BM101: BIOLOGY FOR ENGINEERS

DNA Replication

Instructor: Yashveer Singh, PhD

Slides courtesy: Dr. Durba Pal

17 September 2019
DNA replication summary
▪ All organisms duplicate their
DNA with extraordinary accuracy
before each cell division
▪ Replication machinery achieves
this accuracy, while duplicating
DNA at a speed of 1000
nucleotides per second.
▪ 1 error out of 109 nucleotides
per generation
▪ An overview of replication
process is provided here

Stoker’s Biological Chem

DNA replication summary
▪ Mechanism of chain
elongation in DNA or RNA
involves formation of a
phosphoester bond
between the 3‘-oxygen of
a growing strand and the
5’- phosphate of a
nucleotide triphosphate
(NTP)

Lehninger’s Biochemistry
 DNA replication
▪ DNA replication is semiconservative, which means that one
strand of daughter DNA is from the parent, whereas the
other strand is newly synthesized
DNA replication
▪ DNA replication begins from mostly AT rich
regions (origin or ori site)
▪ DNA helicase binds to AT region and unwinds
the double helix in that region, leading to the
formation of a replication fork
▪ More than one fork are formed
▪ Helicases, and therefore fork, move in both
directions so that complete strand is unwind
 DNA replication
Intrastrand pairing ▪ Re-annealing and formation of intra-strand
structures is prevented by single-strand DNA-
binding (SSB) proteins
▪ SSB coat and straighten out the regions of
single-strand DNA
 DNA replication
▪ DNA polymerase cannot start polymerisation on
its own (de novo) and requires a pre-existing DNA
or RNA strand

▪ DNA primase synthesizes a short RNA or DNA

strand, called a primer, to begin the chain growth
DNA replication
▪ As the two DNA strands are antiparallel,
they have to be copied in 5ʹ-to-3ʹ direction
and 3ʹ-to-5ʹ direction but DNA polymerases
can synthesize only in 5ʹ-to-3ʹ direction
▪ Therefore DNA synthesis is continuous on
one strand (5’-3’, leading strand) and
discontinuous on the other strand (lagging
strand)
▪ Polymerase adds 1000 nucleotides per
second with precision
▪ Fall off of the DNA polymerase from the
template strand is prevented by PCNA
protein, which functions as a sliding clamp
 DNA replication
▪ On the lagging strand, DNA primase synthesises RNA
primer containing a nucleotide with a 3ʹ-OH group at
one end
▪ This primer is elongated by the DNA polymerase at
this end to begin an Okazaki fragment (short
sequences)
▪ The synthesis of each Okazaki fragment ends when
this DNA polymerase runs into the RNA primer
attached to the 5ʹ end of the previous fragment
▪
 DNA replication
▪ RNA primer attached to newly synthesized DNA strand is
removed by RNase H enzyme, which cleaves the RNA strand of
DNA-RNA hybrid
▪ DNA polymerase replace the degraded primer with newly
synthesized DNA strand
▪ The synthesis of new strand starts from a nick (broken
phosphodiester linkage) and as a result of this synthesis nick
moves along the strand (nick translation), which are then
joined by DNA ligases

Lehninger’s Biochemistry
 DNA replication
▪ There is one error out of 109 nucleotides per generation and
it is because of proofreading activity of DNA polymerase
(exonucleolytic proofreading)
▪ Kicks in after an incorrect nucleotide has been covalently
added to the growing chain.
▪ DNA molecules with a mismatched nucleotide at the 3ʹ-OH
end are not stable for extension
▪ DNA polymerase is halted and it moves a step backwards to
clip off any unpaired or mis-paired nucleotides
▪ Minor changes in helix geometry results hydrogen bonding G
and T, a rare isomeric form of C pairs with A, and mutation
 DNA replication

▪ Proper base-pair geometry of a correct incoming deoxyribonucleotide

triphosphate stabilises the polymerase around the base pair
▪ Dissociation of pyrophosphate relaxes the polymerase, allowing
translocation of the DNA by one nucleotide
▪ Mismatch results in unpairing of newly synthesized DNA from the template
and it falls of on the editing (exonuclease) site
 DNA replication

▪ As replication fork moves

along double-strand DNA, it
encounters winding problem
▪ The tension must be relaxed
for fork progression
▪ Type I DNA topoisomerases
releases the strain by cleaving
one strand and allowing free
rotation
DNA replication
▪ The two strands may entangle
during the replication

▪ Type II DNA topoisomerase

removes the entangle by
cleaving one of the strand
Simplified replication fork
 Do it yourself
▪ Read section, DNA Replication, in Chapter 6 – DNA Replication, Repair, and
Recombination from the Essential Cell Biology, B. Alberts, D. Bray, K. Hopkin,
A. Johnson, J. Lewis, M. Raff, K. Roberts, and P. Walter, Garland Science, IV
Edition, 2014

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