DNA Metabolism

Don M. Abrina, M.D.

 The DNA Structure
Watson & Crick DNA is a duplex of 2
strands of polydeoxyribonucleotide chain

Watson & Crick complementary base pairing

1. cytosine forms 3 H bonds with guanine 2.
thymine forms 2 H bonds with adenine

Complementary chains of DNA - opposite

polarity ( 53 direction & 35 direction)
 DNA carrier of genetic information

Genome encodes genetic blueprint for

building that organism

Replication entire genome copied

passed on to a new cell

Replication basis of heredity

 Prokaryotic Replication
DNA double helix when separated each of
the 2 strands serves as a template for
replication of a new complementary strand 2
daughter molecules ( 2 DNA strands with anti
parallel orientaion)

Two postulations of replication:

1. Conservative
2. Semi-conservative
 Conservative Replication of the 2 double
helices formed, one would be entirely of old
material and the other of entirely new material
Semi-conservative replication each strand
of the 2 double helices formed would have
one old and one new strand
Messelson & Stahl proved that the 2nd form
of replication is true growing E.coli in NH4Cl
with N progeny had DNA with N
 Basic Requirements for DNA Synthesis
1. Substrate deoxynucleoside triphosphates
dATP (adenosine triphosphate)
dGTP (guanosine triphosphate)
dCTP (cytosine triphospate)
dTTP (thymidine triphosphate)

2. Template DNA chain that provides precise

information for synthesis of complementary strand
 3. Primer initial portion of a linear
molecule unto which additional units are
added produce the final polymemric chain

Oligoribonucleotides formed with DNA as

the template serve as a primer for
addition of deoxyribonucleotides synthesis
of daughter DNA strands
 4. Enzymes DNA polymerase I, II, III
(extends the polydeoxyribonucleotide chain)

Primase catalyze formation of primer

During elongation the free 3-OH group of

the primer attacks the -phosphorus atom of
incoming deoxyribonucleotide triphosphate.
Central Dogma of Molecular Genetics
 Replication each strand of the parental DNA duplex
is copied by base pairing with complementary
nucleotides
Transcription information contained in DNA is copied
to form complementary sequence of ribonucleotides
Translation information transcribed from DNA into
mRNA ordered polymerization of amino acids protein
synthesis
Reverse transcription RNA can be transcribed into
DNA
e.g. retrovirus RNA
 Steps In DNA Synthesis
A. Separation of complementary strands of DNA

Ori C origin of replication; single unique nucleotide

sequence where DNA replication begins (prokaryotes)

Eukaryotes - Replication begins in multiple sites

Consensus sequence short sequence of AT pairs in

the origin of replication (same order of nucleotide
sequence at each site)
Replication begins at an origin and proceeds bidirectionally
in E. coli
 B. Formation of the Replication Fork:
As the 2 strands unwind and separate form a
Y called the replication fork.
Replication of double stranded DNA bidirectional
1. Proteins required for DNA separation:
a. DnaA protein 20-50 monomers; bind to
specific nucleotide sequences at Ori double
stranded DNA separates forms regions of
single-stranded DNA
 b. Single-stranded DNA-binding (SSB)
proteins helix stabilizing proteins
- bind cooperatively to single strands of
DNA stabilizing the single-stranded state
- protect the DNA from nucleases
- bind to single-stranded DNA preventing
reformation of the double helix
 c. DNA helicases (DnaB protein)
enzyme that bind to single-stranded DNA
near the replication fork moves into
double-stranded region forcing strands
apart unwinding the double helix
Replicating forks may move either bidirectionally or
unidirectionally
 Solving the Problem of Supercoils
Two problems encountered as 2 strands of
the double helix separate:
1. entire chromosome ahead of the
replicating fort rotates
2. accumulates supercoils (supertwists)

For every 10 deoxyribonucleotides added

during replication parental double-helix
makes one complete turn around its axis
 DNA Topoisomerases introduces swivel points
along the double helix avoid need for entire
strand to rotate

Two general classes of topoisomerases:

a. Type I DNA topoisomerase forms a nick
through which the complementary strand passes
- has nuclease and ligase activities
-relaxes negative supercoils in prokaryotes
-relaxes negative and positive supercoils in
eukaryotes
Topoisomerase I
 b. Type II DNA Topoisomerases- bind tightly to the
DNA double helix make transition breaks in
both strands causes a 2nd stretch of DNA
double helix to pass through the break reseals
the break relieves both positive and negative
supercoils

DNA Gyrase introduces negative supercoils into

DNA relieves positive supertwisting in
replication fork
Topoisomerase II function:
 DNA gyrase target of quinolones

Other antibiotics which target topoisomerases:

Nalidixic acid and norfloxacin used for UTI;
inhibits strand-cutting activity inhibit bacterial
DNA
Etoposide and teniposide inhibit human
topoisomerase II; used in treatment of neoplastic
diseases
 Direction of DNA Replication

DNA polymerases reads the parental nucleotide

sequence in the 3 5 direction; synthesizes
new DNA strands in the 5 3 direction
Two different mechanisms on each strand:
1. leading strand strand that is being copied in
the direction of the advancing replication fork
(continuous)
2. lagging strand strand that is being copied in
the direction away from the replication fork
(discontinuous)
 Okasaki fragments short DNA segments
found during DNA replication in the lagging
strand (3 5 direction)
- suggests that chain growth in this region occurs
discontinuously
- 100 to 1000 nucleotides
- later joined to become a single continuous strand
 RNA Primer

Primer short, double stranded region with free

OH group on the 3 end of the shorter strand
initiates synthesis of complementary strand of DNA
OH group first acceptor of a nucleotide by action
of DNA polymerase
Primase (DnaG protein) RNA polymerase
synthesizes short stretches of RNA complementary
and antiparallel to DNA template; uses 5
ribonucleoside triphosphates as building blocks
 Primosone protein complex + primase
Initiates Okazaki fragment formation in the lagging
strand in the 5 3 direction
Recognizes sequences of nucleotides to
synthesize RNA primer
Forms 2 priming sites on each of the separated
complementary strands in the Ori c.
 Chain Elongation

1. DNA Polymerase III catalyzes DNA chain

elongation by using 3-OH group of RNA primer as
acceptor of 1st deoxyribonucleotide
- building blocks are dATP, dTTP, dCTP, and dGTP

2. Inhibition of DNA synthesis by nucleoside

analogs:
- cytosine arabinoside
- adenine arabinoside
- zidovudine and acyclovir
 3. Proofreading of newly synthesized DNA
DNA polymerase III checks to make sure
that the added nucleotide is correctly
matched to its complementary base
- removes misplaced nucleotide and
replaces it withh correct nucleotide
- proofreading activity ( 3 5
exonuclease activity)
 Mutations:
1. missense mutation substitution of one
amino acid in the encoded protein with
another amino acid
2. nonsense mutation - formation of a stop
codon from a codon that corresponds to an
amino acid
3. sense mutation conversion of a stop
codon to one encoding an amino acid
 Excision of RNA primer & replacement with DNA

DNA polymerase I excises the RNA

primer and fills in the gap
Functions:
5 3 polymerase activity
3 5 exonuclease activity
5 3 exonuclease activity
Pol I can remove RNA primer and synthesize DNA to fill the
gap
 DNA Ligase

DNA Ligase joins the DNA chain

synthesized by DNA polymerase III and the
chain made by the DNA polymerase I after
removal of RNA primer
- seals the nick after DNA pol I fills in the
gap
- requires cleavage of ATP to AMP + Ppi
 Human DNA Replication

Similarities to prokaryotic DNA replication

5 classes of DNA polymerases:
DNA polymerase complex
Pol synthesizes RNA primer
Pol elongates leading strand
Pol elongates lagging strand
 Pol excises primers
Pol carries out replication within the
mitochondrion
 DNA Repair

Ultraviolet light yields thymine dimers

prevent DNA polymerase from replicating DNA
beyond the site of dimer formation
Recognition of dimer by UV-specific
endonuclease:
Recognizes the dimer cleaves damaged strand
Excision nuclease (DNA Polymerase I or pol )
recognizes incision made by endonuclease
Gap filled by DNA polymerase I
DNA ligase splice newly formed DNA
 Correction of Base Alterations

Glycosylases cleaves abnormal

bases( uracil occurring in DNA)
apyridmidinic or apurinic site(AP site)

AP endonucleases excision and gap

filling
 DNA damage may arise: (i) spontaneously, (ii)
environmental exposure to mutagens, or (iii)
cellular metabolism.
DNA damage may be classified as: (I) strand
breaks, (ii) base loss (AP site), (iii) base
damages, (iv) adducts, (v) cross-links, (vi)
sugar damages, (vii) DNA-protein cross links.
DNA damage, if not repaired, may affect
replication and transcription, leading to
mutation or cell death.