DNA Replication

2007-2008

DNA Replication
Dr.MAHA SMAISM

STRUCTURE OF NUCLEIC ACIDS

Image by: Riedell
Sugar can be DEOXYRIBOSE (DNA) RIBOSE (RNA)
Built from NUCLEOTIDE SUBUNITS
NITROGEN BASES CAN BE: ADENINEGUANINECYTOSINETHYMINEURACIL
Arrow from: http://www.harrythecat.com/graphics/b/arrow48d.gif


DNA has no URACIL RNA has no THYMINE PURINES (A & G) have 2 RINGS PYRIMIDINES (T, C, & U) have 1 RING
http://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/DNA/DNA/fg4.html http://student.ccbcmd.edu/~gkaiser/biotutorials/dna/fg29.html

Directionality of DNA

You need to number the carbons! it matters!
OH
CH2
O
4 5 3 2 1 PO4
N base
ribose
nucleotide
This will beIMPORTANT!!


The DNA backbone
Made of phosphates and deoxyribose sugarsPhosphate on 5’ carbon attaches to 3’ carbon of next nucleotide OH
O
3 PO4
base
CH2
O
base
O
P
O
C
O
–O CH2
1 2 4 5 1 2 3 3 4 5 5

Double helix structure of DNA

Anti-parallel strands
Nucleotides in DNA backbone are bonded from phosphate to sugar between 3 & 5 carbonsDNA molecule has “direction”complementary strand runs in opposite direction 3 5 5 3


Bonding in DNA
….strong or weak bonds?How do the bonds fit the mechanism for copying DNA? 3 5 3 5 covalent phosphodiester bonds
hydrogen bonds

Base pairing in DNA

Purines adenine (A) guanine (G) Pyrimidines thymine (T) cytosine (C) Pairing A : T 2 bonds C : G 3 bonds


CHARGAFF’s RULES Erwin Chargaff analyzed DNA from different organisms and found A = T G = C Now know its because: A always bonds with T G always bonds with C A Purine always bonds to a Pyrimidine

NUCLEOSOME

CHROMATIN

Semi- Conservative Conservative Dispersive

http://student.ccbcmd.edu/~gkaiser/biotutorials/dna/fg12.html
Starting place = ORIGIN OF REPLICATION Bacteria have one
Bacterial replication
Eukaryotes-multiple origins

Copying DNA

Replication of DNA base pairing allows each strand to serve as a template for a new strand new strand is 1/2 parent template & 1/2 new DNA semi-conservative copy process

Replication: 1st step

Unwind DNA
Dna A protein: bind to specific nucleotide sequences at the origin of replication (rich in AT base pair) by melt the bonds :

helicase enzyme unwinds part of DNA helix stabilized by single-stranded binding proteins

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replication fork
helicase
DNA REPLICATION FORK

5 3 exonuclase removes RNA primer DNA polymerase I full the gaps

But DNA polymerase I still can only build onto 3 end of an existing DNA strand Replacing RNA primers with DNA
5 5 5 5 3 3 3 3 growing replication fork
DNA polymerase I
RNA
ligase



Loss of bases at 5 ends in every replicationchromosomes get shorter with each replicationlimit to number of cell divisions? DNA polymerase III
All DNA polymerases can only add to 3 end of an existing DNA strand Chromosome erosion
5 5 5 5 3 3 3 3 growing replication fork
DNA polymerase I
RNA
Houston, we  have a problem!

Limits of DNA polymerase IIIcan only build onto 3 end of an existing DNA strand Leading & Lagging strands
5 5 5 5 3 3 3 5 3 5 3 3 Leading strand
Lagging strand
Okazaki fragments
ligase
Okazaki
Leading strand continuous synthesis
Lagging strandOkazaki fragmentsjoined by ligase“spot welder” enzyme DNA polymerase III
  3 5 growing replication fork

DNA polymerase III

Replication fork / Replication bubble
5 3 5 3 leading strand
lagging strand
leading strand
lagging strand
leading strand
5 3 3 5 5 3 5 3 5 3 5 3 growing replication fork
growing replication fork
5 5 5 5 5 3 3 5 5 lagging strand
5 3


DNA polymerase III
RNA primer built by primase serves as starter sequence for DNA polymerase III
Limits of DNA polymerase IIIcan only build onto 3 end of an existing DNA strand Starting DNA synthesis: RNA primers
5 5 5 3 3 3 5 3 5 3 5 3 growing replication fork
primase
RNA

DNA Polymerase III

energy
Replication
3 3 5 5

DNA Polymerase III

Replication: 2nd step
Where’s theENERGYfor the bondingcome from? Build daughter DNA strand add new complementary bases DNA polymerase III

energy

ATP
Energy of Replication
Where does energy for bonding usually come from?
ADP
ADP
modified nucleotide
We comewith our ownenergy!
YourememberATP!


ATP
Energy of Replication
Where does energy for bonding usually come from?
AMP
modified nucleotide
energy
We comewith our ownenergy!
And weleave behind anucleotide!
Are thereother energynucleotides?You bet!
DNA replication

Energy of Replication

The nucleotides arrive as nucleoside triphosphatesDNA bases with P–P–PP-P-P = energy for bondingDNA bases arrive with their own energy source for bondingbonded by enzyme: DNA polymerase III ATP
GTP
TTP
CTP
See animation


Adding bases can only add nucleotides to 3 end of a growing DNA strandneed a “starter” nucleotide to bond tostrand only grows 53 DNA Polymerase III
Replication
3 3 5 5 need “primer” bases to add on to


DNA Polymerase III
energy
Replication
3 3 5 5

Replication

3 3 5 5

3 5 5 5 3 need “primer” bases to add on to 3 energy

3 5 Can’t build3’ to 5’direction

3 5 5 5 3 3 3 5 need “primer” bases to add on to

3 5 5 5 3 need “primer” bases to add on to 3 energy
3 5

3 5 5 5 3 need “primer” bases to add on to 3 energy

3 5

3 5 5 5 3 3 3 5

3 5 5 5 3 3 energy
3 5

3 5 5 5 3 3 3 5

ligase
Joins fragments

TELOMERES & TELOMERASE

Image from: AP BIOLOGY by Campbell and Reese 7th edition
Primer removed butcan’t be replaced withDNA because no3’ end available forDNA POLYMERASE Each replicationshortensDNA strand

Replication fork

3’ 5’ 3’ 5’ 5’ 3’ 3’ 5’ helicase
direction of replication
SSB = single-stranded binding proteins
primase
DNA polymerase III
DNA polymerase III
DNA polymerase I
ligase
Okazaki fragments
leading strand
lagging strand
SSB

DNA polymerases

DNA polymerase III 1000 bases/second! main DNA builder DNA polymerase I 20 bases/second editing, repair & primer removal
DNA polymerase III enzyme
Arthur Kornberg 1959
Thomas Kornberg

Fast & accurate!

It takes E. coli <1 hour to copy 5 million base pairs in its single chromosome divide to form 2 identical daughter cells Human cell copies its 6 billion bases & divide into daughter cells in only few hours remarkably accurate only ~1 error per 100 million bases ~30 errors per cell cycle

Editing & proofreading DNA

1000 bases/second = lots of typos! DNA polymerase I proofreads & corrects typos repairs mismatched bases removes abnormal bases repairs damage throughout life reduces error rate from 1 in 10,000 to 1 in 100 million bases

PROOFREADING & REPAIR

Errors can come from:“proofreading mistakes” that are not caught Environmental damage from CARCINOGENS (Ex: X-rays, UV light, cigarette smoke, etc) EX: Thymine dimers http://www.mun.ca/biology/scarr/Thymine-Thymine_Dimers.html
http://www.personal.psu.edu/staff/d/r/drs18/bisciImages/index.html



NUCLEOTIDE EXCISION REPAIR
Cells continually monitor DNA and make repairs NUCLEASES-DNA cutting enzyme removes errorsDNA POLYMERASE AND LIGASE can fill in gap and repair using other strandXeroderma pigmentosum- genetic disordermutation in DNA enzymes that repair UV damage in skin cellscan’t go out in sunlightincreased skin cancers/cataracts http://www.maximilien.asso.fr/images/maxcasque.jpg
http://www.nature.com/jid/journal/v128/n3/images/jid200825i2.jpg