DNA Replication & Repair

Abhishek Dahal

DNA is a molecule that carries Genetic information from generation to next.

Also called as Reserve Bank Of Genetic information.

Central Dogma of life: Flow of information from DNA to RNA to Protein synthesis.

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Nitrogenous Nitrogenous BasesBasesDouble ring Double ring PURINESPURINES

Adenine (A)Adenine (A)Guanine (G)Guanine (G)

Single ring Single ring PYRIMIDINESPYRIMIDINES

Thymine (T)Thymine (T)Cytosine (C)Cytosine (C) T or C

A or G

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Chargaff’s RuleChargaff’s Rule

AdenineAdenine must pair with ThymineThymine GuanineGuanine must pair with

CytosineCytosine The bases form weak hydrogen

bonds

G C

T A

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Antiparallel StrandsAntiparallel Strands

One strand of DNA goes from 5’ to 3’ (sugars)

The other strand is opposite in direction going 3’ to 5’ (sugars)

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DNA DNA ReplicationReplication

copyright cmassengale

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Semi conservative Model of Semi conservative Model of ReplicationReplication

Idea presented by Idea presented by Watson & CrickWatson & Crick TheThe two strands of the parental

molecule separate, and each acts as a template for a new complementary strand

New DNA consists of 1 PARENTAL (original) and 1 NEW strand of DNA

Parental DNA

DNA Template

New DNA

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DNA ReplicationDNA Replication Begins at site called as "Begins at site called as "Origins of Origins of

ReplicationReplication"" Specific Protein Called as dna A binds to

this site causing double strands to separate.

As the 2 DNA strands open at the origin, As the 2 DNA strands open at the origin, Replication Replication BubblesBubbles form form

Prokaryotes (bacteria) have a single bubble Eukaryotic chromosomes have MANY

bubbles Bubbles Bubbles

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DNA ReplicationDNA Replication Two strands open forming Two strands open forming Replication Replication

Forks (Y-shaped region)Forks (Y-shaped region) New strands grow at the forksNew strands grow at the forks

ReplicationReplicationForkFork

Parental DNA MoleculeParental DNA Molecule

3’

5’

3’

5’

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DNA ReplicationDNA Replication

Enzyme Enzyme DNA HelicaseDNA Helicase unwinds and separates the 2 unwinds and separates the 2 DNA strands by breaking the DNA strands by breaking the weak hydrogen bonds.weak hydrogen bonds.

Single-Strand Binding Single-Strand Binding Proteins (Proteins (SSBPSSBP)) attach and keep the 2 DNA strands separated and untwisted

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Replication Replication RequirementRequirement

BeforeBefore new DNA strands can form, there must be RNA primersRNA primers present to start the addition of new nucleotides

PrimasePrimase is the enzyme that synthesizes the RNA Primer

DNA polymerase 3 can then add the new nucleotides and forms new strand.

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DNA ReplicationDNA Replication

DNA polymeraseDNA polymerase can only add can only add nucleotides to the nucleotides to the 3’ end3’ end of the of the DNA DNA

This causes the This causes the NEWNEW strand to be strand to be built in a built in a 5’ to 3’ direction.5’ to 3’ direction.

DNA polymerase DNA polymerase also checks for also checks for incoming nucleotides and act as incoming nucleotides and act as proof reading.proof reading.

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Synthesis of the New DNA Synthesis of the New DNA StrandsStrands

Leading strand synthesized 5’ to 3’ in the direction of the replication fork movement.

It is continuous Requires a single RNA primer

Lagging strand synthesized 5’ to 3’ in the opposite direction.

Discontinuous (i.e., not continuous) Requires many RNA primers , DNA is

synthesized in short fragments.

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Polymerase III

5’ 3

’

Leading strand

base pairs

5’

5’

3’

3’

Supercoiled DNA relaxed by gyrase & unwound by helicase + proteins:

Helicase +

Initiator Proteins

ATP

SSB Proteins

RNA Primer

primase

2Polymerase III

Lagging strand

Okazaki Fragments

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RNA primer replaced by polymerase I& gap is sealed by ligase

DNA polymerase 1 removes the RNA primer The gap left behind is Sealed by Ligase and

new Daughter DNA is formed. Thus process of replication is ended.

DNA repair

The process of replication is extremely accurate but errors occurs sometime and cells posses capacity to repair these errors.

Damaged DNA must be repaired If the damage is passed on to subsequent

generations, then we use the evolutionary term - mutation.

Damage from where?

Consequences of DNA replication errors Chemical agents acting on the DNA UV light imparting energy into DNA molecule Spontaneous changes to the DNA

a) Base-excision repair

Presence of the Uracil ,hypoxanthine and xanthine in DNA is a great example base-excision.

N-glycosylase enzyme replace just the defective base. snip out the defective base 2cut the DNA strand Add fresh nucleotide via DNA Polymerase. Gap is sealed by LIGASE

b)Nucleotide-excision repair

UV light and Ionization radiation causes modification of bases, strand breaks, cross-linkage, etc.

It recognizes more varieties of damage in DNA . Cutting of the defective piece by Exinuclease and its

removal (Degraded). Resynthesis of the cut part by DNA polymerase and

ligase. Defect in this mechanism leads to Xeroderma

pigmentosa

c) Mismatch repair These are normally caused by mismatched bases

i.e. AG and CT. Special enzymes scan the DNA for bulky alterations

in the DNA double helix. GATC endonuclease cuts the strand and the strand

is digested by Exonuclease. These gaps are excised and the DNA repaired by

ligase and polymerase enzyme respectively. Defect in this mechanism causes Lynch syndrome

i.e. patient are of high risk of developing Colon cancer.

d) Double-strand break repair

High energy radiation and free radicals causes DNA breakage and leads to cell death.

Repair mechanism is of 2 type 1) Non-homologous end joining(Yeast) 2) Homologous end joining (mamals) Defect: Cancer and Immunodeficiency

syndrome.

THE END

Thank you……..