DNA REPLICATION

DNA REPLICATION
Ds nature of DNA provide a means of replication
during cell division.
Since the separation of the two DNA strands allows
complementary strands to be synthesised upon
them.
Many enzymes and accessory proteins are required
for in vivo replication. How about in vitro?
In prokaryotes it begins at a region of the DNA
termed the origin of replication.
 DNA REPLICATION
DNA has to be unwound before any of the proteins
and enzymes needed for replication can act, and this
involves separating the double-helical DNA into
single stands.

This separating process is carried out by the enzyme

DNA helicase.

To prevent single strands from re-annealing, small

proteins termed single-stranded DNA binding-
proteins (SSBs) attach to the single DNA strands
DNA HELICASE PRIES APART THE DOUBLE HELIX(AT A
RATE OF 1000 BP/SEC)

(Note: eukaryotic helicase has 6 different subunits)

DNA REPLICATION
SSB HOLDS DNA STRAND BY SUGAR-PHOSPHATE BACKBONE
SINGLE-STRAND DNA BINDING PROTEINS
INITIAL EVENTS AT THE REPLICATION FORK
INVOLVING DNA UNWINDING
DNA REPLICATION IS SEMICONSERVATIVE
(FOLLOW THE YELLOW STRANDS)
DNA Topoisomerase I: Cut & Swivel
(to relaxe wound DNA)
 DNA REPLICATION
On each exposed single strand a short,
complementary RNA chain termed a primer is first
produced, using the DNA as a template.

The primer is synthesised by an RNA ploymerase

enzyme known as a primase, which uses
ribonucleoside triphosphate.

Then DNA polymerase III (DNA pol III) also uses the
original DNA as a template for synthesis of a DNA
strand, using the RNA primer as a starting point.
 DNA REPLICATION

The primer is vital since it leaves an exposed 3’

hydroxyl group for the incoming new nucleotides to
be added by DNA polymerase III only to the 3’ end
and not the 5’ end of a nucleic acid.

Synthesis of the DNA strand therefore occurs only in

a 5’ to 3’ direction from the RNA primer.

This DNA strand is usually termed the leading strand

and provides the means for continuous DNA
synthesis.
 DNA REPLICATION
(c)Ds DNA separates at the origin of replication.
RNA polymerase synthesises short RNA primer
strands complementary to both DNA strands.
 DNA REPLICATION

(b) DNA polymerase III, synthesises new DNA

strands in a 5’ to 3’ direction, complementary to the
exposed, old DNA strands, and continuing from the
3’ end of each RNA primer.
 DNA REPLICATION

(c) As the replication fork moves away from the

origin of replication, DNA polymerase III continues
the synthesis of the leading strand, and synthesises
DNA between RNA primers of the lagging strand.
 DNA REPLICATION

(d) DNA polymerase I removes RNA primers from

the lagging strand and fills the resulting gaps with
DNA. DNA ligase then joins the resulting fragments,
producing a continuous DNA strand.
 DNA REPLICATION
Consequently, DNA synthesis is in the same
direction as DNA replication for one strand (the
leading strand) and in the opposite direction for the
other (the lagging strand). RNA primer synthesis
occurs repeatedly to the allow the synthesis of
fragments of the lagging strand.
 DNA REPLICATION
Since the two strands of double helical DNA are
antiparallel, only one can be synthesised in a
continuous fashion.

Synthesis of the other strand must take place in a

more complex way. The precise mechanism was
worked out by Reiji Okazaki in the 1960s.

Here, the strand, usually termed the lagging strand,

is produced in relatively short stretches of 1-2 kb
termed Okazaki fragments in a 5’ to 3’ direction,
using many RNA primers for each individual stretch.
 SLIDING CLAMP HOLDS POLYMERASE ONTO DNA
(NOTE THAT THIS OCCURS REPEATEDLY ON LAGGING STRAND)

DIMERIC STRUCTURE
LEADING STRAND, LAGGING STRAND, OKAZAKI FRAGMENTS