Dna structure, function

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Rafi M D: Textbook of Biochemistry for Medical Students (2nd Edn)
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Rafi M D: Textbook of Biochemistry for Medical Students (2nd Edn)
DNA Metabolism

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Rafi M D: Textbook of Biochemistry for Medical Students (2nd Edn)
REPLICATION OF DNA
Principles of Replication
1. Copying is accurate
2. Replication is semiconservative
3. Supercoils and DNA topoisomerases
4. Replication fork
5. Replication is simulataneous and
bidirectional
6. DNA synthesis is catalysed by DNA
polymerases

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Rafi M D: Textbook of Biochemistry for Medical Students (2nd Edn)
Replication fork

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Rafi M D: Textbook of Biochemistry for Medical Students (2nd Edn)
REPLICATION IN PROKARYOTES
 Most of the information about the replication
in prokaryotes is obtained from the studies
made on the intestinal bacterium, E.coli.
 The process of replication can be studied
conveniently in three stages.
1. Initiation
2. Elongation
3. Termination

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Rafi M D: Textbook of Biochemistry for Medical Students (2nd Edn)
Initiation
 Helicase enzymes
 Replication fork
 Topoisomerases (also called as DNA gyrases)
 Single-stranded DNA binding (SSB) proteins
 Primer
 Primase
 DNA polymerase III

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Rafi M D: Textbook of Biochemistry for Medical Students (2nd Edn)
Diagrammatic representation of the process of replication

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Rafi M D: Textbook of Biochemistry for Medical Students (2nd Edn)
Elongation
 Okazaki fragments
 DNA polymerase I
 Proof-reading ability

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Rafi M D: Textbook of Biochemistry for Medical Students (2nd Edn)
Termination
 Terminus sequence
 DNA polymerase II

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Rafi M D: Textbook of Biochemistry for Medical Students (2nd Edn)
REPLICATION IN EUKARYOTES
There are five distinct DNA polymerases
identified in eukaryotes. They are numbered by
Greek alphabets.
1. DNA polymerase a synthesises primers for
both leading and lagging strands of DNA.
2. DNA polymerase b has similar function as
that of DNA polymerase II of prokaryotes –
DNA repair.
3. DNA polymerase g exclusively replicates
mitochondrial DNA.
(Continued…

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Rafi M D: Textbook of Biochemistry for Medical Students (2nd Edn)
4. DNA polymerase d is analogous to that of
DNA polymerase III of prokaryotes. It takes
part in the synthesis of both leading and
lagging strands with an intrinsic proof-
reading activity.
5. DNA polymerase e is comparable in its action
to the prokaryotic DNA polymerase I. It is
involved in the removal of RNA primers of
Okazaki fragments on the lagging strand.
Occasionally, it replaces the DNA polymerase
d and performs its function.
…Continues)
(Continued…

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Rafi M D: Textbook of Biochemistry for Medical Students (2nd Edn)
Like bacterial replication, replication in
eukaryotic cells can also be studied in three
stages.
1. Initiation
2. Elongation
3. Termination.
…Continues)

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Rafi M D: Textbook of Biochemistry for Medical Students (2nd Edn)
Initiation
 Helicases
 Topoisomerases
 Replication protein A (RPA)
 Replication fork
 Multiple sites of origin of replication
 Primers

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Rafi M D: Textbook of Biochemistry for Medical Students (2nd Edn)
Overview of DNA replication on the lagging strand in eukaryotes (leading
strand is not shown) (for abbreviations see the text)

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Rafi M D: Textbook of Biochemistry for Medical Students (2nd Edn)
Elongation
 Replication factor C (REC)
 Proliferating cell nuclear antigen (PCNA)
 Sliding clamp
 DNA polymerase d
 Okazaki fragments
 RNase H
 FENI (flap endonuclease I)
 DNA polymerase e

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Rafi M D: Textbook of Biochemistry for Medical Students (2nd Edn)
Termination
 Telomeres
 Telomerases
 RNA dependent DAN synthesis
 Reverse transcriptase

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Rafi M D: Textbook of Biochemistry for Medical Students (2nd Edn)
● The differences between the DNA replication
in bacteria and human cells give scope for
developing antibiotics that target bacterial
replication but do not affect human cells.
Several drugs (e.g. ciprofloxacin, nalidixic
acid ) act by inhibiting the enzymes of
bacterial replication.
INHIBITORS OF REPLICATION
(Continued…

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Rafi M D: Textbook of Biochemistry for Medical Students (2nd Edn)
Drugs targeting human replication enzymes
such as topoisomerases and telomerases
arrest new DNA synthesis and thus the cell
division. E.g. doxorubicin, adriamycin,
etoposide. Certain nucleotide analogues such
as 6-mercaptopurine and 5-fluorouracil
inhibit human DNA polymerases. They are
also used as anti-cancer drugs.
…Continues)

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Rafi M D: Textbook of Biochemistry for Medical Students (2nd Edn)
CELL CYCLE
• S (Synthetic) phase
• M (mitotic) phase
• Gap1 (G1) and gap2 (G2)
• Dormant phase (G0)
• Cyclins
• Cyclin-dependent kinases (CDKs)

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Rafi M D: Textbook of Biochemistry for Medical Students (2nd Edn)
Cancer and the cell cycle
Cell cycle in mammalian cells (for abbreviations see the text)

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Rafi M D: Textbook of Biochemistry for Medical Students (2nd Edn)
Apoptosis
A programmed cell suicide is referred to as
apoptosis

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Rafi M D: Textbook of Biochemistry for Medical Students (2nd Edn)
The exchange of genetic information between
various DNA segments is known as
recombination. Apart from rearranging genetic
information, it also repairs damaged DNA.
There are two types of recombinations.
RECOMBINATION
1. Homologous recombination: The exchange of
genetic information takes place between
identical DNA sequences (parts of
chromosomes). The most common example is
recombination between the maternal and
paternal chromosomes of a pair. (Continued…

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Rafi M D: Textbook of Biochemistry for Medical Students (2nd Edn)
2. Non-homologous recombination: In this
type, the exchange of genetic information
occurs randomly (need not be between
identical sequences). Genetic rearrangement
usually takes place by this process
(transposition). It also involves the
experimental installation of foreign DNA into
a host's chromosome.
…Continues)

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Rafi M D: Textbook of Biochemistry for Medical Students (2nd Edn)
A diagrammatic representation of homologous recombination
HOMOLOGOUS RECOMBINATION

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Rafi M D: Textbook of Biochemistry for Medical Students (2nd Edn)
Three models have been proposed to explain
homologous recombination.
i) Holliday model
ii) Meselson–Radding model
iii) Double-strand break model

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Rafi M D: Textbook of Biochemistry for Medical Students (2nd Edn)
Holliday model of homologous recombination (the areas shown in dotted
boxes are heterologous regions)

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Rafi M D: Textbook of Biochemistry for Medical Students (2nd Edn)
Three types of non-homologous
recombination have been identified.
i) Transposition
ii) Site-specific recombination
iii) Non-homologous end joining.
NON-HOMOLOGOUS RECOMBINATION

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Rafi M D: Textbook of Biochemistry for Medical Students (2nd Edn)
A diagrammatic representation of retrotransposition

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Rafi M D: Textbook of Biochemistry for Medical Students (2nd Edn)
The types of damage inflicted on the DNA
molecule by various physical and chemical
agents (mutagens) are broadly classified into
four categories
DNA DAMAGE
(Continued…

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Rafi M D: Textbook of Biochemistry for Medical Students (2nd Edn)
TYPES OF DNA DAMAGE
…Continues)

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Rafi M D: Textbook of Biochemistry for Medical Students (2nd Edn)
REPAIR OF DNA
Four major repair systems are involved in
rectifying the DNA damage.
1. Mismatch repair
2. Base excision repair
3. Nucleotide excision repair
4. Double–strand break repair.

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MECHANISMS OF DNA REPAIR

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Rafi M D: Textbook of Biochemistry for Medical Students (2nd Edn)
● Even though replication occurs with high
fidelity, defects do occur during copying.
Mismatch repair corrects errors involving
single base pair or a small region of
unpaired DNA
MISMATCH REPAIR

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Rafi M D: Textbook of Biochemistry for Medical Students (2nd Edn)
A diagrammatic representation of mismatch repair

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Rafi M D: Textbook of Biochemistry for Medical Students (2nd Edn)
A diagrammatic representation of base excision repair
BASE EXCISION REPAIR

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Rafi M D: Textbook of Biochemistry for Medical Students (2nd Edn)
● Xeroderma pigmentosum
NUCLEOTIDE EXCISION REPAIR

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Rafi M D: Textbook of Biochemistry for Medical Students (2nd Edn)
● Double-strand breaks are common during
immunoglobulin gene rearrangement which
are repaired forthwith. Otherwise, double-
strand breaks occur due to the free radicals,
radiation and chemotherapeutic agents.
DOUBLE-STRAND BREAK REPAIR