Dna replication lec (2)

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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.

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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.

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DNA Replication

Learning Outcomes

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

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

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Why replicate DNA?????

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

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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????

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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

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

melting

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Initiation

replicon.

Initiation…..

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

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• Separation of dsDNA replication forks

• Two replication forks move in opposite directions

bidirectional

replicon.

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

Initiation…..

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4. DNA Gyrase /Topoisomerase: prevents formation of supercoils

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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)

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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

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6. Primase: RNA Polymerase

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

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II. Elongation

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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

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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

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Elongation…..

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III. Termination:

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

• gaps-filled with deoxyribonucleotides

• sealed by the enzyme ligase.

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

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

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• 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 

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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.

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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.

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