Cell and Molecular Biology

BS 4th Semester M/E

Course Code: ZOOL-303 Credit hours: 3(3-0)

Dr. Lubna Rasool

Department of chemistry
University of Sahiwal
 Objective

• To provide understanding about DNA replication in cell

• To provide understanding about DNA for the ultimate

maintaining and growth of organism and cell

• models of DNA replication
• DNA Polymerases
• Basic rules of replication
Mechanism of DNA replication
Initiation

▪ Proteins bind to DNA and open up double helix

▪ Prepare DNA for complementary base pairing

Elongation

▪ Proteins connect the correct sequences of nucleotides into

a continuous new strand of DNA

Termination

▪ Proteins release the replication complex

 DNA Replication
3’ end has a free deoxyribose
5’ end has a free phosphate

▪ Only build the new strand in the 5’

to 3’ direction
▪ Scans the template strand in 3’ to
5’ direction
Starts at origin
Initiator proteins identify specific base sequences on DNA called

sites of origin

Prokaryotes – single origin site e.g E.coli - oriC

Eukaryotes – multiple sites of origin (replicator) e.g. yeast - ARS

(autonomously replicating sequences)

Eukaryotes

Prokaryotes
……Continued
Replication forks move in one or opposite directions
DNA Replication
Initiation

• Primase (RNA polymerase) builds an RNA primer (5-10 nt )

• DNA polymerase attaches onto the 3’ end of the RNA primer

DNA polymerase
……. Continued

• Helicase is the first replication enzyme to bind at the origin of

replication.

• It move the replication forks forward by "unwinding" the DNA

• SSB coat the separated strands of DNA near the replication fork,

keeping them from coming back together into a double helix.

DNA Replication

Elongation

• DNA polymerase uses each strand as a

template in the 3’ to 5’ direction to

build a complementary strand in the 5’

to 3’ direction DNA polymerase

…. Continued
• E. coli, has a single origin of replication on its chromosome.

• The origin is about 245 base pairs long and has mostly A/T rich
base pairs

• DNA unwind, two Y-shaped structures called replication

forks are formed, or replication bubble.

• The replication forks will move in opposite directions as

replication proceeds.
Leading Strand

▪ Topisomerase unwinds DNA

▪ Helicase breaks H-bonds

▪ DNA primase

▪ DNA polymerase slides along the leading strand in the 3’ to 5’

▪ The RNA primer is degraded by RNase H and replaced with DNA

nucleotides by DNA polymerase

▪ DNA ligase
Lagging Strand
▪ Topisomerase Helicase
▪ DNA primase creates RNA primers in spaced intervals
▪ DNA polymerase slides along the leading strand in the 3’ to 5’
direction synthesizing the matching Okazaki fragments in the 5’
to 3’ direction
▪ The RNA primers are degraded by RNase H
▪ DNA ligase connects the Okazaki fragments to one another
(covalently bonds the phosphate in one nucleotide to the
deoxyribose of the adjacent nucleotide)
 Replication machineries
• Replication machineries consist of factors involved in DNA
replication appearing on template ssDNAs.
• It Includes
– replication enzymes;
– DNA polymerase
– DNA helicases
– DNA clamps
– DNA topoisomerases,
– and replication proteins; e.g. single-stranded DNA binding
proteins (SSB).
 Replisomes
▪ Most of the bacteria, all of the factors involved in DNA

replication are located on replication forks and the complexes

stay on the forks during DNA replication. These replication

machineries are called replisomes or DNA replicase systems.

▪ In eukaryotic and some bacterial cells the replisomes are not

formed.
 Termination

• Termination requires that the progress of the DNA replication

fork must stop or be blocked.

• Termination at a specific locus

(1) A termination site sequence in the DNA, and

(2) A protein which binds to this sequence to physically

stop DNA replication.

• Terminus site-binding protein, or Ter protein named in various

bacteria.
… continued

• Bacteria have circular chromosomes

• It occurs when the two replication forks meet each other on the

opposite end of the parental chromosome.

 Differences Between
Eukaryotic and Prokaryotic
Cells
• Prokaryotic cells are quite simple in structure.

• Have no nucleus, no organelles

• A single, circular chromosome

• Eukaryotic cells have a nucleus,

• Multiple organelles

• More DNA arranged in multiple, linear chromosomes.

 Similarities Between
Prokaryotic and Eukaryotic
DNA Replication
• steps for DNA replication are generally same

• Unwinding the DNA is accomplished by an enzyme DNA

helicase

• New DNA strands is produced by polymerases.

• Both follow semi-conservative Replication pattern

 Continued….
• DNA produced in different directions

• Making of a leading and a lagging strand.

• Lagging strands are created by the production of small DNA

fragments called Okazaki fragments that are eventually joined

together.

• Both types of organisms also begin new DNA strands with a small

primer of RNA.
 Differences Between
Prokaryotic and Eukaryotic
DNA Replication
▪ Site of replication of prokaryotes is cytoplasm while in eukaryotes

is nucleus

▪ Length of Okazaki fragments is1500 bases in eukaryotes and 150

bases in prokaryotes

▪ Replication rate is 50 bases/ s in eukaryotes and 500 bases/s in

prokaryotes
Continued…
Continued…
 Key points

• Plasmid

• Haploid

• Histone protein

• Non coding sequence

• Repetitive DNA sequence

Please study the topics from Campbell Biology

Thank you very much