Gene Expression

Lesson 10
DNA STRUCTURE
 Nucleic acid Double Helix (2
strands of DNA)
DNA STRUCTURE
• Nucleotide – sugar, phosphate group and a
base

Complementary strands pair up

(Adenine & Thymine, Cytosine &
Guanine)
• Base pairs held together by hydrogen bonds

Strands are antiparallel (5’ - 3’ and

3’ – 5’)
DNA REPLICATION
DNA
REPLICATION

+ Basic Concept: create a new

strand by matching nucleotides
to an existing strand
+ Purpose: cells need to make a
copy of DNA before dividing so
each daughter cell has a complete
copy of genetic information
3 proposed Models of Replication
Meselson and Stahl Experiment
DNA + Replication is semi-conservative
(one strand is old, one strand new)
REPLICATION
Semi-conservative
Parental DNA strands

Each of the parental strands serves as a

template for a daughter strand

Daughter DNA strands

Anti-parallel strands

+ Nucleotides in DNA backbone are bonded from

phosphate to sugar between 3 & 5 carbons
+ DNA molecule has “direction”
+ complementary strand runs in opposite
direction
THIS WILL CAUSE A PROBLEM FOR
REPLICATION
DNA Replication
Makes logical sense how DNA replication happens:
1. DNA has two strands with identical information
2. Must open up
3. Exposing unpaired bases
4. Bases are matched perfectly (compliment)
5. Forming two double helixes from one
 Replication
continued
+ DNA is read in parts
+ It does not start at the
beginning and go all the way
to the end, that would take too
long
+ Replication can start at many
points in eukaryotes
Large team of enzymes coordinates replication
E. coli Origin of Replication
chromosome

oriC

AT-rich region
5′ –GGA T CC T GGGT A T T AAAAAGAAGA T C T AT T TA T T T AGAGA T C T G T T C T AT
CC T AGGACCC A T A A T T T T T C T T C T AGA T AA AT AAA T CTCT AGAC AAGA T A
1 DnaA box 50
T G T GA T C T CT T A T T AGGA T CGC AC T GCCCT GT GGA T AACA AGGA T CGGCT
AC AC T AGAGA A T A A TCCT AGCGT GAC GGGACACCT A T TGT T CC T AGCC GA
51 DnaA box 100
T T T A AGA TCA ACA ACCTGGA AAGGA T C AT T AA CTG T GAAT GA T CGG T GAT
A A A T T C T AGT T GT T GGACC T T T CC T AGT AA T T GAC ACT T AC T AGCC AC T A
101 DnaA box 150
CC T GGACCGT A T A AGCTGGGA T C AGA A TGAGGGT T A TACA CAGC TC A A AA
GGACC T GGCA T A T T CGACCC T AGT C T T ACT CCCAA T ATGT GT C GAGT T T T
151 DnaA box 200
AC T GA AC AACGGT T GT TCT T TGGA T A ACTACCGGT T GA T CCA AGCT T CCT
T GAC T T GT T GCC A ACAAGA A ACCT A T T GAT GGCCA ACT AGGT T C GA AGGA
201 DnaA box 250
GAC AGAGT TA T CCA CAGTAGA TCGC –3′
CT GT C T C A AT AGGT GTCAT C T AGC G
251 275
 5′ 3′
3′ 5′

AT-rich region DnaA boxes

DnaA proteins bind to DnaA boxes and to
each other. Additional proteins that cause
the DNA to bend also bind (not shown).
This causes the region to wrap around
the DnaA proteins and separates the
AT-rich region.

AT- DnaA protein

rich
region

How the origin 5′

3′ 3′
5′

sequence initiates DNA helicase (DnaB protein) binds to the

origin. DnaC protein (not shown) assists
this process.

replication DNA helicase

5′
3′ 3′
5′
DNA helicase separates the DNA in both
directions, creating 2 replication forks.

3′
5′ Fork Fork
3′ 5′
 + Unwind DNA
+ helicase enzyme
+ unwinds part of DNA helix
+ stabilized by single-stranded binding proteins
Replication: 1st step + PREVENTS DNA MOLECULE FROM CLOSING!
+ DNA gyrase
+ Enzyme that prevents tangling upstream from the
replication fork

helicase gyrase

single-stranded binding proteins replication fork

Replication: 2nd step
+ RNA Primase
+ Adds small section of RNA (RNA
primer) to the
3’ end of template DNA
+ Why must this be done?
+ DNA polymerase 3
(enzyme that builds new
DNA strand) can only add
nucleotides to existing
strands of DNA
 Replication: 3rd step
 Build daughter DNA strand
 add new complementary bases
 With the help of the enzyme
DNA polymerase III

DNA
Polymerase III
Replication: 4th step
+ Replacement of RNA primer by DNA
+ Done by DNA polymerase I
Leading & Lagging strands Okazaki

Limits of DNA polymerase III

 can only build onto 3 end of
an existing DNA strand 5


rag ments
ki f
Okaza 5
3 5 5 3
3
5 Lagging strand
3
ligase
growing 3
replication fork
5
Leading strand

Lagging strand
3
5

3
DNA polymerase III
 Okazaki fragments
 joined by ligase Leading strand
 “spot welder” enzyme  continuous synthesis
DNA replication on the lagging strand
RNA primer is added
 built by primase
 serves as starter sequence for DNA polymerase III

HOWEVER short segments called Okazaki fragments

are made because it can only go in a 5 3 direction

5

3 5 3
5
3
3 5

growing 3 primase
replication fork DNA polymerase III
5

RNA 5

3
Replacing RNA primers with DNA
NEXT DNA polymerase I
 removes sections of RNA DNA polymerase I
primer and replaces with 5

DNA nucleotides 3

3
5 ligase
growing 3
replication fork
5

RNA 5

3

STRANDS ARE GLUED

TOGETHER BY DNA LIGASE
 Replication fork
DNA
polymerase III lagging strand
DNA
polymerase I
3’
Okazaki primase
fragments 5’
5’ ligase
3’ 5’ SSB

3’ helicase

DNA
polymerase III
5’ leading strand
3’
direction of replication
SSB = single-stranded binding proteins
DNA Replication

Notes:

1. Enzymes and their functions

2. Direction of replication
- Always 5’ to 3’
DNA Replication

3. Difference
between leading
and lagging strands
 SSBs
Helicase DNA
Polymerase III

Leading Strand

DNA DNA Ligase

Polymerase I
Primase DNA Pol
Primer III Lagging
Strand
 MAJOR PROTEINS INVOLVED IN DNA
REPLICATION
+Replication begins with DNA untwisting itself and opening up
+ DNA Helicase
+ Responsible for unwinding the molecule
+The old DNA strands = templates
+New nucleotides come and bond to the template creating a new
strand = Complementary Strand
+ DNA Polymerase
+ Responsible for adding nucleotides
+ Forms covalent bond between nucleotides
+ DNA ligase binds the two new strands
 Functions of key proteins involved with DNA
replication
• DNA helicase breaks the hydrogen
Origin
bonds between the DNA strands. Single-strand
binding protein
• Topoisomerase alleviates positive DNA helicase DNA polymerase III 3′
5′
supercoiling. Topoisomerase II

• Single-strand binding proteins keep Leading

RNA
primer
the parental strands apart. RNA primer
strand

• Primase synthesizes an RNA Replication fork

DNA polymerase III
Okazaki fragment
primer.
Primase
5′ DNA
3′ Parental DNA Lagging strand
• DNA polymerase III synthesizes a ligase
3′
daughter strand of DNA. 5′

• DNA polymerase I excises the

RNA primers and fills in with Direction of fork movement
Linked Okazaki
fragments
DNA (not shown).

• DNA ligase covalently links the

Okazaki fragments together.
Error Correction
DNA Polymerase – can also “proofread” the newly formed strand:
1. Excise bases that are “mismatched”

2. Replace with the correct base

3. Then move forward to next base

 DNA Replication

DNA from cell to cell, has to be replicated with fidelity

Mistakes – mutations, cancer, etc.

Only one mistake in a billion nucleotides is made by the replication machinery

DNA replication is a result of the coordination of > 10 protein complexes and

enzymes
 DNA is a double helix

Bases in middle make up sequence

Summary of Semi-conservative replication
DNA •
•
Strands separate
Each one is copied
Replication • One “lagging” and one “leading”
• Forming two double helixes from one
• Proteins do all the copying
• Incredibly accurate
 Practice Replication
◦T T G C T A G

◦T A G C G C T

◦A C C G T C A

◦G C T A T G T