DNA & DNA Replication

 DNA
◦ Comprised of genes
◦ In non-dividing cell nucleus as
chromatin
 Protein/DNA complex
◦ Chromosomes form during
cell division
 Duplicate to yield a full set in
daughter cell
 Nucleic acids are polymers
◦ Monomers are called nucleotides
◦ Nucleotides = base + sugar + phosphate
 Base = purine or pyrimidine
 Purines = adenine, guanine
 Pyrimidines = thymine, cytosine, uracil
 Sugar = deoxyribose or ribose
 Phosphate, a single phosphate in DNA
◦ Sugar of nt 1 is linked to the phosphate of nt 2 by
a phosphodiester bond
 Nucleotides
◦ A, G, T, C
 Sugar and
phosphate form the
backbone
 Bases lie between

the backbone
 Held together by

H-bonds between
the bases
◦ A-T – 2 H bonds
◦ G-C – 3 H bonds
 Base-pairing rules
◦ AT only (AU if DNA-
RNA hybrid)
◦ GC only
 DNA strand has
directionality – one end
is different from the
other end
 2 strands are anti-
parallel, run in opposite
directions
◦ Complementarity results
◦ Important to replication
 We must start to think of the nucleotides –
A, G, C and T as part of a special language –
the language of genes that we will see
translated to the language of amino acids in
proteins
 A gene is the sequence of nucleotides
within a portion of DNA that codes for a
peptide or a functional RNA
 Sum of all genes = genome
 Semiconservative
 Daughter DNA is a

double helix with 1

parent strand and 1
new strand
 Found that 1 strand

serves as the
template for new
strand
 Each strand of the parent DNA is used as a
template to make the new daughter strand
 DNA replication makes 2 new complete double
helices each with 1 old and 1 new strand
 Site where replication
begins
◦ 1 in E. coli
◦ 1,000s in human
 Strands are separated
to allow replication
machinery contact with
the DNA
◦ Many A-T base pairs
because easier to break 2
H-bonds that 3 H-bonds
 Note anti-parallel
chains
 Bidirectional movement of the DNA replication machinery
 An enzyme that
catalyzes the addition
of a nucleotide to the
growing DNA chain
 Nucleotide enters as

a nucleotide tri-PO4
 3’–OH of sugar
attacks first
phosphate of tri-PO4
bond on the 5’ C of
the new nucleotide
◦ releasing
pyrophosphate (PPi) +
energy
 Bidirectional synthesis of the DNA double
helix
 Corrects mistaken base pairings
 Requires an established polymer (small

RNA primer)
primer before addition of more
nucleotides
 Other proteins and enzymes necessary
 Original theory
◦ Begin adding nucleotides at origin
◦ Add subsequent bases following pairing rules
 Expect both strands to be synthesized simultaneously
 This is NOT how it is accomplished
Correction: Refer to
Figure 6-15 on page 205
of your textbook for
“corrected” figure. This
figure fails to show the
two terminal phosphate
groups attached on the 5’
end of the nucleotide
strand located at the top
of this figure.
 Actually how DNA is synthesized
◦ Simple addition of nucleotides along one strand,
as expected
 Called the leading strand
 DNA polymerase reads 3’  5’ along the leading
strand from the RNA primer
 Synthesis proceeds 5’  3’ with respect to the new
daughter strand
 Remember how the nucleotides are
added!!!!! 5’  3’
 Actually how DNA is synthesized
◦ Other daughter strand is also synthesized 5’3’
because that is only way that DNA can be
assembled
◦ However the template is also being read 5’3’
 Compensate for this by feeding the DNA strand
through the polymerase, and primers and make
many short segments that are later joined (ligated)
together
◦ Called the lagging strand
 Base pairing rules must be maintained
◦ Mistake = genome mutation, may have
consequence on daughter cells
 Only correct pairings fit in the
polymerase active site
 If wrong nucleotide is included
◦ Polymerase uses its proofreading ability to
cleave the phosphodiester bond of improper
nucleotide
 Activity 3’  5’
◦ And then adds correct nucleotide and
proceeds down the chain again in the 5’  3’
direction
 DNA polymerase can only ADD nucleotides
to a growing polymer
 Another enzyme, primase,
primase synthesizes a
short RNA chain called a primer
◦ DNA/RNA hybrid for this short stretch
◦ Base pairing rules followed (BUT A-U)
◦ Later removed, replaced by DNA and the backbone
is sealed (ligated)
 Simple addition of
primer along leading
strand
◦ RNA primer synthesized
5’  3’, then
polymerization with DNA
 Many primers are
needed along the
lagging strand
◦ 1 primer per small
fragment of new DNA
made along the lagging
strand
◦ Called Okazaki
fragments
 Other enzymes needed to excise (remove)
the primers
◦ Nuclease – removes the RNA primer nucleotide
by nucleotide
◦ Repair polymerase – replaces RNA with DNA
◦ DNA ligase – seals the sugar-phosphate backbone
by creating phosphodiester bond
 Requires Mg2+ and ATP
 Helicase opens double helix and helps it
uncoil
 Single-strand binding proteins (SSBP)
keep strands separated – large amount
of this protein required
 Sliding clamp
◦ Subunit of polymerase
◦ Helps polymerase slide along strand
 All are coordinated with one another to
produce the growing DNA strand
(protein machine)
 One polymerase complex apparently synthesizes
leading/lagging strands simultaneously
 Even more complicated in eukaryotes
 For the rare mutations occurring during
replication that isn’t caught by DNA
polymerase proofreading
 For mutations occurring with daily

assault
 If no repair

◦ In germ (sex) cells  inherited diseases

◦ In somatic (regular) cells  cancer
 Mismatch repair
◦ Enzyme complex recognizes mistake and
excises newly-synthesized strand and fills in
the correct pairing
 Eukaryotes
“label” the
daughter strand
with nicks to
recognize the
new strand
◦ Separates new
from old
 Depurination – removal of a purine base
from the DNA strand
 Deamination is the removal of an amine

group on Cytosine to yield Uracil

◦ Could lead to the insertion of Adenine rather
than Guanosine on next round
 Caused by exposure to UV light
 2 adjacent thymine residues

become covalently linked

 Different enzymes
recognize, excise
different mistakes
 DNA polymerase

synthesizes proper
strand
 DNA ligase joins

new fragment with

the polymer