Apr 17, 2023 1

Apr 17, 2023 2

Apr 17, 2023 3

Dr Farah Deeba Khan

www.wiley.com/college/pratt/.../animations/dna_replication/index.html

Learning Objectives• To understand the process of DNA replication.

• To differentiate between Prokaryotic & Eukaryotic DNA replication.

• To determine its importance.

• To learn DNA repair mechanism.

Apr 17, 2023 4

Apr 17, 2023 5

DNA Replication

Transcription Translation

ReplicationThe process of making an identical copy of a section of double-stranded DNA, using existing DNA as a template for the synthesis of new strands.

Apr 17, 2023 6

DNA Replication

Learning Outcomes

Learning OutcomesStudents should be able to answer…………..

Apr 17, 2023 7

Apr 17, 2023 8

Learning Outcomes:Students should be able to answer…………..

Apr 17, 2023 9

Why replicate DNA?????

• If DNA never replicated, • mitosis would slowly halve the size of the genome until each cell would die. 

Apr 17, 2023 10

CELL CYCLEStage Activity Duration

G1 Growth & increase in cell size 10 hr

S DNA synthesis 8 hr

G2 Post-DNA synthesis 5 hr

M Mitosis 1 hrApr 17, 2023 11

When DNA replicates?

How Replication Works????

Apr 17, 2023 12

Apr 17, 2023 13

Models of Replication

one each of the old and new strands

Sections of the old and new pieces duplex dispersed randomly

Old duplex is conserved with completely new strand

Requirements

• DNA template

• RNA Primer (Free 3' -OH group )

• dNTP (dATP, dGTP, dCTP, dTTP)

• Proteins & Enzymes of DNA Replication– DNA Helicases – DNA single-stranded binding proteins (SSB-protein)– DNA Gyrase /topoisomerase– Primase – DNA Polymerase  – DNA Ligase Apr 17, 2023 14

Sequential ActionsInitiation:• Recognition of the site of start

(short sequence rich in AT base pairs)

• Unwinding & Separation of dsDNA• Primer synthesis

Elongation:

• Add dNTPs to the existing strand

• Form phosphodiester bonds

• Correct the mismatch bases

• Extend the DNA strand

Termination:

• Stop the replicationApr 17, 2023 15

Apr 17, 2023 16

I. Initiation1. DnaA protein binding with AT rich regions in DNA

melting

Apr 17, 2023 17

Apr 17, 2023 18

Initiation

replicon.

Initiation…..

Apr 17, 2023 19

2. Helicases: bind to dsDNA and stimulate separation of the two strands.

Apr 17, 2023 20

• Separation of dsDNA replication forks

• Two replication forks move in opposite directions

bidirectional

replicon.

Apr 17, 2023 21

3. Single-stranded binding proteins: bind, stabilize and keep the ssDNA separated.

Initiation…..

Apr 17, 2023 22

4. DNA Gyrase /Topoisomerase: prevents formation of supercoils

Apr 17, 2023 23

Topoisomerase

Type I • negative supercoils in bacteria• negative & positive in eukaryotes• ATP not required

Type II• requires ATP• for interlocked supercoils after Chromosomal Replication

(Prokaryotes & eukaryotes)

Apr 17, 2023 24

Apr 17, 2023 25

5. RNA Primer:• strands of short ~ 10 bp, double stranded regions

• have RNA base pairs with a free –OH on 3’-end

• serve as starting point for DNA synthesis

Apr 17, 2023 26

Apr 17, 2023 27

6. Primase: RNA Polymerase

adds (ATP, UTP, CTP, GTP) one at a time to 5’3’, anti-parallel to parent DNA strand

Apr 17, 2023 28

Apr 17, 2023 29

II. Elongation

Apr 17, 2023 30

Direction of replication5’3’

Elongation…

7. DNA Polymerase III: adds (dATP, dTTP, dCTP, dGTP) one at a time to RNA primer, 5’3’, antiparallel to parent DNA strand

Apr 17, 2023 31

Elongation….

DNA Polymerase Type III

•  read in the 3' → 5' direction, • adds dNTPs at rate ~800 dNTPs/S• Pol III discriminate between correctly paired bases and incorrectly paired bases proofreading

Apr 17, 2023 32

Elongation…..

Apr 17, 2023 33

Apr 17, 2023 34

Apr 17, 2023 35

III. Termination:

• The RNA primers degraded by RNAse H &DNA Polymerase I (exonuclease),

• gaps-filled with deoxyribonucleotides

• sealed by the enzyme ligase.

Apr 17, 2023 36

Apr 17, 2023 37

Apr 17, 2023 38

Apr 17, 2023 39

Apr 17, 2023 40

Termination….In prokaryotes two replication forks meet at one point Ter

Apr 17, 2023 41

In linear eukaryotic chromosomes, • DNA replicated until meets another origin of replication • Termination same

Apr 17, 2023 42

• non-coding part of DNA at the ends of Ch…. • composed of several thousands repeats of hexameric sequence AGGGTT

• Problem occurs at end of a linear chromosome is reached.

• In this gap DNA is extended by telomerase. • This extension is called a telomere 

Apr 17, 2023 43

Mechanism of Action of Telomerase

Functions of telomeres • Prevent the termini from entangling & sticking• structural integrity• complete replication of chromosomes.

• functional organization of chromosomes within the nucleus.

• regulation of gene expression.

• Serves as a molecular clock that controls the replicative capacity of human cells and their entry into senescence.

Apr 17, 2023 44

Apr 17, 2023 45

Telomere Length and Aging

• Most human somatic cells lack telomerase activity but present in over 90% of cancerous and in vitro immortalized cells.

• Shorter telomeres are associated with cellular senescence and death.

• Diseases causing premature aging are associated with short telomeres.

Apr 17, 2023 46