NUCLEIC ACIDS

Tagadiuc Olga,
PhD, associated professor
 Types (1)

DNA
Location – nucleus, mitochondria
Function – preservation and transmission of
genetic information between generations
Chemical composition-
1. Nitrogenous bases – A, G, C, T
2. Pentose – deoxyribose
3. Phosphate
 Types (2)

RNA
Location – nucleus, cytoplasm, ribosomes
Function – realization of genetic information
Chemical composition –
1. Nitrogenous bases – A, G, C, U
2. Penroza – riboza
3. Restul fosfat
Levels of structural organization
of DNA:

1. Primary – the nucleotide

sequence of the polynucleotide
strand
2. Secondary – double helix
3. Tertiary – DNA super helix
4. Quaternary - nucleosome
The primary structure of DNA and RNA
DNA STRUCTURE
DNA STRUCTURE

Zamudio N M et al. Reproduction 2008;136:131-146

(Source: http://www.nature.com/scitable/topicpage/dna-packaging-nucleosomes-and-chromatin-310) - See more at:
http://yonderbiology.blogspot.com/2013/05/fun-fact-unraveled-dna-from-cells-of.html#sthash.B1R8FbIg.dpuf
 RNA levels
of structural organization:

1. Primary – the nucleotide

sequence of the polynucleotide
strand
2. Secondary -
3. Tertiary - according to
4. Quaternary - the RNA type
 tRNA structure

1. Primary – the nucleotide

sequence of the polynucleotide
strand
(average length 95 nucleotides)
1. Secondary – has the shape of the
„clover leaf ”
2. Tertiary – has the shape of the
„letter L”
 Secondary
structure of
the tRNA
 Tertiary
structure of the
tRNA
rRNA structure
 Ribosome structure
Structura ribozomilor
Ribosome structure
 Genetic information flow

1. Replication or DNA biosynthesis

2. Transcription or RNA biosynthesis
3. Reverse transcription
4. RNA replication
5. Translation or protein biosynthesis
 DNA Replication
in Prokaryotic Cells
 REPLICATION
1. Location – cytoplasm
Prokaryotes do not have
nucleus and DNA is
present as a DNA-protein
complex called nucleoid.
2. In prokaryotes, DNA
replication is the first
step of cell division
3. Template - DNA
double helix
4. Semiconservative
 REPLICATION

3. Substrates:
• For DNA strands synthesis –
deoxyribonucleotide triphosphates (dATP,
dGTP, dCTP, dTTP)
• For primer synthesis – ribonucleotide
triphosphates (ATP, GTP, CTP, UTP)
• Are high-energy compounds – source of
energy for 3´,5 ´-phosphodiesther bond
synthesis
 REPLICATION
4. Enzyme complex :
 Helicase
 Primase or initiation DNA-dependent RNA-
polymerase
 DNA-polymerases I, II and III
 Ribonuclease H
 DNA-ligase
 Topoisomerases I and II
 Replication stages

1. Initiation
2. Elongation
3. Termination
Replication stages - Initiation

1. Prokaryotic DNA is organized into circular

chromosomes.
2. The prokaryotic DNA molecules contain a
single origin of replication (ORI) and a
single replicon.
3. The origin sites are generally longer than
eukaryotic origin sites (in E. Coli – 245 b.p.)
Replication stages - Initiation
4. ORI sequence has mostly A/T b. p. (which are
held together by fewer hydrogen bonds than
G/C base pairs), making the DNA strands easier
to separate.
 dnaA attaching site – 5’-TTATCCACA-3’
 dnaB attaching site – 5'-GATCTNTTNTTTT-3
Replication stages – Initiation
1. dnaA, dnaB and dnaC proteins
2. Helicase
3. Gyrase or topoisomerase
4. SSB proteins
5. Primase
6. RNA-polymerase
Replication stages – Initiation
1. Mediated by dnaA, dnaB and dnaC proteins
2. Dna A protein recognizes and binds up to four 9bp
repeats in ORI
3. Dna A protein subunits then successively melt three
tandemly repeated 13bp segments in the presence of
ATP (open complex).
4. The Dna A protein then guides a Dna B - Dna C complex
into the melted region to form a so called prepriming
complex. The Dna C is subsequently released. Dna B
further unwinds open complex.
 Replication stages –
Initiation
5. DNA gyrase, single stranded binding protein (SSB) and
Helicase bound to prepriming complex – the new
complex is called priming complex.
6. In the presence of gyrase and SSB, helicases further
unwinds the DNA in both directions so as to permit
entry of primase and RNA polymerase.
 RNA polymerase forms primer for leading strand
synthesis
 primase in the form of primosome synthesis
primer for lagging strand synthesis.
7. DNA polymerase III binds to the above complex and
forms replisome.
Replication
stages –
initiation –
replication
fork
formation
Replication
stages –
initiation –
replication
fork
formation
Replication stages – ELONGATION
Elongation - synthesis of leading strand
 The DNA daughter strand that is synthesized
continuously on 5'-->3' template is called leading
strand.
 DNA pol-III synthesizes DNA by adding 5'-P of
deoxynucleotide to 3'-OH group of the already
presenting fragment. Thus chain grows in 5'-->3'
direction.
 The reaction catalyzed by DNA pol-III is very fast -
9000 nucleotides per minute at 37°C.
 The RNA primer that was initially added by RNA
polymerase is degraded by RNase.
Replication stages – ELONGATION
 Elongation - Synthesis of lagging strand

1. The daughter DNA strand which is synthesized in

discontinuous is called lagging strand.
2. It occurs in the following steps:
 Synthesis of Okazaki fragment: To the RNA
primer synthesized by primosome, 1000-2000
nucleotides are added by DNA pol-III to synthesis
Okazaki fragments.
 Excision of RNA primer: When the Okazaki
fragment synthesis was completed up to RNA
primer, then RNA primer was removed by DNA pol-I
using its 5'-->3' exonuclease activity.
 Elongation - Synthesis of lagging strand

 Filling the gap (Nick translation): The gap

created by the removal of primer, is filled up by
DNA pol-I using the 3'-OH of nearby Okazaki
fragment for its polymerizing activity.

 Joining of Okazaki fragment (Nick sealing):

The nick between the fragments are sealed by
DNA ligase which catalyze the formation of
phosphodiester bond between a 3'-OH at the end
of one fragment and a 5' - phosphate at the other
end of another fragment. The enzyme requires
NADH for this reaction.
 Replication stages –
ELONGATION

http://themedicalbiochemistrypage.org/images/replicationforkdetail.jpg
 Replication stages –
TERMINATION

1. Termination occurs when the two replicating

forks meet each other on the opposite side of
circular DNA.
2. Termination sites like Ter A, B, C, D, E and F are
found in DNA.
 Ter A terminates the counter-clockwise
moving fork
 Ter C terminates the clockwise moving
forks.
 The other sites are backup sites.
 Replication stages –
TERMINATION

 ter - the termination sites of the prokaryote replication

 Replication stages –
TERMINATION

 Termination at these sites are possible

because, at these sites TUS protein
(Termination utilizing substance) will
bound to Dna B protein and inhibits its
helicase activity.
 Dna B protein is released and termination
results.
 Replication stages –
TERMINATION
TUS-protein blocks replication by binding to the ter site
 Replication stages –
TERMINATION
 After the complete synthesis, two duplex
DNA are found to be catenated (knotted).
 This catenation removed by the action of
topoisomerase.
 Finally, from single parental duplex DNA, two
progeny duplex DNA synthesized.
REGULATION OF PROKARYOTIC
REPLICATION:

 Especially initiation of replication is

regulated.
 Dna A protein regulates the initiation of
replication.
 DNA replication is regulated also by the level
of OriC methylation.
Prokaryotic vs eukaryotic dna
replication
 Telomerase
Function
 In Linear eukaryotic chromosome, once the
first primer on each strand is remove, then
it appears that there is no way to fill in the
gaps, since DNA cannot be extended in the
3′–>5′ direction and there is no 3′ end
upstream available as there would be in a
circle DNA.
 If this were actually the situation, the DNA
strand would get shorter every time they
replicated and genes would be lost forever.
 Telomerase
Function
Elizabeth Blackburn and
her colleagues have
provided the answer to fill
up the gaps with the help The Nobel Prize in Physiology
of enzyme telomerase. So, or Medicine 2009
that the genes at the ends, was awarded jointly to
are conserved. Elizabeth H. Blackburn,
Carol W. Greider and Jack W.
Szostak "for the discovery of
https://www.nobelprize.org/nobel_priz how chromosomes are
es/medicine/laureates/2009/advanced protected by telomeres and
-medicineprize2009.pdf the enzyme telomerase".
 Telomerase Function
1. Telomerase is a ribonucleoprotein (RNP) i.e. it has
RNA with repetitive sequence.
2. Repetitive sequence varies depending upon the
species.
 For example Tetrahymena thermophilia RNA
has AACCCC sequence
 Oxytrica has AAAACCCC.
 In human it has AAUCCC repeats.
3. Telomerase otherwise known as modified
Reverse Transcriptase. This enzyme was also
known as telomere terminal transferase.
 Telomerase Function
1. The 3′-end of the lagging strand template has base pairs with a
unique region of the telomerase associated RNA.
2. Hybridization facilitated by the match between the sequence at
the 3′-end of telomere and the sequence at the 3′-end of the
RNA.
3. The telomerase catalytic site then adds deoxy ribonucleotides
using RNA molecule as a template, this reverse transcription
proceeds to position 35 of the RNA template.
4. Telomerase then translocates to the new 3′-end by pairing with
RNA template and it continues reverse transcription.
5. When the G-rich strand sufficiently long, Primase can make an
RNA primer, complementary to the 3′-end of the telomere’s G-
rich strand.
6. DNA polymerase uses the newly made primer to prime
synthesis of DNA to fill in the remaining gap on the progeny
DNA. The primer is removed and the nick between fragments
sealed by DNA ligase.
 DNA repair
Mechanisms used by cells to correct replication
errors and fix DNA damage, includ:
 Proofreading, which corrects errors during
DNA replication
 Mismatch repair, which fixes mispaired
bases right after DNA replication
 DNA damage repair pathways, which detect
and correct damage throughout the cell cycle
 Proofreading
 Most DNA
polymerases can “check
their work” with each
base that they add.
 This process is
called proofreading.
 If the polymerase
detects that a wrong
(incorrectly paired)
nucleotide has been
added, it will remove
and replace the
nucleotide before
continuing with DNA
synthesi
 Mismatch repair
 happens right after new DNA has been made,
 removes and replace mis-paired bases (ones that were not fixed
during proofreading).
 can also detect and correct small insertions and deletions that
happen when the polymerases "slips," losing its footing on the
template
 Mechanism:
1. a protein complex (group of proteins) recognizes and binds to
the mispaired base.
2. a second complex cuts the DNA near the mismatch, and more
enzymes chop out the incorrect nucleotide and a surrounding
patch of DNA.
3. a DNA polymerase then replaces the missing section with
correct nucleotides,
4. a DNA ligase seals the gap
Mismatch repair
 DNA damage repair mechanisms
Repair processes that help fix damaged DNA include:
1. Direct reversal: Some DNA-damaging chemical reactions
can be directly "undone" by enzymes in the cell.
2. Excision repair: Damage to one or a few bases of DNA is
often fixed by removal (excision) and replacement of the
damaged region.
 In base excision repair, just the damaged base is
removed.
 In nucleotide excision repair, as in the mismatch
repair we saw above, a patch of nucleotides is removed.
3. Double-stranded break repair: Two major pathways, non-
homologous end joining and homologous recombination, are
used to repair double-stranded breaks in DNA (that is, when
an entire chromosome splits into two pieces).
DNA damage repair mechanisms
Repair processes that help fix damaged DNA include:

Direct reversal
 DNA damage repair mechanisms
 Base excision repair is
used to detect and
remove certain types of
damaged bases.
 For example, a chemical
reaction called
deamination can convert
a cytosine base into
uracil, a base typically
found only in RNA.
 During DNA replication,
uracil will pair with
adenine rather than
cytosine, so an
uncorrected cytosine-to-
uracil change can lead to
a mutation^55start
superscript, 5, end
superscript.
 DNA damage repair mechanisms
 Nucleotide excision
repair is used to
remove and replace
damaged bases that
distort the DNA double
helix.
 The most common type
of damage is a thymine
dimer, that consists of
two thymine bases that
react with each other
and become chemically
linked
 Thymine
dimer
formation
 Xeroderma pigmentosum
 Xeroderma pigmentosum (XP) is an inherited condition
characterized by an extreme sensitivity to ultraviolet (UV) rays from
sunlight.
 This condition mostly affects the eyes and areas of skin exposed to
the sun. Symptoms typically develop by the time a child is 2 years old.
 Xeroderma pigmentosum is caused by mutations in genes that are
involved in repairing damaged DNA. Inherited mutations in at least
nine genes have been identified.
 The condition is inherited in an autosomal recessive manner.
 People with XP need total protection from sunlight. This includes
protective clothing, sunscreen, and dark sunglasses when out in the
sun.
 To prevent skin cancer, medications like retinoid creams may be
prescribed. Skin cancers that do develop should be treated using
standard practices.
Xeroderma pigmentosum