2. OVERVIEW
DNA is the reserve bank of genetic information. DNA is the chemical basis of
heredity. DNA is not only present in chromosomes of eaukaryotes ,but also in
the single chromosome of prokaryotes.
The genetic information present in DNA is copied and transmitted to
daughter cells.
3. PROKARYOTES
In prokaryotes DNA replication is much simpler than in eukaryotes but
involves the same type of mechanisms.
Proteins involved :-
● DnaA protein
● DNA helicases
● DNA primase
● Single stranded Dna binding protein
● Topoisomerses
● DNA polymerase
● DNA ligase
4. DnaA
It initiates replication by binding with specific nucleotide sequences within
oriC
Binding causes AT-rich region in origin to melt
Melting results in localised region of ssDNA
5. DNA helicases
it binds to ssDNA and forces the strands apart it requires energy provided by
ATP hydrolysis
Unwinding at replication fork results in supercoiling in other regions
It has a hexameric structure
6. DNA primase
Primase is an enzyme that is capable to synthesize short stretches of
RNA sequences which are complementary and antiparallel to template
,known as a primer.
Primers are an integral part of DNA replication. These primers serve as
an initiating site for the addition of nucleotides by DNA polymerase.
7. Single stranded DNA binding protein
Binds to ssDNA binding is cooperative. The SSB are not enzymes but rather
proteins that shift equilibrium of dsDNA and ssDNA in the direction of single
stranded forms
8. Topoisomerases
As the two strands of double helix are separated there is appearance of positive
supercoils in the region of DNA ahead of replication fork and negative supercoils
behind. Toposiomerases are the enzymes which solve this problem by cleaving one
or both strands of DNA.
Type 1 - Reversibly cleave one strand of the double helix.Don’t require ATP .Each
time a nick is created in one DNA strand the intach strand is passed through before
resealing
9. Type 2 - bind tightly with DNA double helix and make transient breaks in both
strand.Then the second stretch of DNA passes through break and reseals it.
10. DNA polymerase
DNA polymerase III elongates a new DNA strand by adding
deoxyribonucleotides one at a time to 3’ end of growing chain , this is the 5’-3’
polymerase function.
This also possess a proofreading function as each nucleotide is added
polymerase III checks if the added nucleotide has complementary base to the
template strand , if not it is removed by 3’-5’ activity of polymerase III.
11. DNA ligase
DNA ligase is a specific type of enzyme that facilitates the joining of DNA
strands together by catalyzing the formation of a phosphodiester bond.
This enzyme joins the 3′ hydroxyl group of one nucleotide with the 5′
phosphate end of another nucleotide at an expense of ATP
12. EUKARYOTES
DNA replication in eukaryotes closely follow prokaryotes.
Proteins involved :-
● Origin recognition complex(ORC)
● Minichromosome maintenance complex(MCM)
● Replication Protein A (RPA)
● Polymerases
● Proliferating Cell Nuclear Antigen (PCNA)
● RNase H
13. Origin recognition complex(ORC)
Multi-subunit DNA binding complex. It binds to the Origin of replication site
in ATP dependent manner
Minichromosome maintenance
complex(MCM)
Minichromosome maintenance complex(MCM) is a DNA helicase
essential for genomic DNA replicatio
14. Replication Protein A (RPA)
Single-stranded DNA binding protein called replication protein A (RPA) binds
to the exposed single-stranded template in lagging strand
The RPA has a function to protect ssDNAA.
15. Polymerases
DNA polymerase αcontains primase and initiates DNA synthesis
DNA polymerase δ elongates okazaki fragments of the lagging strand.
Involved in proof reading
DNA polymerase ε elongates the leading strand
16. Proliferating Cell Nuclear Antigen (PCNA)
The assembly of proliferating cell nuclear antigen (PCNA) is catalysed by
Replication Factor C (RFC) which serves as clamp loader
The PCNA functions as sliding clamp
Rnase H & Flap Endonuclease 1 (FEN 1)
Rnase H and FEN 1 together carry out RNA primer removal
