Molecular Basis of Inheritance

DNA Replication and Genes

LECTURE 4
Dr. Sachin Kapur
M.Phil, Phd

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Molecular Basis of Inheritance
DNA Replication and Genes
LECTURE 4
 THE QUESTION ISN’T WHO IS
GOING TO LET ME, IT’S WHO IS
GOING TO STOP ME.

LET’S CRACK IT!

DNA Replication

Machinery and Enzymes

Which of the following first suggested the conservative model of DNA replication?

(DPMT2007)
A. Cairns
B. Meselson and Stahl
C. Watson and Crick
D. Taylor
Machinery and Enzymes

➢ DNA replication is a complex multistep process that requires a

number of enzymes, protein factors and metal ions.

DNA Polymerase

DNA ligase

Primase

Topoisomerase

Helicase
DNA Replication

DNA Polymerase

➢ DNA-dependent DNA polymerase is the main enzyme which takes part in combining
deoxyribose nucleotides to form new DNA strands.

Polymerase α Initiation of replication.

Polymerase β DNA repair

Polymerase γ Mitochondrial DNA repair.

Polymerase δ Replication activity.

Polymerase ε Polymerase activity of He La cells.

DNA Replication

DNA ligase

➢ DNA ligase enzyme joins newly synthesized fragments of DNA.

➢ DNA ligase was discovered by Khorana.
DNA multiplication is called:
(BHU 2006)
A. Translation
B. Replication
C. Transduction
D. Transcription
DNA Replication

Replication Mechanism
DNA has genetic properties was revealed for the first time by: (HP-PMT 2008)

A. Griffith
B. Avery
C. Wilkins
D. Chargaff
DNA Replication

Origin of Replication

Activation of Deoxyribonucleotides

Exposure of Parent DNA Strands

Formation of RNA Primer

Base Pairing

New Strand Formation

Proofreading
DNA Replication

Origin of Replication

➢ Replication begins at a particular spot called origin of replication or ori.

A sequential expression of a set of human genes occurs, when a steroid molecule binds
to the:
(CBSE 2007)
A. Messenger RNA
B. DNA sequence
C. Ribosomes
D. Transfer RNA
Which particular process was used by Meselson and Franklin in order to study the semi-
conservative replication of DNA:

(HP-PMT 2008)
A. Centrifugation
B. Chromatography
C. Density gradient centrifugation
D. Buoyant density centrifugation
DNA Replication

Activation of Deoxyribonucleotides

➢ Deoxyribonucleotides occur freely inside the nucleoplasm.

➢ They are first phosphorylated and changed to active forms which have three
phosphate residues instead of one.
➢ Enzymes phosphorylase is required alongwith energy.
DNA Replication

Exposure of Parent DNA Strands

➢ Enzyme helicase (unwindase) acts over the ori site and unwinds the two strands of
DNA by destroying hydrogen bonds.
➢ Unwinding of DNA molecule into two strands results in the formation of Y-shaped
structure, called replication fork (Y fork).
DNA Replication

Exposure of Parent DNA Strands

➢ The exposed single strands are stabilised with the help of single strand binding
proteins (SSBP).
➢ Due to unwinding, a supercoiling gets developed on the end of DNA opposite to
replicating fork.
➢ This tension is released by enzyme topoisomerase.
➢ In prokaryotes, DNA gyrase has topoisomerase activity.
DNA Replication

Formation of RNA Primer

➢ At the free 3' end of one strand and fork end of the second strand a small strand of
RNA is synthesized with the help of DNA-dependent enzyme RNA polymerase or
primase.
➢ The synthesized RNA is called RNA primer. (It is 4-12 nucleotides long)
DNA Replication

Base Pairing

➢ The two separated DNA strands in the area of replication fork now function.
➢ Their nitrogenous bases attract complementary phosphorylated nucleotides.
➢ Enzyme pyrophosphatase removes two phosphates from phosphorylated nucleotides
and change them into monophosphate state.
➢ It releases energy which is used in building hydrogen bonds.
DNA Replication

New Strand Formation

➢ It requires DNA polymerase III in prokaryotes and polymerase δ/ε in eukaryotes.

➢ In the presence of Mg2+, ATP (GTP), TPP and DNA polymerase III, the adjacent
nucleotides establish phosphodiester bonds and get linked to form replicated DNA
strand.
DNA Replication
DNA Replication

New Strand Formation

➢ As replication proceeds, new areas of parent DNA duplex unwind and separate so
that replication proceeds rapidly.
➢ RNA primer is removed and the gap filled with complementary nucleotides by means
of DNA polymerase I.
DNA Replication

New Strand Formation

➢ Two strands of DNA run in antiparallel direction, so the two templates provide
different ends for replication.
➢ Replication over the two templates proceeds in opposite direction.
➢ One strand with polarity 3' -, 5' forms its complementary strand continuously because
3 'end of the latter is always open for elongation.
➢ It is called leading strand.
DNA Replication

New Strand Formation

➢ Replication is discontinuous on the other template with polarity 5' → 3' because only
a short segment of DNA strand can be built in 5' → 3' direction.
➢ Short segments of replicated DNA are called Okazaki fragments.
➢ Each of them has 1000- 2000 bp in prokaryotes and 100-200 bp in eukaryotes.
Okazaki is known for his contribution to the understanding of:
(DUMET 2010)

A. Transcription
B. Translation
C. DNA replication
D. Mutation
DNA Replication

New Strand Formation

➢ RNA primer is also required every time a new Okazaki fragment is to be built.
➢ After replacing RNA primer with deoxyribonucleotides and their polymerisation,
Okazaki fragments are joined together by means of enzyme, DNA ligase.
➢ DNA strand built up of Okazaki fragments is called lagging strand.
➢ As one strand grows continuously while the other strand is formed discontinuously,
DNA replication is semi discontinuous.
The Okazaki fragments in DNA chain growth :
(CBSE 2007)
A. Polymerize in the -3' to -5' direction and forms replication fork
B. Prove semiconservative nature of DNA replication
C. Polymerize in -5' to -3' direction and explain -3' to -5' DNA replication
D. Result in transcription
 Differences between Leading Strand and Lagging Strand

Leading Strand Lagging Strand

It is a replicated strand of DNA which grows Lagging strand is a replicated strand of DNA
continuously without any gap. which is formed in short segments called
Okazaki fragments. Its growth is discontinuous.

It does not require DNA ligase for its growth. DNA- ligase is required for joining Okazaki
fragments.

The direction of growth of the leading strand The direction of growth of the lagging strand is
is 5’ → 3’. 3’ → 5’ though in each Okazaki fragment it is 5’
→ 3’ direction.
 Differences between Leading Strand and Lagging Strand

Leading Strand Lagging Strand

Only single RNA primer is required. Starting of each Okazaki fragment requires a
new RNA.

Formation of leading strand is quite rapid. Formation of lagging strand is slower.

Its template opens in 3’ → 5’ direction. Its template opens in 5’ → 3’ direction.

Formation of leading strand begins Formation of lagging strand begins a bit later
immediately at the beginning of replication. than that of leading strand.
DNA Replication

Proofreading

➢ A wrong base is sometimes introduced during replication.

➢ The frequency is one in ten thousand.
➢ DNA polymerase III is able to sense the same.
➢ It goes back, removes the wrong base, allows addition of proper base and then
proceeds forward.
DNA Replication

Proofreading

➢ DNA polymerase III is unable to distinguish uracil from thymine so that it is often
incorporated in place of thymine.
➢ DNA polymerase I removes the mismatched or wrong nucleotides if present and
synthesises a correct replacement by using the intact strand as template.
➢ The newly formed segment is sealed by DNA ligase.
 Differences between Prokaryotic DNA Replication & Eukaryotic DNA Replication

Prokaryotic DNA Replication Eukaryotic DNA Replication

It occurs inside the cytoplasm. It occurs the nucleus.

There is single origin of replication. Origin of replication are numerous.

DNA polymerase III carries out both initiation Initiation is carried out by DNA polymerase ɑ
and elongation. while elongation by polymerase δ and ε.

DNA repair and gap filling is done by DNA The same is performed by DNA polymerase β.
polymerase I.
 Differences between Prokaryotic DNA Replication & Eukaryotic DNA Replication

Prokaryotic DNA Replication Eukaryotic DNA Replication

RNA primer is removed by DNA polymerase I. RNA primer is removed by DNA polymerase β.

Okazaki fragments are large, 1000-2000 Okazaki fragments are short, 100-200
nucleotides long. nucleotides long.

Replication is very rapid, some 2000 bp per Replication is slow, some 100 nucleotides per
second. seconds.

DNA gyrase is needed. DNA gyrase is not needed.

E.coli about to replicate was placed in a medium containing radioactive thymidine for
five minutes. Then it was made to replicate in a normal medium. Which of the following
observation will be correct?
(AIIMS 2007)
A. Both the strands of DNA will be radioactive
B. One strand radioactive
C. Each strand half radioactive
D. None is radioactive
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