1.

3 Iniciación de la replicación
y control de la iniciación
DNA REPLICATION
 DNA Replication
• New DNA strands are synthesized by using the existing (parent)
strands as templates in the formation of new, daughter strands that
are complementary to the parent strands.
• Could proceed by either a conservative or a semiconservative
mechanism:
– If a conservative mechanism were used, the two daughter
strands would form a new double-stranded DNA molecule and
the parent duplex would remain intact.
– If a semiconservative mechanism were used, the parent strands
would be permanently separated and each would form a duplex
molecule with the newly synthesized daughter strand base-
paired to it.
– Definitive evidence that duplex DNA is replicated by a
semiconservative mechanism came from a now classic
experiment conducted by M. Meselson and W. F. Stahl.
 DNA is anti-parallel
• Two strands run
parallel to each other
but with opposite
alignments
(directions)
• McGraw-Hill DNA
• Why is being anti-
parallel an advantage
to the DNA
molecule?
 DNA REPLICATION
• Semi-conservative =
each one of the
parent DNA strands
is passed to the
daugher DNA + one
new strand for each
• Semi-conservative
DNA (30 secs)
SNEAK PREVIEW:
DNA REPLICATION
PLAYERS (enzyme
review)
 Question:
When and where does DNA Replication take
place?

Synthesis Phase (S phase)

• S phase in interphase of the cell cycle.
• Nucleus of eukaryotes
S
DNA replication takes phase
place in the S phase.
G1 interphase G2

Mitosis
-prophase
-metaphase
-anaphase
-telophase
 DNA copying

• Each cell division cell must copy its entire DNA

• So each daughter cell gets a complete copy
• Rate of synthesis
– Bacteria = 1000 bases per second
– Mammals = 100 bases per second
• Problem - with a single replication origin in DNA
– Bacteria genome is 4 x 10E6. Takes 20 minutes to copy.
– Human is 3.2 x 10E9. Would take 10,000 times longer.
 Initiation
• This process is initiated at particular points in the
DNA, known as "origins", which are targeted
by initiator proteins.
• In E. coli this protein is DnaA.
• In yeast, this is the origin recognition complex.
• Sequences used by initiator proteins tend to be "AT-
rich" (rich in adenine and thymine bases), because A-
T base pairs have two hydrogen bonds (rather than
the three formed in a C-G pair) and thus are easier to
strand-separate.
• Once the origin has been located, these initiators
recruit other proteins and form the pre-replication
complex, which unwinds the double-stranded DNA.
 Fill out DNA Replication
Enzymes CHART
• Lots of enzymes are
needed to start each step
 Enzyme Topoisomerase
also called DNA gyrase
• Unwinds double helix
Enzyme Enzyme

DNA
 Topoisomerase
• Topoisomerase Youtube I and II (1:45)
• Topoisomerase Animation (2:16)
Enzyme Helicase:
separates (breaking H-
bonds) double helix at the
replication fork

YOU TUBE DNA replication (1:05)

Helicase
 DNA Helicase
• The enzyme is unwinding the chain and
breaking the H-bonds between the
complementary base pairs (A-T, G-C).
 Enzyme:
Primase
Helicase Primase
Helicase
= the enzyme
that makes RNA
nucleotides
into a primer
 RNA Primer
• Nucleotides for the starting point
for DNA replication
• Short strands of RNA
 DNA replication is initiated at many
points in eukaryotic chromosomes.
• Called Replication Bubbles
• They will eventually all meet to form whole
replicated strand
DNA Replication Bubble: DNA
duplicates in both directions
EM of DNA replication
 Origins of Replication
• sites along the DNA molecule where
enzymes start the DNA replication - then
proceeds in both directions to form
“bubbles”
 Replication Forks
Y-shaped regions of replicating DNA
molecules where new strands are
growing.
 SSB’s
single strand binding proteins
• Stabilize the DNA strands as they
are being replicated
• Prevents rejoining of DNA strands
 DNA Polymerases Require a
Primer to Initiate Replication

• DNA is synthesized from deoxyribonucleoside 5′-

triphosphate precursors (dNTPs).
• DNA synthesis always proceeds in the 5′→3′ direction
because chain growth results from formation of a
phosphoester bond between the 3′ oxygen of a growing
strand and the α phosphate of a dNTP.
• DNA polymerases cannot initiate chain synthesis de
novo; they require a short, preexisting RNA or DNA
strand, called a primer, to begin chain growth.
 DNA Polymerases
• DNA Polymerase I
• Cuts off RNA primers and fills in with
DNA (between Okazaki fragments) –
lagging strand
• Can proofread
• DNA Polymerase III
• Elongates the strand by adding DNA
nucleotides on leading strand
• Also proofreads and corrects the
DNA strand
Anti-parallel strand builds in
the opposite direction (but
always in 5’ to 3’ direction)
Leading Strand Lagging Strand
• Template strand of
DNA
• Continuous addition
of nitrogenous bases
• in 5’ to 3’ direction
• McGraw-Hill Replication Fork
• Other DNA strand
• Forms short strands
of Okazaki fragments
(that will be joined
later)
• in the 5’ to 3’ direction
• DNA Replication You Tube (1:35)
• OKAZAKI FRAGMENTS
• The short strands of newly made DNA
fragments on the lagging strand are called
Okazaki fragments after the Japanese
Biochemist Reiji Okazaki.
•
 Enzyme: DNA Ligase
a linking enzyme joins the strands

Example: joining two Okazaki

fragments together.

DNA ligase
Okazaki Fragment 1 Okazaki Fragment 2
5’ 3’

3’ Lagging Strand
5’
 DNA LIGASE
is the enzyme that
joins the Okazaki
fragments (sugar -
phosphate backbone)
with covalent bonds

DNA REPLICATION
(look for ligase) 2:00
 SUMMARY
DNA Replication (5:45) –
shows all the enzymes
• Summary Youtube of DNA replication (4:11)
• Good explanation of the 5’ to 3’ strands
and leading and lagging strands
 Includes all your friendly
enzymes
• DNA Replication (3:56) Great animation
 How Fast?

• Prokaryotic DNA polymerase can work

at about 1000 bases per second.

• Eukaryotic DNA polymerase can work at

about 50 bases per second.
 Animation: DNA Replication
• DNA makes DNA
• *DNA with enzymes cartoons
All Together Now
• McGraw Hill Replication Fork animation
 DNA Replication Easy Version (3:11)

• DNA Replication Youtube (7:48)

• Good CLICK and REVIEW

 What if there is a mistake?

Typically
about one in
a billion
nucleotides
is incorrectly
paired
 Proofreading

Initial base-pairing errors

are usually corrected by
DNA polymerase I.
 Telomeres
• At the ends of each
chromosome is a
protective cap
called a telomere.
• Each time a cell
divides, the
telomeres are
snipped shorter,
 Telomerase
• enzyme which adds DNA
sequence repeats
("TTAGGG" in all
vertebrates) to the 3' end of
DNA strands (an overhang)

• McGraw Hill Telomeres

Animation
Telomeres Added to ends of
chromosomes
Rutgers Telomere Animation
 QUIZ
• Activity (choose your enzymes and
proteins)
 MOVIE
• Media Showcase (cool animation)

• Replication Overview Movie