Gena Relication

dr. Evi Kurniawaty, M.Sc

 Review of NUCLEIC ACIDS

• RNA
• DNA – Carries information
– Genetic material from the nucleus to
– In the nucleus the site where
proteins are made
– Double stranded
– Single stranded
– A,T
– A,U
– C,G
– C,G

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http://tigger.uic.edu/classes/phys/phys461/phys450/ANJUM04/RNA_sstrand.jpg
 DNA is found in the nucleus

Chromosomes
are made of
Humans have 23 DNA
pairs of
chromosomes

http://games.goadrich.com/images/find-dna.jpg
 The sides of
the ladder are
sugar and
phosphates;
the rungs of
the ladder are
the nitrogen
bases A,T,C,G

http://www.mariemontschools.org/halsall/images/dna_molecule.gif
How DNA is the Master Control
DNA can make copies of itself=
REPLICATION
DNA
nucleotides

REPLICATION
REpeats
 DNA Replication
• The structure of DNA provided an insight to
Watson and Crick for how DNA replicates
– each strand in a duplex of DNA is complementary to each
other, each can form a template when separated.
– The order of bases on one strand can be used to add in
complementary bases and therefore duplicate the pairs of
bases exactly.
 Model for DNA Replication
Watson and Crick’s model: semiconservative replication

Copyright © 2005 Pearson Education, Inc., publishing as Benjamin Cummings

 Model for DNA Replication
Watson and Crick’s model: semiconservative replication

before after

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 Three models of DNA replication

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 Three models of DNA replication

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 Fig 16.10 Three models of DNA replication

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 Experiment which supports the
Semiconservative Model

• Matthew Meselson and Franklin Stahl

– experiments supported the semiconservative
model
– labeled the nucleotides of the old strands with a
heavy isotope of nitrogen (15N) while any new
nucleotides would be indicated by a lighter
isotope (14N).
– Replicated strands could be separated by density
in a centrifuge.
Meselson-Stahl Experiment supports
the Semiconservative Model
– Each model: the semi-conservative model, the
conservative model, and the dispersive model,
made specific predictions on the density of
replicated DNA strands.

Semi-
Conservative Dispersive
Conservative

one heavy both

one light intermediate
Meselson-Stahl
experiment

Conclusion:
DNA replication follows
semiconservative model

Conservative and Dispersive

models were disproven in
their experiment.

Copyright © 2005 Pearson Education, Inc.,

publishing as Benjamin Cummings
 DNA Replication

• More than a dozen enzymes and other

proteins participate
– E. coli can replicate 4.5 x 106 base pairs bp) in
less than an hour
– human cells can replicate 6 x 109 bp in only a few
hours
• DNA replication is very accurate
– less than 1 error per billion nucleotides!!
 DNA Replication Start Sites

• Where does DNA replication start?

– special sites termed origins of replication
– single site in bacterial chromosome
– multiple sites in eukaryotic chromosome
• Enzymes (helicases) separate the two strands
– forms a replication “bubble”
– other proteins (single strand binding proteins -
ssb) bind to keep strands separated
 Origins of Replication

replication bubbles fuse

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 Enzymes of DNA Replication
• DNA polymerases
– synthesize DNA by adding a nucleotide that is
complementary to the base in the template strand
– Rate of synthesis
• Bacteria - 500 nucleotides / sec
• Human cells - 50 nucleotides / sec
 Incorporation of a nucleotide

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 Fig 16.14 DNA Synthesis

• leading strand is
synthesized continuously
• lagging strand is synthesized
discontinuously in short
segments called
Okazaki fragments
DNA ligase joins the
fragments
Replication Fork

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 Initiating DNA Synthesis
• After separation of the DNA strands
– DNA Polymerase cannot initiate DNA syn.
– Needs a 3’ OH to add nucleotide to.
– synthesizing a new chain requires a primer, a short
segment of RNA
– Primase (an RNA Polymerase) adds about 10
nucleotides complementary to template
Priming DNA
Synthesis

Note: RNA primer is

removed from DNA
by another DNA
Polymerase

Copyright © 2005 Pearson Education, Inc., publishing as Benjamin Cummings

Priming DNA
Synthesis

Copyright © 2005 Pearson Education, Inc., publishing as Benjamin Cummings

 Table 16.1 Bacterial DNA Replication
Proteins

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 Bacterial DNA Replication Proteins

• Helicase
– Unwinds parental double helix at replication forks
• ssb protein
– binds to and stabilizes ssDNA
• Topoisomerase
– Corrects “overwinding” ahead of replication forks
– breaks, swivels, and rejoins DNA strands
 Bacterial DNA Replication Proteins

• Primase
– synthesizes single primer for leading strand
– synthesizes RNA primer for each lagging strand
• DNA pol III
– continuous synthesis of leading strand
– discontinuous synthesis of lagging strand
 Bacterial DNA Replication Proteins

• DNA pol I
– removes primer (RNA) from DNA strand and
replaces it with DNA
• DNA Ligase
– joins 3’ end of fragment with 5’ end of adjacent
fragment
DNA Helicase
DNA double helix are tightly
coupled. High temperature is
needed to break them (95oC)
DNA replication Fork
 Summary of DNA Replication

Copyright © 2005 Pearson Education, Inc., publishing as Benjamin Cummings

• Mutation: A change in the base sequence of
the DNA
• Mutations are changes in the genotype which
may or may not affect the phenotype
 Causes of mutations
• Spontaneous mutations
– Occur in the absence of mutation causing agents
– Due to occasional mistakes in DNA replication
• Induced mutations
– Caused by mutagens, agents such as chemicals
and radiation which induce mutations
 Types of Repair of DNA

• Bases may be damaged by chemical and/or

physical agents
– UV light, reactive chemicals, radiation, etc.
• Some mismatched bases may be missed by
proofreading activity of DNA pol
• Must be corrected to ensure high fidelity of
DNA sequence
 Types of mutations

• Base substitutions
– The most common type of mutation
– A single base pair is replaced by another
• Frame shift mutations
– One or more base pairs are inserted or deleted in
the DNA
– Results in a change in the reading of codons
 Chemical mutagens
• Example: Nitrous acid alters adenine such that it pairs
with cytosine instead of thymine
 Radiation
• Ionizing radiation e.g., Xrays and gamma rays
Causes the formation of ions that can react with
nucleotides (causing base changes) and the
deoxyribose-phosphate backbone (causes
chromosomes to break).

• UV radiation
Induces formation of
covalent bonds between
adjacent thymines to form
thymine dimers which can not be replicated
 Consequences of base substitutions
• Silent mutation: base change results in no
change of the amino acid sequence of the
translated protein
• Silent mutations have no effect on phenotype
• A result of the fact that multiple codons can
code for the same amino acid
– E.g., AGU and AGC both code for Serine
 Consequences of base substitutions
• Missense mutation: base change results in the
change of an amino acid in the translated protein
 Consequences of missense mutations
• The amino acid substitution induced by the
missense mutation may have no effect on the
function of the protein OR
• It may abolish the activity of the protein or
alter its function having an effect on
phenotype
Example: sickle cell disease in humans is due
to a missense mutation in the gene for globin.
As a result the shape of red blood cells is
altered affecting their movement through
capillaries.
 Consequences of base substitutions
• Nonsense mutation: base change generates a stop
codon in place of that coding for an amino acid
• Results in production of a truncated protein. Usually
results in a non-functional protein
 Consequences of frameshift mutations
• Frameshift mutation: addition or deletion of one or more
bases
• Results in misreading of the codons (changed reading
frame)
• Almost always results in long stretches of altered amino
acids and the production of inactive protein
 Nucleotide Excision Repair
• In nucleotide excision
repair, a nuclease cuts
out a segment of a
damaged strand.
– The gap is filled in by
DNA polymerase and
ligase.

Copyright © 2005 Pearson Education, Inc., publishing as Benjamin Cummings

 Xeroderma Pigmentosum
• Individuals with this
genetic disease have
defective repair
enzymes
– can’t remove thymine
dimers caused by UV
light
– very sensitive to sunlight
and often get skin
cancers
From: Concepts of Genetics, 6th edition,
Klug and Cummings, Prentice Hall

Note the moles on/near the boy’s nose

 Royal Hemophilia

• X linked
• Queen Victoria (Great Britain) was the
carrier
• Gene passed on to many other royal
families in Europe
– Russian, Prussian and Spanish affected
– But not the British royal family…
• Genetic warfare?
Hemophilia in the British Royal Family
 Translation: Making Proteins
These are the abbreviations for the amino acids A group of three
nucleotides in
messenger RNA
codes for a
certain amino
acid to be placed
in a protein.
Each group of
three
nucleotides is
called a CODON.

http://www2.hawaii.edu/~johnb/micro/micr230/micr230_lectures/lecture9_files/image001.gif
 DNA Repair
• Spontaneous DNA damage
• Pathways to remove DNA damage
• Damage detection
• The repair of Double-strand break
• DNA repair enzymes
Mutation Generation passed on to daughter
DNAs
Emergency DNA Repair for Double helix break