• DNA Replication,

Repair, and
Recombination

Indwiani Astuti
Dept of Pharmacology & Therapy
Fac Of Medicine
Universitas Gadjah Mada
[email protected]
 DNA Maintenance

• Mutation rate are

extremely low
• 1 mutation out of 109
nucleotides per
generation
DNA replication
Separation, Base pair
The Chemistry of DNA
replication
DNA Synthesis by DNA polymerase
DNA Polymerase
Nucleotide polymerizing enzyme, first discovered in 1957
DNA replication with two forks
This Doesn’t Work!
DNA replication Fork
DNA Proofreading
Structures of DNA polymerase during polymerizing and editing
E: exonucleolytic; P: polymerization
Why 5’->3’?
The need for accuracy
Site-directed mismatch repair in eucaryotes
In procaryotes, old DNAs are usually methylated on A while newly synthesized ones are not. So
Cells can distinguish old and newly synthesized DNAs and mutate mismatches on new ones.
 DNA Proofreading
RNA usually doesn’t have this. Why?
• Pairing, correct nucleotide has higher affinity
binding to the moving polymerase
• Un-Paired nucleotide is easier to be off before
covalent ligation, even after binding.
• Exonucleotic proofreading
• Strand directed mismatch repair
DNA Primer synthesis
On Lagging strand
DNA Replication at the Lagging strand
DNA Ligase
DNA Helicase
DNA double helix are tightly coupled. High
temperature is needed to break them (95oC)
DNA Binding Protein
SSB: Single Strand DNA-binding Proteins, also called helix destabilizing proteins
SSB Proteins

DNA
DNA Clamping Protein
Cycle of DNA Polymerase/Clamping Protein loading and unloading
At the lagging strand (how about leading strand?)
Protein machinery for DNA replication
A Moving Replication
Structure of the Moving Complex
DNA winding
DNA topoisomerase I
DNA topoisomerase II
DNA topoisomerase II
Mammalian replication Fork
(eucaryote, DNA polymerase (primase) a synthesize RNA/DNA, DNA polymerase delta is
the real polymerase)
 Summary
• DNA replication 5’->3’
• DNA proof reading
• Lagging strand, back-stitching, Okazaki fragment
• Proteins involved:
1. DNA polymerase, primase
2. DNA helicase and single-strand DNA-binding
protein (SSB)
3. DNA ligase, and enzyme to degrade RNA
4. DNA topoisomerases
DNA Replication in Chromosome
DNA replication in Bacterial Genome
Initiating Proteins for DNA replication

1.Initiator protein,
2.helicase binding to initiator protein,
3.helicase loading on DNA,
4.helicase opens the DNA and binds to
primase,
5.RNA primer synthesis,
6.DNA polymerase binding and DNA
synthesis
Regulation for DNA replication
In Bacteria, hemimethylated origins are resistant to initiation, delayed methylation leads
to delayed initiation at the second phase
DNA replication in eucaryotes
Multiple replication origin
50 nucleotides/second, autoradiography
The four standard phases of a eucaryotic cell
DNA replication occurring at S Phase (DNA synthesis phase)
G1 and G2, gap between S and M
Different regions of a chromosome are replicated at different times
Arrows point to the replicating regions at different times
 Some facts about Replication in
eucaryotes
• Multiple replication origins occurring inclusters (20-80)
(replication units)
• Replication units activated at different times
• Within replication units, replication origins are
separated 30,000-300,000 pairs apart.
• Replication forks form in pairs and create a replication
bubbles moving in opposite directions
• Different regions on the same chromosome are
replicated at distinct times in S phase
• Condensed Chromatin replicates late, while less
condensed regions replicate earlier
A close look at an origin of replication in yeast
ORC: origin recognition complex
B1, B2, B3: other regions binding to required proteins
The replication origins of human genes are more complex
Even far distant DNA sequences could be important
Histone remains associated with DNA
In vitro experiments
DNAs with different sizes are replicated. Only the daughter DNAs replicated from
parental DNA with histones showed histone binding
Addition of new histones
Chromatin assembly factors (CAFs) help to add
and assemble new nucleosomes
Bacteria DNAs are circular, not a problem
There is a problem for eucaryote DNAs: ???

Hint: Telomere
Telomerase Structure

Reverse transcriptase with RNA

template to bind to DNA strands
Telomerase and its function
T-loops at the end of
mammalian chromosomes
 Summary
• Specific DNA sequence determine replication
origin, recruiting proteins to form replication
machinery. relatively complex in eucaryotes
• Bacteria has single replication origin. Eucaryotes
have multiple origins and less defined.
• Replication forks are activated at different times
in eucaryotes
• Telomere and telomerase
MECHANISMS OF DNA REPAIR
 DNA Repair
• Spontaneous DNA damage
• Pathways to remove DNA damage
• Damage detection
• The repair of Double-strand break
• DNA repair enzymes
 DNA DAMAGE

1- Tautomerisation: spontaneous

Guanine: Keto form: pairs with Cytosine

Enol form: mispairs to Thymine

Thymine: Keto and Enol forms

Adenine and Cytosine: Amino and Imino forms

1- Tautomerisation:

2- Deamination:

spontaneous base degradation

Base Pairing Deamination Mispairing

C G U A
A T Hypoxanthine C
G C Xanthine C
• 3- Oxidation:damaging bases by free radicals of
oxygen
• 4- Alkylation: addition of alkyl groups to bases or
backbone DNA
• 5-Depurination: loss of Adenine or Guanine
bases: 10 000 bases per day per cell
• Other DNA hydrolysis reactions
• 6-Pyrimidine dimers: by sunlight
• ( UV ).
• 7-Single or double strand break.
 DNA REPAIR MECHANISMS

Three kinds:

A-Damage reversal

B-Damage removal

C-Damage tolerance
A-Damage reversal

may be divided in three different kinds:

1- Photo reactivation: most simple way for

DNA repair : a single step reaction.
Photolyase enzyme can split pyrimidine dimers:
breaks the covalent bond
Existence in mammalian not yet proved.
A-Damage reversal
1- Photo reactivation:

2-Most of damaged bases are repaired by one

enzyme, removes mismatched base restoring the correct
one. Without the need to break the DNA backbone:
- Uracil, product of cytosine deamination, is detected,
removed by uracil N-glycosylase, and replaiced by
cytosine.
-Hypoxanthine, product of adenine deamination, is
recognized, removed by hypoxanthine N-glycosylase
then replaced by adenine

-Alkylation is repaired by enzymatic transfer of methyl

group by Methyl Guanine-DNA MethylTransferase ( M G
M T ), and we will have Guanine

-Demethylation is used to repair 1-methyl adenine and

3-methyl cytosine.
A-Damage reversal

1- Photo reactivation:

2-Most of damaged bases

3-Ligation of simple strand breaks:

Simple breaks in one strand are repaired by DNA ligase.
A-Damage reversal

B-Damage removal:

Is the most common repair mechanism. It includes: base

excision repair, nucleotide excision repair, and mismatch
repair.
1-Base excision repair ( B E R ):

It repairs small, non bulky DNA lesions: methylated,

oxidized, reduced bases. It is estimated to occur 20 000
times a day in each cell in our body:damaged or
inappropriate base is removed from its sugar linkage and
replaced.
Different steps of BER:

a- removal of the damaged base by a DNA

glycosylase. Eight enzymes,each one responsible for
identifying and removing a specific kind of base
damage.

b- removal of its deoxyribose phosphate in the

backbone, producing a gap: an AP site.Two genes
encoding enzymes with this function.
Different steps of BER:

c- replacement with the correct nucleotide. Done

by DNA polymerase beta ( one of at least 11 DNA
polymerases encoded by our genes ), using the
other strand as a template.

d- ligation of the break in the strand. Two

enzymes are known that can do this.
1-Base excision repair ( B E R ):

2-Nucleotide excision repair ( N E R ):

The process of NER is biochemically complicated, 30 distinct
proteins that function as a large complex called the nucleotide
excision repairosome.
- The most important DNA repair pathway,
- The sole repair system for bulky DNA lesions, which creates a
block to DNA replication and transcription.
- Can also repair many of the same defects that are corrected by
direct repair, base excision and mismatch repair
( N E R ):
It probably recognizes lesions that distort the DNA

Steps in NER are:

a- Recognition of damage by one or more protein
factors.
b- Assembly of repair complex: nucleotide excision
repairosome.
c- Double incision of the damaged strand several
nucleotides away from the damaged site, on both sides,
by an endonuclease.
( N E R ):

d- Removal of the short segment ( about 24 to 32

nucleotides ) containing the damaged region, by an
exonuclease.
e- Filling in of the resulting gap by a DNA polymerase:
synthesizes DNA using the opposite strand as a template.
f- Ligation: a DNA ligase binds the synthesized piece into
the backbone.
1-Base excision repair ( B E R ):

2-Nucleotide excision repair ( N E R ):

3- Mismatch repair ( M M R ):

This process occurs after DNA replication as a last

"spellcheck" on its accuracy.
( M M R ):

Incorrect bases incorporated as a result of mistakes

during DNA replication ( base mispairs, short insertions
and deletions ) are excised as single nucleotides by a
group of repair proteins which can scan DNA and look
for incorrectly paired bases (or unpaired bases) which
will have aberrant dimensions in the double helix.
Synthesis of the repair patch is done by a DNA
polymerase .
A-Damage reversal

B-Damage removal

C-Damage tolerance

It is not truly repair but a way of coping with damage:

C-Damage tolerance:

1- Double strand break ( D S B ):

Naturally occurring reactive oxygen molecules and

ionizing radiation are prevalent sources of such damage.
DSB’s are a major cytotoxic lesion : even a single
unrepaired DSB can be a lethal event.
There are two different mechanisms of repair:
1- Double strand break:

a- End joining repair of DSB’s:

Joins broken chromosome ends in a manner that does
not depend on sequence homology and may not be error
free. incorrect ends may be joined, and repair mechanism
causes sequence errors.
a- End joining repair of DSB’s:
Three steps in end joining repair of DSB's:
1- Recognition of broken ends.
2- Unwinding of short stretch of DNA to uncover short
regions of homology "microhomologies"
3- Removal of unpaired regions and ligation of products.

This mechanism: called non homologous end-joining.

a- End joining repair of DSB’s:

b- Recombination repair: homologous recombination:

It is an important and preferred mechanism of repair
since it is least likely to result in mutations:
broken ends are repaired using the information on the
intact homologous chromosome.

Requires an extensive region of sequence homology

between the damaged and template strands.
C-Damage tolerance:

1- Double strand break ( D S B ):

a- End joining repair of DSB’s:

b- Recombination repair:

2- Error prone repair:

It is used when all else fails + + +
2- Error prone repair:

Involves the replication machinery bypassing sites of

base damage, allowing normal DNA replication and gene
expression to proceed downstream of the (unrepaired)
damage.

Involves low-fidelity DNA polymerases that are able to

bypass DNA lesions that stall the high-fidelity
polymerases required for DNA replication.
2- Error prone repair:

To overcome the block, these 'sloppy copiers' add

nucleotides to the replicating strand opposing the
DNA lesion,
-allowing replication to continue,
-and introducing mutations into the newly
synthesized sequence.
SEQUENTIAL EXPRESSION OF REPAIR SYSTEMS:

- Damage reversal

- Excision-resynthesis ( E B R, then N E R, then

MMR)

- Recombination

- And finally error prone repair.

DNA DAMAGE, DNA REPAIR,
AND AGING:

DNA damage, particularly oxidative lesions, is thought to

contribute to aging. In fact, some scientists believe :
accumulation of uncorrected DNA damage over years is a
major cause of aging.
following observations:
* Animals with fastest rates of DNA repair have longest life
spans.
* Animals with highest rates of oxidative damage by free
radicals generally have shortest life spans.
* In lower life forms, anti-oxidant supplements, which can
correct and prevent DNA damage, increase life span.
* Exposure to external causes of DNA damage decreases
life span.
* Humans who have genetic diseases resulting in greater
spontaneous DNA damage or inefficient DNA repair often
show signs of premature aging.
PREVENTING DNA DAMAGE AND
IMPROVING THE CAPACITY OF DNA REPAIR:
These two goals seem possible by:
1- insuring enough intake of antioxidants as vitamins C
and E and beta-carotene.
2- caloric restriction ( reduction of total daily calorie
intake by about 35% for animals ).
3- avoiding exposure to UV and ionizing radiations,
and toxic chemicals.
4- fighting infections
Emergency DNA Repair for Double helix break
 DNA Repair Summary
• Spontaneous DNA damage: spontaneous
alteration of bases, depurination and
deamination, thymine dimer
• Pathways to remove DNA damage: base
excision repair, nucleotide excision repair
• Damage detection: base flipping
• The repair of Double-strand break:
nonhomolous end joining, homologous end
joining
• DNA repair enzymes: heat shock proteins
CONCLUSION
DNA:many kinds of damage: base damage, mispairing,
single or double strand break
Many repair mechanisms:
- Damage reversal (Photoreactivation, ligation…)
- Damage removal: BER, NER, and MMR
- Damage tolerance: end joining repair, recombination
repair, and error prone repair.
These mechanisms are not always error free
DNA damage contribute to aging
Preventing DNA damage and improving capacity of
DNA repair may delay aging.
 DNA Recombination

• General recombination
• Site specific recombination
General DNA Recombination
Heteroduplex joint
 General Recombination
• Two homologous DNA molecules cross over
• The site of exchange can occur anywhere
• A strand of one DNA molecule has become
base-paired to a strand of the second DNA
to create heteroduplex joint
• No nucleotide sequences are altered
The procedure of general recombination

DNA synapsis: base pairs form between

complementary strands from the two DNA molecules
DNA Hybridization
The initial step for DNA
recombination
RecA protein-mediated DNA synapsis
Rec A has multiple DNA binding sites, hence can hold a single strand
and a double helix together
Rec A is also a DNA-dependent ATPase
DNA Branch Migration
Holiday Junction for DNA recombination
Exchange of the first single strand between two different DNA double helices is slow and
difficult, then intermediate state Holiday Junction, then complete exchange
Holiday Junction for DNA
recombination
and its resolution
 Summary for General
Recombination
• General recombination allows large fraction of
genetic information to move from one
chromosome to another.
• General recombination requires the breakage of
double helices, beginning with a single strand
breakage.
• General recombination is facilitated by Rec A in
bacteria and its homologs in eucaryotes.
• Holiday junction is the intermediate state of
general recombination
 Site-specific recombination
• Moves specialized nucleotide sequence (mobile
genetic elements) between non-homologous sites
within a genome.
• Transpositional site-specific recombination
• Conservative site-specific recombinatinon
 Transpositional site-specific
recombination
• Modest target site selectivity and insert mobile
genetic elements into many sites
• Transposase enzyme cuts out mobile genetic
elements and insert them into specific sites.
Three of the many types of mobile genetic elements found in bacteria
Transposase gene: encoding enzymes for DNA breakage and joining
Red segments: DNA sequences as recognition sites for enzymes
Yellow segments: antibiotic genes
Cut and Paste Transposition
DNA-only
The structure of the central intermediate formed by transposase (integrase)
Replicative Transposition
Retrovirus-based Transposition
Retroviral-like retrotransposition
Reverse Transcriptase

RNA
Non-retroviral retrotransposition
Conservative Site Specific Recombination
Integration vs. inversion
Notice the arrows of directions
Bacteriophase Lambda
Genetic Engineering to control Gene expression
Summary
• DNA site-specific recombination
• transpositional; conservative
• Transposons: mobile genetic elements
• Transpositional: DNA only transposons,
retroviral-like retrotransposons,
nonretroviral retrotransposons
THANK YOU

[email protected]
IA