DNA- R eglication,

Recombination, and Repair

The hereditary molecule, DNA speaks
Ireplicate and recombinate,
24
To penit the cell to proliferate,
Environnental insults try to danage me,
But Iprotect mryself uithadequate repairs"

eoxyribonucleic acid (DNA) is a macro The central dogma of life

molecule that carries genetic information
The biological information flows from DNA
Irom generation to generation. It is responsible to RNA, and from there to proteins. This is the
to preserve the identity of the species over
central dogma of life (Fig.24. 1). It is ultimately
millions of years. DNA may be regarded as a the DNA that controls every function of the cell
reserve bank of genetic information or a through protein synthesis.
memory bank.
As the carrier of genetic information, DNA in
Asingle mammalian fetal
cell contains only a a cell must be duplicated (replicated),
It is surprising
ew picograms (10-12 g) of DNA. maintained and passed down accurately to the
that this litle quantity of DNA stores intormaton
and every
daughter cells. Three distinct processes are
differentiation designed for this purpose. The 'three Rs of
nat will determine the
Tunction of an adult animal. DNA-replication, recombination, and repair, are
dealt with in this chapter. There are certain
common features betweern the three Rs.
Why did DNA evolve as genetic
material?
the
principle, can performDNA.
KNA molecules. in carried out by DNAnisunplon DAA ranslaticn +Proiein
cellular functions that are RNA as the genetic
contain
In fact, many viruses stable than Roplication
is more
material. Chemically, DNA course evolution,
of
RNA. Hence, during the molecule Fig. 24.1: The central dogma of lite.
suitable
a more information.
DNA is preferred as of genetic
for long-term repository 523
 524 BIOCHEMISTRY
These sites mostly consist ol a short sequence of
( They act on the same substrate (DNA). A
A-T base pairs. A specific protein called doa
They are primarily concerned with the making (20-50 monomers) binds with the site of origin
and breaking of phosphodiester bonds (the double-strandod
for replication. This causes the
backbone of DNA structure). DNA to separate.
Enzymes used in the three processes are
mostly similar/comparable. Replication bubbles
The two complementary strands of DNA
separate at the site of replication to form a
REPLICATION OF DNA bubble. Multiple replication bubbles are formed
in eukaryotic DNA molecules, which is essential
DNA 0s the genetic material. When the cell for a rapid replication process (Fig.24.3).
divides, the daughter cells receive an identical
copy of genetic information from the parent cell. RNA primer
Replication is aprocess in which DNA copies For the synthesis of new DNA, a short
itself to produce identical daughter molecules fragment of RNA (about 5-50 nucleotides,
of DNA. Replication is carried out with high
fidelity which is essential for the survival of the variable with species) is required as a primer.
The enzyme primase (a specific RNA
species. Synthesis of a new DNA molecule is a polymerase) in association with single-stranded
complex process involving a series of steps.
binding proteins forms a complex called
The salient features of replication in primosome, and produces RNA primers. A
prokaryotes are described first. This is followed constant synthesis and supply of RNA primers
by some recent information on the eukaryotic should occur on the lagging strand of DNA. This
replication. is in contrast to the leading strand which has
almost a single RNA primer.
REPLICATION IN PROKARYOTES
Vheplication is semiconservative
The parent DNA has two strands complemen
tary to each other. Both the strands undergo
simultaneous replication to produce two
daughter molecules. Each one of the. newly
synthesized DNA has one-half of the parental
DNA (one strand from original) and one-half of
new DNA (Fig.24.2). This type of replication is
known as semiconservative since half of the
original DNA is conserved in the daughter DNA.
The first experimental evidence for the
semiconservative DNA replication was provided
by Meselson and Stahl (1958).
Initiation of replication
The initiation of DNA synthesis occurs at a
site called origin of replication. In case of
Daughter DNA Parent DNA Daughter DNA
prokaryotes, there is a single site whereas in
eukaryotes, there are multiple sites of origin. Fig. 24.2 : DNA replication semiconsevative.
Chapter 24: DNA-
-REPLICATION, RECOMBINATION, AND REPAIR 525

proteins. They possess no enzyme activity. SSB

proteins bind only to single-stranded DNA
(separated by helicases), keep the two strands
separate and provide the tenplate for new DNA
synthesis. It is believed that SSB proteins also
protect the single-stranded DNA degradation by
nucleases.

DNA synthesis catalysed

by DNA polymerase Il
The synthesis of a new DNA strand, catalysed
by DNA polymerase II, occurs in 5'3'
direction. This is antiparallelto the parent
template DNA strand. The presence of all
Fig. 24.3: Schematic representation of muitple
replication bubbles in DNA replication. the íour deoxyribonucleoside triphosphates
(dATP, dGTP, dCTP and dTTP) is an essential
prerequisite for replication to take place.
DNA synthesis is semidiscontinuous and The synthesis of two new DNA strands,
bidirectional simultaneously, takes place in the opposite
direction-one is in a direction (5'’3) towards
The replication of DNA Occurs in 5' to 3'
direction, simultaneously, on both the strands of the replication fork which is continuous, the other
in a direction (5'3)) away from the replication
DNA. On one strand, the leading (continuous or fork which is discontinuous (Fig.24.4).
forward) strandthe DNA synthesis is The incoming deoxyribonucleotides are
continuous. On the other strand, the lagging
added one after another, to 3' end of the growing
(discontinuous or retrograde) strand-the
synthesis of DNA is discontinuous. Short pieces DNA chain (Fig.24.5). A molecule of pyro
phosphate (PPi is removed with the addition of
Gf DNA (15-250 nucleotides) are produced on
each nucleotide. The template DNA strand (the
the lagging strand. parent) determines the base sequence of the
synthesis
In the replication bubble, the DNA from newly synthesized complementary DNA.
DCCurs in both the directions (bidirectional)
the point of origin. Polarity problem
Replication fork and DNA synthesis The DNA strand (leading strand) with its
3'-end (3'-OH) oriented towards the fork can be
The separation of the twO strands of parent elongated by sequential addition of new
replication
DNA results in the formation of a nucleotides. The other DNA strand (lagging
1ork. The active synthesis of DNA OCcurs in this
along the strand) with 5-end presents some problem, as
region. The replication fork moves there is no DNA polymerase enzyme (in any
molecules are
Darent DNA as the daughter DNA organism) that can catalyse the
addition of
synthesized. nuclcotides to the 5 end (i.e. 3’5 direction) of
DNA helicases : These enzymes bind to both however is
the growing chain. This problem a series of
the DNA strands at the replication tork. solved by synthesizing this strand as
helicases move along the DNA helix and are made in the
small fragments. These piecesand later joined
Their function normal 5 3 direction,
Separate the strands.
Comparable with a zip opener. Helicases are together.
dependent on ATP for energy supply. of the
: Okazaki pieces : The small fragments
Single-stranded DNA binding (SSB) proteins synthesized DNA are called
known as DNA helix-destabilizing discontinuously
ese are also
 BIOCHEMISTRY
526

sized by DNA polymerase | and the

produced on the
Okazaki pieces. These are
DNA. Okazaki small fragments of DNA produced by DNA
lagging strand of the parent polymerase I. This process nick sealing requires
a continuous breakdown of ATP
pieces are later joined to form DNA energY, provided by the
I and
strand of DNA. DNA polymerase (details AMP and PPi.
process
ligase are responsible for this
given later). dna A protein

Proof-reading function 3

of DNA polymerase Ill Native DNA

Fidelity of replication is
the most important for the dna A protein
very existence of an 5
organism. Besides its 5'3
directed catalytic function,
DNA polymerase ll also Replication bubble
has proof-reading
activity. It checks the Leading
incoming nucleotides and Lagging strand
strand 5
allows only the correctly
matched bases (i.e. 5 Lagging
complementary bases) to Leading strand
strand
be added to the growing Origin of replication
DNA strand. Further, DNA
polymerase edits its
mistakes (if any) and Leading
strand
removes the wrongly RNA primer
placed nucleotide bases.
-DNA polymerase Ill
Replacement of RNA
primer by DNA
The synthesis of new
DNA strand continues till it Newly
is in close proximity to synthesized
DNA
RNA primer. Now the DNA 3
polymerase ComeS into
DNA helicase
picture. It removes the RNA
primer and takes its
position. DNA polymerase
SSB
I catalyses the synthesis
(5’3' direction) of a
fragment of DNA that
replaces RNA primer Lagging
strand Okazaki pieces
(Fig.24.6).
The enzyme DNA ligase Replication fork
catalyses the formation of a
phosphodiester linkage bet Fig. 24.4 : Overview of DNA replication process
ween the DNA synthe (SSB-Single-stranded binding proteins).
Chapter 24 DNA-REPLICATION, RECOMBINATION, AND REPAIR
527

DNA
G A
T G T

omprom

OH OH 3

RNA primer Entering dATP

Growing DNA

Fig. 24.5 : DNA replication with a growing complementary strand.

Another enzyme-DNA polymerase I-has at least five distinct DNA polymerases are known
been isolated. It participates in the DNA repair in eukaryotes. Greek letters are used to number
process. these enzymes.
1. DNA polymerase a is responsible for the
Supercoils and DNA topoisomerases synthesis of RNA primer for both the leading
As the double helix of DNA separates from and lagging strands of DNA.
one side and replication proceeds, supercoils are 2. DNA polymerase B is involved in the
formed at the other side. The formation of
repair of DNA. Its function is comparable with
supercoilscan be better understood by DNA polymerase Ifound in prokaryotes.
comparing DNA helix with two twisted ropes
tied at one end. Hold the ropes at the tied end 3. DNA polymerase y participates in the
replication of mitochondrial DNA.
in a fixed position. And let your friend pull the
formation of 4. DNA polymerase 8 is responsible for the
ropes apart from the other side. The
Supercoils is clearly observed. replication on the leading and lagging strands of
Type I DNA topoisomerase cuts the singie DNA. It also possesses proof-reading activity.
DNA strand (nuclease activity) to overcomne the
problem of supercoils and then reseals the strand 3
5 Newly synthesized DNA
gase activity). Type Il DNA topoisomerase (also
3
strands and -3 DNA template
Known as DNA gyrase) cuts both
reseals them to overcome the problem of
Supercoils. DNA topoisomerases are targeted by Excised
RNA primer
-DNApolymerase l
Grugs (campthoterin for topoisomerase I,
and 5
dsacrime and etoposide for topoisomerase I,
and amsacrime and etoposide for topoisomerase -Nick sealed
Il) in the treatment of cancers. by DNA ligase
5' Daughter DNA
REPLICATION IN EUKARYOTES
3 Parent DNA
Replication of DNA in eukaryotes closely
differences, the aclion of DNA
resembles that of prokaryotes. Certain Fig. 24.6: Oveniew of
polymerase land DNA ligase.
however, exist. Multiple origins of replication is
eukaryotic cell. Further,
d characteristic feature of
528 BIOCHEMISTRY
5. DNA polymerase [ is involved in prool catalyses the assembly of proliferating cell
reading function of DNA replication. nuclear antigen (PCNA) molecules. The DN
polymerase 8 binds to the sliding clamp and
The differences in the DNA replication elongates the Okazaki fragment to a final lenoth
between bacteria and human cells, attributed to
of about 150-200 bp. By this elongation, tho
the enzymes, are successfully used in replication complex approaches the RNA primer
antibacterial therapy to target pathogen (bacterial) of the previous Okazaki fragment.
replication and spare the host (human) cells.
The RNA primer removal is carried out by a
PROCESS OF REPLICA TION pair of enzymes namely RNase H and flan
endonuclease I (FEN). This gap created by RNA
IN EUKARYOTES
removal is filled by continued elongation of the
The replication on the leading (continuous) new Okazaki fragment (carried out by
strand of DNA is rather simple, involving DNA polymerase S, described above). The small nick
polymerase 8 and a sliding clamp called that remains is finaly sealed by DNA ligase.
proliferating cell nuclear antigen (PCNA). PCNA Eukaryotic DNA is tightly bound to histones
is so named as it was first detected as an antigen
in the nuclei of replicating cells. PCNA forms (basic proteins) to íorm nucleosomes which, in
turn, organize into chromosomes. During the
ring around DNA to which DNA polymerase S
binds. Formation of this ring also requires another course of replication, the chromosomes are
factor namely replication factor C (RF). relaxed and the nucleosones get lo0sened. The
DNA strands separate for replication, and the
The replication on the lagging (discontinuous) parental histones associate with one of the
strand in eukaryotes is more complex when parental strands. As the synthesis of new DNA
compared to prokaryotes or even the leading strand proceeds, histones are also
produced
strand of eukaryotes. This is depicted in simultaneously, on the parent strand. At the end
Fig.24.7, and briefly described hereunder. of replication, of the two daughter chromosomal
The parental strands of DNA are separated by DNAs ormed, one contains the parental histones
the enzyme helicase. A single-stranded DNA while the other has the newly synthesized
binding protein called replication protein A histones.
(RPA) binds to the exposed single-stranded
template. This strand has been opened up by the INHIBITORS OF DNA REPLICA TION
replication fork (a previously formed Okazaki Bacteria contain
fragment with an RNA primer is also shown in
a specific type
topoisomerase namely gyrase. This enzyme cuts
Fig.24.4). and reseals the circular DNA (of bacteria), and
The enzyme primase forms a complex with thus overcomes the problemn of supercoils.
DNA polymerase a which initiates the synthesis Baclerial gyrase is inhibited by the antibiotics
of Okazaki fragments. The primase activily of ciprofloxacin, novobiocin and nalidixic acid.
pol d-primase complex is capable of producing These are widely used as antibacterial agents
10-bp RNA primer. The enzyme activity is then since they can effectively block the replication
switched from primase to DNA polymerase a of DNA and multiplication of cells. These
which elongates the primer by the addition of antibacterial agents have aimost no effect on
20-30 deoxyribonuclcotides. Thus, by the action human enzymes.
of pol -primase complex, a short stretch of
DNA attached to RNA is formed. And now the Certain compounds that inhibit human
complex dissociates from the DNA. topoisomerases are used as anticancer agents
The next step is the binding of replication e.g. adriamycin, etoposide, doxorubicin. The
nucleotide analogs that inhibit DNA replication
factor C (RFC) to the elongated primer (short are also used as anticancer drugs e.8
RNA-DNA). RFC serves as a clamp loader, and
6-mercaptopurine, 5-fluorouracil.
Chapter24
:DNA-REPLICATION.
RECOMBINATION, AND REPAIR 529

Previous
3 Okazaki fragment
5 Termplate for
RPA lagging strand
DNA polymerase a
primase complex
RNA primer
ANA primor

PCNA
RFC

DNA polymerase &

RNase H
EENI

DNA ligase
New strand
of DNA
3
5 5

(APA-Replication protein A:
replication on the lagging strandin eukaryotesH-Abonuclease H: FEN-Elan
DNA
Pig. 24.7: An outline ofnuclear AFC-Replication factor CFNase
PCNA-Proliterating cell antigen: strand not shown).
endonuclease ,: Note : Leading