNA is the sugar, with the 2-
deoxyribose in DNA being replaced by the alternative pentose sugar ribose in RNA.

The DNA double helix is stabilized by hydrogen bonds between the bases attached to the
two strands. The four bases found in DNA are adenine (abbreviated A), cytosine (C),
guanine (G) and thymine (T). These four bases are attached to the sugar/phosphate to
form the complete nucleotide.

DNA REPLICATION BEGINS AT SPECIFIC CHROMOSOMAL SITES

Most important decision every cell has to make is whether, and when, to replicate
its DNA. DNA replication is controlled at the initiation step. Control would be most
efficient if there are specific sites on chromosomes at which DNA replication always
begins in vivo.

Molecular studies indicate that replication of these DNAs actually begins at a

defined sequence of base pairs near the center of these bubbles, called the replication
origin .

REPLICATION ORIGIN - Stretch of DNA that is necessary and sufficient for

replication of a circular DNA molecule, usually a plasmid or virus, in an appropriate host
cell.

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E. COLI REPLICATION ORIGIN

The E. coli replication origin oriC is an ≈240-bp DNA segment present at the start site for
replication of E. coli chromosomal DNA.

oriC contain repetitive 9-bp and AT-rich 13-bp sequences, referred to as 9-mers (dnaA
boxes) and 13-mers, respectively, these are binding sites for the DnaA protein that
initiates replication.

THREE COMMON FEATURES OF REPLICATION ORIGINS

1. Replication origins are unique DNA segments that contain multiple short repeated
sequences.

2. These short repeat units are recognized by multimeric origin-binding proteins. These
proteins play a key role in assembling DNA polymerases and other replication enzymes
at the sites where replication begins.

3. Origin regions usually contain an AT-rich stretch. This property facilitates unwinding
of duplex DNA because less energy is required to melt A·T base pairs than G·C base
pairs. Origin-binding proteins control the initiation of DNA replication by directing
assembly of the replication machinery to specific sites on the DNA chromosome.

GENES AND PROTEINS IN DNA REPLICTION

Genes Proteins
• DnaA
• dnaA
• DnaB or Helicase
• dnaB
• DnaC
• dnaC
• Primase
• dnaG
• SSB

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 Fig. The proteins at a DNA replication fork. The major types of proteins that act at a DNA
replication fork are illustrated, showing their positions on the DNA.

DnaA PROTEIN INITIATES REPLICATION IN E. COLI

Genetic studies first suggested that initiation of replication at oriC most likely
depended on the protein encoded by a gene designated dnaA. Subsequent genetic studies
with recombinant E. coli further pinpointed the DnaA protein as a prime candidate for
interaction with oriC. In vitro studies showed that pure DnaA protein binds to the four 9-
mers in oriC, forming an initial complex that contains 10-20 protein subunits.
Binding of DnaA to the oriC 9-mers facilitates the initial strand separation, or
"melting," of E. coli duplex DNA, which occurs at the oriC 13-mers. This process
requires ATP and yields a so-called open complex.

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 Model of initiation of replication at E. coli oriC

The 9-mers and 13-mers are the repetitive sequences

 Multiple copies of DnaA protein bind to the 9-mers at the origin and then
"melt" (separate the strands of) the 13-mer segments.
 The role function of DnaC is to deliver DnaB, which is composed of six
identical subunits, to the template.
 One DnaB hexamer clamps around each single strand of DNA at oriC,
forming the prepriming complex. DnaB is a helicase, and the two molecules then
proceed to unwind the DNA in opposite directions away from the origin.

DnaB Is an E. coli Helicase that Melts Duplex DNA

Further melting of the two strands of the E. coli chromosome to generate unpaired
template strands is mediated by the protein product of the dnaB locus, a helicase that is
essential for DNA replication. One molecule of DnaB, a hexamer of identical subunits,
clamps around each of the two single strands in the open complex formed between DnaA
and oriC. This binding requires ATP and the protein product encoded by the dnaC locus,
which "escorts" DnaB to the DnaA proteins, yielding the prepriming complex.

Helicases constitute a class of enzymes that can move along a DNA duplex
utilizing the energy of ATP hydrolysis to separate the strands. In E. coli, the separated
strands are inhibited from subsequently recoiling by a single-strand-binding protein (SSB
protein), which binds to both separated strands.

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Helicases like DnaB bind to a single-stranded segment of DNA, then move along that
strand melting the hydrogen bonds that link it to its complementary strand. Like many
proteins that bind to DNA, helicases exhibit a directionality with respect to the unwinding
reaction.
DnaB moves along the single strand of DNA to which it binds in the direction of
its free 3 end, and in this sense it is said to unwind DNA in the 5 '- 3' direction.

E. coli Primase Catalyzes Formation of RNA Primers for DNA Synthesis

As noted earlier, DNA polymerases can only elongate existing primer strands of
DNA or RNA. The primers used during DNA replication in both prokaryotes and
eukaryotes are short RNA molecules whose synthesis is catalyzed by the RNA
polymerase primase. Primase is usually recruited to a segment of single-stranded DNA
by first binding to a DnaB hexamer already attached at that site. The term primosome is
now generally used to denote a complex between primase and helicase, sometimes with
other accessory proteins. In initiation of E. coli DNA replication, a primosome is formed
by binding of primases to DnaB in the prepriming complex. After the bound primases
synthesize short primer RNAs complementary to both strands of duplex DNA, they
dissociate from the single-stranded template.

At a growing fork, one strand is synthesized from multiple primers

(a) The overall structure of a growing fork.

Synthesis of the leading strand, catalyzed by DNA polymerase III, occurs by sequential
addition of deoxyribonucleotides in the same direction as movement of the growing fork.

(b) Steps in the discontinuous synthesis of the lagging strand.

This process requires multiple primers, two DNA polymerases, and ligase, which joins
the 3 - hydroxyl end of one (Okazaki) fragment to the 5 -phosphate end of the adjacent
fragment.

(c) DNA ligation. During this reaction, ligase transiently attaches to the 5 phosphate of
one DNA strand, thus activating the phosphate group.

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 Figure: A β -subunit dimer tethers the core of E. coli DNA polymerase III to DNA.

The Leading and Lagging Strands Are Synthesized Concurrently

Once the prepriming complex and an RNA primer are formed at the E. coli replication
origin, chain elongation to yield the leading strand proceeds with little difficulty. As
we've seen, however, lagging-strand synthesis proceeds discontinuously from multiple
primers. Two molecules of core DNA polymerase III are bound at each growing fork;
one adds nucleotides to the leading strand, and the other adds nucleotides to the lagging
strand. Coordination between elongation of the leading and lagging strand is essential;
otherwise one template strand would be incorporated into a duplex with a newly
synthesized complementary strand while large parts of the other template strand would
remain single-stranded.

1. A single DnaB helicase moves along the lagging-strand template toward its 3 end,
thereby melting the duplex DNA at the fork.
2. One core polymerase (core 1) quickly adds nucleotides to the 3 end of the leading
strand as its single-stranded template is uncovered by the helicase action of DnaB. This
leading-strand polymerase, together with its b -subunit clamp, remains bound to the
DNA, synthesizing the leading strand continuously.

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3. A second core polymerase (core 2) synthesizes the lagging strand discontinuously as
an Okazaki fragment. The two core polymerase molecules are linked via a dimeric t
protein.

4. As each segment of the single-stranded template for the lagging strand is uncovered, it
becomes coated with SSB protein and forms a loop. Once synthesis of an Okazaki
fragment is completed, the lagging-strand polymerase dissociates from the DNA but the
core remains bound to the t -subunit dimer. The released core polymerase subsequently
rebinds with the assistance of another b clamp in the region of the primer for the next
Okazaki fragment.

E. coli DNA Polymerase III Catalyzes Nucleotide Addition at the Growing Fork

Three DNA polymerases (I, II, and III) have been purified from E. coli. In addition to its
role in filling the gaps between Okazaki fragments, DNA polymerase I probably is the
most important enzyme for gap filling during DNA repair. DNA polymerase II functions
in the inducible SOS response discussed later; this polymerase also fills gaps and appears
to facilitate DNA synthesis directed by damaged templates. Our discussion here focuses
on DNA polymerase III, which catalyzes chain elongation at the growing fork in E. coli.

Termination of replication

Because bacteria have circular chromosomes, termination of replication occurs

when the two replication forks meet each other on the opposite end of the parental
chromosome. E coli regulate this process through the use of termination sequences
which, when bound by the Tus protein, enable only one direction of replication fork to
pass through. As a result, the replication forks are constrained to always meet within the
termination region of the chromosome.

Eukaryotes initiate DNA replication at multiple points in the chromosome, so

replication forks meet and terminate at many points in the chromosome; these are not
known to be regulated in any particular manner. Because eukaryotes have linear
chromosomes, DNA replication often fails to synthesize to the very end of the
chromosomes (telomeres), resulting in telomere shortening. This is a normal process in
somatic cells — cells are only able to divide a certain number of times before the DNA
loss prevents further division. (This is known as the Hayflick limit.) Within the germ cell

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line, which passes DNA to the next generation, telomerase extends the repetitive
sequences of the telomere region to prevent degradation. Telomerase can become
mistakenly active in somatic cells, sometimes leading to cancer formation.

Summary of DNA replication

A self-correcting DNA polymerase catalyzes nucleotide polymerization in a 5'-to-3'

direction, copying a DNA template. Since the two strands of a DNA double helix are
antiparallel, this 5'-to-3' DNA synthesis can take place continuously on only one of the
strands at a replication fork (the leading strand). On the lagging strand short DNA
fragments are made by a "backstitching" process. Because the self-correcting DNA
polymerase cannot start a new chain, these lagging-strand DNA fragments are primed by
short RNA primer molecules that are subsequently erased and replaced with DNA.

DNA replication requires the cooperation of many proteins, including (1) DNA
polymerase and DNA primase to catalyze nucleoside triphosphate polymerization, (2)
DNA helicases and single-strand binding proteins to help open up the DNA helix so that
it can be copied, (3) DNA ligase and an enzyme that degrades RNA primers to seal
together the discontinuously synthesized lagging-strand DNA fragments, (4) DNA
topoisomerases to help relieve helical winding and tangling problems, and (5) initiator
proteins that bind to specific DNA sequences at a replication origin and catalyze the
formation of a replication fork at that site.

At a replication origin a specialized protein-DNA structure is formed that subsequently

loads a DNA helicase onto the DNA template; other proteins are then added to form the
multienzyme "replication machine" that catalyzes DNA synthesis.

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Figs. 1-7: Diagrammatic drawings showing different progressive stages of DNA
replication in E. coli.

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Enzymes of Dna Replication

• Helicases: Helicases are proteins that unwind DNA using energy of ATP and
create two potential replication forks.
• SSB protein (single-stranded DNA–binding protein): SSB proteins prevent
reanealing of single stranded DNA.
• DNA topoisomerases:
Topoisomerase I: DNA molecules can coil and bend in space, leading to
changes in topology, including formation of negative or positive supercoils. Local
unwinding of a DNA duplex whose ends are fixed causes stress that is relieved by
supercoiling. Any enzyme that cleaves only one strand of a DNA duplex and then reseals
it is classified as a type I topoisomerase (Topo I). The Topo I from E. coli acts on
negative, but not positive, supercoils.
Topoisomerase II or DNA gyrase:
Topo II enzymes have the ability to cut both strands of a double-stranded DNA
molecule, pass another portion of the duplex through the cut, and reseal the cut in a
process that utilizes ATP.
• DNA ligase: It catalyzes formation of a phosphodiester bond between a 3’
hydroxyl at end of one DNA strand and a 5’ phosphate at end of another strand.
• Primase: Primase synthesizes a short RNA primer, which remains base-paired to
template DNA that is extended at 3’ end by DNA polymerase.
• DNA polymerases: DNA polymerases are main polymerizing enzymes that add
deoxyribonucleotides to 5’ end of RNA primer.

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