Lecture 15- DNA Replication

Question 1

Review DNA Structure
(structure relates to function)
Nucleotide Structure

Answer 1

Phosphate group + pentose sugar + nitrogenous base (A,T,C,G)

Phosphodiester bond formed between 5’ phosphate group of one nucleotide and 3’-OH group of another

A and T are held together by two hydrogen bonds

G and C are held together by three hydrogen bonds

Question 2

DNA Replication
Duplication of DNA molecule=?
in which phase

Answer 2

Duplication of DNA molecule = DNA replication
– S phase of cell cycle

Question 3

• Virtually the same process in all organisms:

Answer 3

– In all dividing cells, a double-stranded DNA molecule
–> two double-stranded daughter DNA molecules,
identical to each other except for rare mutations

Question 4

• Understanding how DNA is replicated is important because?

Answer 4

How cells and organisms produce offspring
Key experimental methods in biology

Question 5

DNA: Semiconservative Replication
what happens during replication?
each parental strand serves as a template for the synthesis____
each parental strand serves as a template for the synthesis of a new ____ strand
the cell will replicate both?

Answer 5

During replication, the two strands of the parental molecule separate

Each parental strand serves as a template for the synthesis of a new daughter strand

-Each antiparallel strand is complementary to the other
-The cell will replicate both complementary strands of
DNA, each forming a template for replication

Question 6

DNA: Semiconservative Replication
The order of bases on one strand is used to add complementary
bases to make a new strand
what is the result?

Answer 6

Result - the two strands will be duplicated exactly

Each daughter DNA molecule each made up of one parental
strand and one new strand = semiconservative

Question 7

Semiconservative

Answer 7

Each daughter DNA molecule is made up of one parental
strand and one new strand = semiconservative

Question 8

DNA Replication Steps

*Steps in DNA replication are universal

Answer 8

Same steps occur regardless of whether DNA is
replicated in a cell or test tube or whether DNA is
just a segment or entire chromosome

Question 9

Speed of DNA replication

Answer 9

DNA replication is relatively slow
– Eukaryotes – 50 nucleotide/sec
* In largest human chromosome replication would take 2
months!
* In reality only takes a few hour

Question 10

Where does Replication begin?

What happens at the origin sites? (The DNA strands ____ forming a _____ bubble -replication forks at each end

What direction does replication proceed?

Answer 10

Replication begins simultaneously at many origins of
replication = specific sequence of nucleotides

At the origin sites, the DNA strands separate, forming a
replication bubble
-Replication forks at each end

Replication proceeds in both directions until the entire
molecule is copied (bidirectional replication)

Question 11

DNA Replication
Eukaryotic Chromosome

Answer 11

At each replication fork, the new
strand with free 3’ end = leading
strand; new strand with 5’ end is
lagging strand

Replication bubbles
grow as replication
continues

When two
replication bubbles
meet, they fuse to
make larger bubble

Question 12

DNA Replication: Prokaryotes
In Prokaryotice chromosomes(including mitochondria and chloroplast) the replication starts at the origin and moves around ______ in both directions

Answer 12

Prokaryotic chromosome (as well as mitochondria and
chloroplast)
circular chromosome
origin of replication–> replication starts at the origin and moves around the circular chromosome in both directions

Question 13

DNA Replication – The Proteins
Several proteins involved in the replication process:

Helicases

Answer 13

untwist and separate parental strands at
replication fork

Question 14

DNA Replication – The Proteins
Several proteins involved in the replication process:

Single-stranded binding proteins (SSBs)

Answer 14

stabilize separated strands

Question 15

DNA Replication – The Proteins
Several proteins involved in the replication process:

Topoisomerase II

Answer 15

relieves strain ahead of replication fork due to untwisting

Question 16

DNA Replication – The Proteins
Several proteins involved in the replication process:

RNA Primase

Answer 16

synthesizes RNA primers to initiate new DNA strand

Question 17

DNA Replication – The Proteins
Several proteins involved in the replication process:

DNA Polymerases

Answer 17

– extend the RNA primer (5-10 nucleotides
with free 3’ OH); removes primer and replaces it with DNA
*In E. coli – DNA polymerases III and I respectively

Question 18

DNA Replication – The Proteins
Several proteins involved in the replication process:

DNA Ligase
Ligase(glue)

Answer 18

joins the DNA fragments fragments(Okazaki fragments)

Question 19

DNA Replication-Unwinding DNA

Answer 19

-DNA polymerase extends an RNA primer (red)
-Helicase unwinds the parental DNA strand
-Topoisomerase II relives the stress of unwinding
-Single-strand binding protein stabilizes single strands of DNA
-The DNA polymerase complex acts at the site of the growing chain to increase the chain length one DNA subunit at a time

refer to Lecture 15 pg 13

Question 20

Synthesizing a New DNA Strand: Primers
DNA polymerase
A new DNA strand can only be synthesized in ___’—>___” free
3’ OH needed
DNA polymerase cannot ____ of a DNA strand by themselves?

Answer 20

A new DNA strand can only be synthesized 5’—>3’
– Free 3’ OH group needed

– DNA polymerases cannot initiate synthesis of a DNA
strand by themselves

Question 21

DNA Polymerase can only…
add nucleotides to the ___’ end of an existing nucleoitde chain that is base paired with template strand?

Answer 21

DNA polymerases can only add nucleotides to the 3’
end of an existing nucleotide chain (DNA or RNA) that
is base-paired with the template strand

Question 22

Synthesizing a New DNA Strand: Primers

A primer must be synthesized first
Enzyme that makes this primer is?

RNA primase synthesizes a short piece of RNA, ______ to the DNA template

Answer 22

The enzyme that makes this primer is RNA primase(RNA primase is an RNA polymerase)

RNA primase synthesizes a short piece of RNA,
complementary to the DNA template

So, all new DNA strands have a short stretch of RNA at
their 5’ end

Since newly synthesized DNA can only be elongated at
the 3’ end, the two daughter strands are made
differently

Question 23

Continuous vs. Discontinuous Replication

Continuous replication is leading strand
and leading strand has its 3’ end pointing toward? and synthesized as one long?
Leading strand has only ____ primer?
the lagging strand has its 3’ end pointing away from? and is synthesized in ______ bits=okazaki fragments
lagging strand has many______?

Answer 23

-The leading strand has its 3’ end pointing toward the
replication fork, and so is synthesized as one long,
continuous polymer as the parental strand is unwound.
-Leading strand has only one primer
-The lagging strand, has its 3’ end pointed away from the
replication fork, and is synthesized in short bits =
Okazaki fragments
-Lagging strand has many primers

Question 24

DNA polymerase can only synthesize DNA which way?

Synthesis of both strands must happen in which direction?
DNA polymerase synthesis top strand ____’ to ____’ bottom strand cannot be synthesized in a 3’ to 5’ direction
the only way DNA polymerased can synthesize bottom(lagging) strand is by?

Answer 24

DNA polymerase can only synthesize DNA 5’ –> 3’

Synthesis of both strands must happen in the direction of the replication fork

DNA polymerase synthesizes
top strand 5’ to 3’, bottom strand cannot be
synthesized in a 3’ to 5’
direction

The only way DNA
polymerase can synthesize
the bottom (lagging) strand
5’ to 3’ is by making a short
fragment, backing up and
making a new one

These fragments are called
Okazaki fragments

Question 25

Primers are required for?
primer for leading strand
primer for lagging strand

Answer 25

Primers are required for both the leading and the lagging strand
-Leading strand only needs one primer

– For lagging strand, each Okazaki fragment must be
primed separately
* Result: multiple primers and multiple Okazaki fragments not joined together…

Question 26

Synthesizing a New DNA Strand: Elongating the Primer
once primer is synthesized what happens?

Answer 26

Once primer(s) synthesized,
DNA polymerase elongates
primer i.e. adds DNA
nucleotides to 3’ end of
growing strand

Question 27

Recall Nucleotide Structure

Answer 27

• Deoxyribose sugar
• phosphate group (5’ carbon)
• Nitrogenous base (1’carbon)
• Free OH group (3’ carbon)

Question 28

Synthesizing a New DNA Strand:
Elongating the Primer
How are nucleotides added on?
elongation 3’OH

Answer 28

1. Incoming nucleotides are accepted if they correctly base pair with the template
2. The 3’ OH of the growing strand attacks the high- energy phosphate bond of the incoming nucleotide to initiate the synthesis reaction

Question 29

Synthesizing a New DNA Strand:
Elongating the Primer

Answer 29

Nucleotides come in as
nucleoside triphosphates
* Phosphate tails highly reactive
* As these monomers join DNA strand, two phosphate groups lost as pyrophosphate
* Hydrolysis of pyrophosphate into two inorganic phosphates is a coupled exergonic reaction that drives the polymerization reaction
* Enzyme that catalyzes the
reaction is DNA polymerase

Question 30

Synthesizing a New DNA Strand:
Elongating the Primer

Answer 30

On the leading strand DNA
keeps adding nucleotides
continuously
* The lagging strand is
synthesized in Okazaki
fragments
* When DNA polymerase
encounters primer of adjacent
Okazaki fragment, it falls off
* Different DNA polymerase
removes primers and replaces
RNA with DNA
* DNA ligase links the DNA
fragments together

Question 31

Synthesizing a New DNA Strand:
Elongating the Primer

Answer 31

(As each new primer for an Okazaki fragment is synthesized, the lagging strand forms a loop that persists until the new lagging strand encounters the previous Okazaki fragment)

Synthesis of leading and
lagging strands occurs at
same pace
* Positioning of polymerases
requires that the lagging
strand be looped so both
leading and lagging strand
pass through in same
direction

Question 32

Proofreading

Answer 32

*Very rarely, an incorrect nucleotide is added
*when this happens, the proofreading function of DNA polymerase removes the incorrect nucleotide
*Then the correct nucleotide is added to replace the incorrect one

Most DNA polymerases can correct their own mispairing
* As a nucleotide comes into the growing DNA strand, it is temporarily held in place by hydrogen bonds
* Connect nucleotide in new strand to template strand
* If improper hydrogen bonds form (i.e. if wrong base inserted) DNA polymerase detects the mispairing by activating a cleavage function
* removes the incorrect nucleotide, and inserts the correct one in its place
* When done, reactivates the polymerase
function and continues adding bases

Question 33

Eukaryotic DNA Replication:
Lagging Strand Problems

Answer 33

-The leading strand replicates the whole template strand
-The last RNA primer on the lagging strand sits near the end of the template strand
-The RNA primer is removed and a section of template DNA remains unreplicated
-In the next round of replication the shortened template results in a shorter chromosome
-If this pattern were allowed to persist the chromosomes would be severely shortened after several generation

Question 34

Eukaryotic DNA Replication:
Lagging Strand Problems

Answer 34

On the leading strand, there is only one
primer and replication continues to the
end; primer is eventually replaced by
DNA
* But lagging strand requires multiple
RNA primers and the final primer is
added about 100 nucleotides from the
3’ end of the template
* When it is removed, the new daughter
strand is shortened by about 100
nucleotides
* If this were to continue each time
replication occurred, the DNA would
eventually be nibbled away to nothing

Question 35

Eukaryotic DNA Replication:
Telomerase to the Rescue!

Answer 35

-The terminal part of the telomere in the template DNA strand remains unreplicated
-The telomerase enzyme contains an RNA template that allows the shortened 3’ end of the template strand to be restored by the addition of more telomere repeats
-A new segment of lagging strand can then be formed so that the original telomere in the template strand is completely restored

Question 36

Eukaryotic DNA Replication:
Telomerase to the Rescue!

Answer 36

Eukaryotes have evolved a
mechanism to solve the problem of shortened ends
* Each end of a eukaryotic
chromosomes is capped by a
repeating sequence called the telomere
* In humans, the telomere consists of the
sequence 5’-TTAGGG-3’ repeated around 1500-3000 times
* In some cell types, before the next replication round begins the enzyme telomerase replaces the lost repeats from the previous round

Question 37

Eukaryotic DNA Replication: Telomeres
They protect?

Answer 37

The repeating sequences of bases in telomeres don’t code for
proteins
* They protect the genetic information on the ends of linear
chromosomes from being lost
* Telomerase = enzyme containing RNA template used to lengthen
the template strand, which allows replication of functional part
of daughter strand to be completed

Question 38

Telomerase

Answer 38

enzyme containing RNA template used to lengthen
the template strand, which allows replication of functional part
of daughter strand to be completed