molbio ch2

Bacterium

only a single circular
chromosome is present,

nucleic acid-based activities

all cellular activities, including DNA replication, recombination and gene
expression

Cell division

a parental cell must duplicate its entire genome before dividing. Subsequently, each of the two new daughter
cells that are created then receives one copy of the genome through a process called

DNA replication

process to ensure that the DNA is duplicated before cell division so that each offspring cell receives chromosome(s) identical
to the parent's

Multicellular organisms; increase the size and complexity

in \____________, cell division does not automatically result in the creation of the
new organism. Instead, they \_________ and \________ of the original organism.

S Phase

multiple origins

In each cell cycle, eukaryotic cells
must replicate large amounts of genomic DNA distributed on multiple chromosomes. To accomplish this feat in a reasonable
period of time, replication initiates throughout \________ at \________along each
chromosome.

Chromosome segregation

DNA replication must also be coordinated with \_________________
to ensure that each daughter cell receives a complete and unaltered complement of genetic information. Mistakes in either
DNA replication or chromosome segregation can result in loss or duplication of this genetic information, events that can
play an important role in the genesis of cancer cells or diseases.

Remember

DNA can either be obtained from a parent cell for division, or from another cell of the same generation. It is
then expressed within that cell, or transferred to another.

semiconservative model,

the conservative model, and

the
dispersive model

three DNA replication models were proposed

two parental strands separate, allowing each separated strand to serve
as a template for the synthesis of a complementary strand

each double-stranded
daughter DNA molecule will have a conserved DNA strand that is derived from the parental DNA and a newly synthesized
strand.

each contain
one old strand (green) and one new
strand (red).

semiconservative model

two strands of the double helix unwind at the replication site to the extent needed for
synthesizing new base sequence.

two original strands remain entwined after replication so that one of the two DNA
molecules present after replication contains both original strands and the other DNA molecule is made of the two new strands

One contains
two old strands (green), the other
contains two new strands (red).

conservative model

shares some of the features of the conservative model but predicts that each strand
of the daughter DNA molecules and parental DNA molecules has interspersed sections of both old and new DNA

mixtures of old and new material

Dispersive model

1958,

Matthew Meselson and Franklin Stahl

equilibrium density gradient centrifugation

semiconservative

In \____, \____ and \______used equilibrium density gradient centrifugation experiments to demonstrate
that DNA replication is semiconservative.

E.coli Cells

15NH4Cl

14NH4Cl

In their experiments (Meselson and Stahl), they cultured \___________ for many generations
in a growth medium containing \_______ as the sole source of nitrogen.

all nitrogenous bases in
DNA were labeled with the heavy isotope 15N

Subsequently, they transferred the bacteria to a new growth medium containing
\________ as the sole source of nitrogen.

Centrifugation

cesium chloride
equilibrium density gradient

Bacterial cells were removed at various times for DNA extraction, followed by \________ for the \____________

two bands

\[15N/15N]
\[14N/14N]

If the replication mechanism is conservative, there should be \________ in the gradient after
one generation, one band representing \_____parental duplex, and the other band representing \_____ newly synthesized
DNA

two [15N/15N]

template

\[14N] strand

\[15N/14N] hybrid
band

If the replication follows a semiconservative mechanism, the \_________ parental strands will
separate to serve as the \________ for replication, and each will be supplied with a new\________

One \_________ would appear in the gradient.

E. coli growing
in 15N medium

Transfer to
14N medium

Continue growth
in 14N medium

Centrifuge


Before transfer
to 14N \> One generation after
transfer to 14N \> Two generations after
transfer to 14N

Meselson/Stahl Experiment Flow

Meselson/Stahl experimental procedure

or

Equilibrium density gradient centrifugation experiment

involves the use of a technique referred to as sedimentation equilibrium centrifugation or buoyant
density gradient centrifugation.

a density gradient of cesium chloride

15N-DNA

14N-DNA

Samples are forced by centrifugation through a \______________. Molecules
of DNA will reach equilibrium when their density equals the density of the gradient medium. As such, \______ will
reach the point at a position closer to the bottom of the tube than will \__________.

conservative hypothesis

results showed that there is one DNA band representing [15N/14N] in the gradient for the cells incubated in the
15NH4Cl-containing medium after one generation of incubation in the 14NH4Cl-containing medium. As such, this result
rules out the \________ which should have two bands

hybrid product with one-fourth [15N] and three fourths
\[14N].

\[15N/14N] and [14N/14N] DNAs


\[15N/14N]

\[14N/14N].

semiconservative mechanism.

In two rounds of replication, the dispersive mechanism would give one \_______________

On the other hand, the semiconservative hypothesis predicts that [15N/14N] would separate and serve as the
template for replication and produce \__________

It should give 1:1 ratio for these two DNA molecules.
After two generations of incubation in the 14NH4Cl-containing medium, Meselson and Stahl observed that there are two
bands in the gradient in which one represents \_______ and the other one represents \__________ Therefore, this experiment
proves that DNA replication follows the semiconservative mechanism.

initiation, elongation, and termination

DNA replication can be divided into three phases

origins of replication

replication bubble

DNA replication
starts at specialized sites called\_____ moves away from an origin in both directions, creating a
structure known as a \_________

origins of replication

replication forks

DNA double helix is opened at the \____________and
unwound on both sides of the origin to form two structure called \________ that unwind the double helix in
opposite directions.

replication forks

re the sites at which single-stranded DNA is exposed, and at which DNA
synthesis occurs.

replication enzymes and proteins

Once the DNA double helix is opened, \____________ are loaded to the single strand, and these will
form the templates for the daughter strands that are to be synthesized

Elongation Phase

After the replication machinery is in place,
and the DNA has been opened up, replication enters the elongation phase.

During this phase, the replication
machinery moves along the parent DNA strands and forms the daughter strands as it proceeds.

Termination of DNA

ter

occurs when the two replication forks moving in opposite directions meet, and the replication complexes are
disassembled.

In bacteria, this occurs at a specific site called \____, whereas in eukaryotes, no specific termination sequence is
required.

(1) Helicase (orange rings) is recruited to the origin
Replication bubble allows both strands to be copied
in opposing directions

(2) Helicase recruits primase, which will synthesize
RNA primers (green) on both strands. For simplicity,
only leading strand replication is shown.

(3) Sliding clamp is recruited and binds

(4) DNA polymerases (blue) are recruited. This interacts
with the sliding clamp to elongate 3! end of primers.

(5) Both replication forks travel in opposing directions,
helicase works to unwind the DNA, allowing synthesis
of new strands to occur. Replication of the leading strand
is continuous, but replication of the lagging strand is
discontinuous, and produces short fragments of DNA that
are joined later (not shown).

(6) Replication is complete. RNA primers are removed
and DNA polymerases fill in nucleotides. DNA ligases
(not shown) seal any gaps that remain.

DNA REPLICATION PROCESS

5' -\> 3' direction.


5'-triphosphate

3'-
hydroxyl group

Due to the nature of the DNA synthesis reaction, all synthesis of nucleotide chains occurs in the

In a growing
chain, the incoming nucleotide has a \___________on its sugar. The last nucleotide added to a growing chain has a \__________ on the sugar.

nucleophile
alpha phosphate group

DNA polymerase

Because this 3'-hydroxyl group is a \__________, it attacks the\______, leading
to the elimination of pyrophosphate and the formation of a new phosphodiester bond, which links two nucleotides together
and allows DNA strand synthesis to occur.

This is catalyzed by

5'-\>3' direction.

All known DNA
polymerases so far can only add nucleotides in a \_______ As such, DNA replication can only occur in the \_______

fork

During replication, the double helix DNA would unwind to create a \____

t

strand synthesis
occurs in opposite directions for the two templates in a double helix. (t/f)

Okazaki and his colleagues

Following the fact that DNA synthesis can only make
one direction 5'-\>3', \______ and \____proposed a semi discontinuous replication mechanism

Semidiscontinuous replication mechanism

In this model, both
strands could not replicate continuously.

leading strand

same

DNA polymerase could make one strand which is the \________ continuously
in the 5'-\>3' direction at the replication fork on the exposed 3'-\>5' template strand .

Its direction of synthesis is
\____ as the direction in which the replication fork is moving.

Lagging strand

Okazaki fragments

would have to be made discontinuously in small fragments—fragments are typically 1000-2000 nucleotides long

Opposite

The discontinuity of synthesis
of the lagging strand is because its direction of synthesis is \_______ to the moving direction of the replication fork

REMEMBER

The 5'
end of each of these Okazaki fragments is closer to the replication fork than the 3' end

DNA ligase

The small Okazaki fragments of the
lagging strand are then linked together by an enzyme called \___________, and together these will constitute the second
daughter strand.

Eukaryotic DNA replication

more complex than prokaryotic DNA, due to several features of eukaryotic DNA

eukaryotic cells contain much more DNA, and their DNA is complexed with nucleosomes

eukaryotic chromosomes are linear rather than circular

Why eukaryotic DNA is more complex than Prokaryotic DNA?

minutes to hours.

multiple replication origins.

replication from a single origin
on a typical eukaryotic chromosome would take days to complete. However, replication of entire eukaryotic genomes
is usually accomplished in a matter of \_____________. To facilitate the rapid synthesis of large quantities of DNA,
eukaryotic chromosomes contain \____________

Eukaryotic replication origins not only act as sites of replication
initiation, but also control the timing of DNA replication.Eukaryotic replication origins not only act as sites of replication
initiation, but also control the timing of DNA replication.

act as sites of replication

control the timing of DNA replication.

Eukaryotic replication origins not only \_________
initiation, but also \_______________

Replisome

DNA replication is carried out in all organisms by a multiprotein complex called

eplicon

Replication fork

DNA replication begins at
the\___________, where the two DNA strands unwind at the r\___________

initiation phase

During the \__________, the double helix parental DNA is opened up in order to give replication enzymes and other
proteins access to the single strands that will form the template for the daughter strands that are to be synthesized. In fact,
a specific initiator protein can recognize the origin of replication and recruit helicase to the origin of replication

Helicase

is the enzyme that harnesses the chemical energy from the ATP hydrolysis to separate the two DNA strands at the replication
fork

Single-strand DNA binding proteins (SSBs)

nucleases.

bind selectively
to single-stranded DNA as soon as it forms.

its binding can stabilize the single-stranded DNA so they will not anneal to reform the double helix and protect the single-stranded DNA from hydrolysis by \________

This also allows
enzymes to attach to the newly opened single strand and initiate elongation

SSBs are removed as DNA synthesis
proceeds.

negatively supercoiled, closed circle

In prokaryotes, DNA exists in a \_______________ form

positive supercoils

a replisome

When two DNA strands are separated during
replication, \_____________ are introduced ahead of the replication fork. This is because the strands of duplex DNA
unwind to allow each single strand of DNA to serve as a template for the synthesis of a complementary strand; \__________
that is prevented from rotating will accumulate positive supercoils in front of the replication fork further twisting
the DNA onto itself.

DNA gyrase (Type
II topoisomerase)

negative supercoils

If the replication fork continued to move, the torsional strain of the positive supercoils would eventually
make further replication impossible, because the DNA would be too compacted. At this moment, \________________fights these positive supercoils by putting \____________ ahead of the replication fork.
Thus, the
torsional strain is released, and replication can continue.

REMEMBER

General features of the replication fork. DNA gyrase and helicase work to unwind the DNA so that it can be replicated. The newly unwound,
single strands are coated with ssDNA-binding protein, SSB for short. Replicative DNA polymerase is bound to the template strand (either leading or
lagging) via the "- Beta subunit. Downstream, DNA polymerase II and DNA ligase remove the RNA primers, replace them with the correct DNA fragments
and also ligate (seal together) the Okazaki fragments on the lagging strand.

Supercoiling is formed ahead of the replication fork. As the helicase unwinds DNA, the twist ahead of the fork increases in tension. This is
similar to twisting a rubber band, then using two fingers to separate the strands. The supercoil is resolved by topoisomerase I or II, allowing the continuous
movement of helicase.

cannot synthesize a new DNA strand from the very start of the parent
strand. can only add nucleotides to the 3' end of a nucleotide fragment that already exists

One unusual feature of DNA polymerase is that

Remember

'It is necessary to have a short
strand of RNA termed primer for the beginning of DNA replication. The primer must have a free 3-hydroxyl to which the
growing chain can attach.

Primase

s responsible for copying a short stretch of the DNA template strand to produce the RNA
primer sequence

one

one primer each

The leading strand
requires \______ primer. On the other hand, each Okazaki fragment in the lagging strand would need \_________

hydrogen-bonded

The Primer is \___________ to the template so it can provide a stable framework to which the nascent chain starts to grow

10-30 bases

Primase

DNA Polymerase III

In bacteria,
a short RNA primer of around \____ - \______ is synthesized by a \_______, followed by the replication directed by \___________

four subunits

primase; alpha catalytic
subunit and accessary subunit.

eukaryotic primase complex contains\_____

Two of them function as a \______

then there is an\_________
subunit and an \______

DNA polymerase

Primases and alpha catalytic subunit bind in a complex with the \___________

polymerase-alpha subunit-primase complex

10-30

polymerase switching.

Normally,
the \___________ synthesizes a stretch of \______- \_____nucleotides of RNA. Subsequently, alpha catalytic
subunit continues to synthesize a short stretch of DNA before the DNA polymerase takes over the replication process. This
phenomenon is called \________

DNA Polymerase III

Once the replication is initiated, the synthesis of new DNA strand is directed by one of the polymerases -

3'-hydroxyl

The first nucleotide is added to the \_____ of the RNA primer, and synthesis proceeds from the 5'
end to the 3' end on both the leading and the lagging strand.

RNA Primer

DNA polymerase I's exonuclease
activity and polymerization activity.

Okazaki maturation process

As the replication fork moves, the \____ is removed
and is replaced by newly formed deoxynucleotides. These reactions are performed by \_____________ This is a so-called \________

DNA polymerase
I

DNA
ligase

Just after RNA removal, \_______uses its polymerase activity to fill in the gap left by the RNA with new DNA. When it reaches the beginning of
the next DNA segment, the DNA polymerase cannot seal the final nick in the DNA backbone that remains. Finally, \_______ is the enzyme responsible for sealing the nick between the new strands synthesized by polymerase III and polymerase
I.

Properties of DNA Polymerases of E.coli

Mass (kDa)

Structural gene

Polymerization (5'-\>3')

Exonuclease(5'-\>3')

Exonucelase (3'-\>5')

Number of subunits:

103

polA

yes

yes

yes

1

Polymerase I


Mass (kDa)

Structural gene

Polymerization (5'-\>3')

Exonuclease(5'-\>3')

Exonucelase (3'-\>5')

Number of subunits:

90

polB

Yes

No

Yes

More than 4

Polymerase II


Mass (kDa)

Structural gene

Polymerization (5'-\>3')

Exonuclease(5'-\>3')

Exonucelase (3'-\>5')

Number of subunits:

830

polC

Yes

No

Yes

More than 10

Polymerase III


Mass (kDa)

Structural gene

Polymerization (5'-\>3')

Exonuclease(5'-\>3')

Exonucelase (3'-\>5')

Number of subunits:

polymerization activity

exonuclease activity.

All three DNA polymerases have the 5'-\> 3' \_______ and 3'-\> 5- \_________

The 5'-\> 3' polymerization activity

3'-\> 5' exonuclease activity

\_____________ can add nucleotides to a
growing chain during DNA synthesis. On the other hand, the \___________ is part of proofreading function
which can remove incorrect nucleotides one at a time in the process of replication and replace them with the correct one.

DNA polymerase I

Only \__________ has 5'-\> 3' exonuclease activity which can remove short stretches of nucleotides during repair.

DNA polymerase I

nick translation.

After polymerase III has produced the new polynucleotide chain, \___________removes RNA primer through 5'-\> 3'
exonuclease activity. It then fills in behind it with its polymerase activity (Fig. 2.11).

This is a cut-and-patch process which
is also called \______

Polymerase I also uses nick translation in the repair process

A) Two DNA fragments
present, the newly synthesized
DNA contains RNA primer and is separated by a nick (short break)

B) The nick serves as a DNA
polymerase I binding site

(C) DNA polymerase I acts
as both a polymerase and
an exonuclease. It removes
the primer and fills in the gap
that is left. Degraded
primer is left behind.

(D) DNA ligase forms
a phosphodiesterbond
between the DNA fragments,
sealing the remaining nick.

FIG. 2.11 RNA primers are removed before DNA nicks are fixed.

T

The movement of polymerases is coordinated to allow the polymerases
on both strands to travel together in the direction of fork movement. Therefore, if the leading strand replication
stalls, lagging strand replication will also be halted. (T/F)

replication complex, or replisome

The coupling of DNA replication on the leading and lagging strands in bacteria is achieved by physically associating the
proteins replicating each strand into one large protein called the \__________




The replisome associates
with the DNA, and this complex moves together as the replication fork moves. Protein within the complex can engage
and disengage from the DNA itself, but remains associated with the fork.

DNA polymerase
III holoenzyme or the replisome

In E. coli, the multisubunit assembly of DNA polymerase, sliding clamp, and clamp loader is called the \________

DNA polymerase
III holoenzyme or the replisome

alpha subunit

epsilon subunit

theta subunit

contains two copies of the multisubunit DNA polymerase III, which
contains the\______(the DNA polymerase activity), the \___________ (the 30-50 proofreading exonuclease), and the \___________
(which stimulates the exonuclease) together with sliding clamps and a clamp loader that continually reloads sliding clamps
on the lagging strand.

Clamp loader and sliding clamp

play an important role in recruiting DNA polymerase to the appropriate location on the
DNA template.

localize specifically at the region where DNA synthesis needs to commence

Fig 2.12

Fig 2.12

Sliding clamp

Clamp loader

\_________is responsible for holding catalytic cores onto their template strands

\________places the clamp
on DNA.

tau (t).

One polymerase replicates the leading strand and one replicates the lagging strand as the fork moves along the double
helix. Polymerases are linked together by a protein called \__________

Tau protein

replication complex

associated with the clamp loader and links
this polymerases-clamp loader complex to the helicase.

Therefore, the \__________ keeps the lagging strand polymerase
associated with the fork even as it is being released from the DNA at the end of each Okazaki fragment.

Furthermore,
through this association of polymerase, clamp loader, and sliding clamp, the loading of a new clamp and a
polymerase onto DNA at the beginning of each Okazaki fragment can be efficiently coordinated with the progress of
the polymerase on the leading strand.

bidirectionally
replicon

Most eukaryotic and bacterial DNAs replicate \_______.

The DNA under the control of the origin of replication
is called a \________

the origin of replication.

The circular E. coli chromosome has a single replicon because it replicates from a single
starting point—\____________

bubble

Two

DNA replication begins with the creation of a \_______, which is a small region
where the parental strands have separated and progeny strands have been synthesized

\____ forks arise from this bubble
and move in opposite directions. As the bubble expands, the replicating DNA begins to take on the theta shape until both
replication fork meet on the other side of the circle

Eukaryotic

bidirectionally

telomere.

\____chromosomes have many replicons, and the
replication in these replicons begins simultaneously. Again, the replication forks move \______, and they keep
moving until they reach the end of the chromosome, which is known as the \_____

rolling circle mechanism

Some circular DNAs are replicated by a \_________ instead of the theta mode

bacteriophage.

Replication by rolling
circles is common among \________

oIX174

bacterial phage named \______ has a single stranded circular DNA genome and adopts a simple form of rolling circle replication

free 3'-OH end

DNA polymerase

the 5' end

Basically, replication of only one strand is used to generate copies of
some circular molecules. For example, a double-stranded replicative form gives rise to many copies of a single-stranded
progeny DNA.


One strand of a double-stranded DNA is nicked and the\_________ generated by the nick is extended by
the \________. The chain is elongated around the circular strand template until it reaches the starting point. Subsequently,
the newly synthesized strand displaces the original parental strand. Meanwhile, the displaced strand is free as a
circle. This displaces the

counterclockwise and trailing out

duplex circle

phage virion

During the replication process, the intermediates that have the double-stranded part of the
replicating DNA can be considered to be rolling \______ and \_______the progeny single-stranded DNA.

During the replication phase of viral infection, it may be used as a template to synthesize the complementary strand.
The \_______ may then be used as a rolling circle to generate more progeny. During phage morphogenesis, the displaced
strand is packaged into the \_______.

rolling circle mechanism

circular DNAs

intact DNA strand

displaced strand

can be used to replicate double-stranded DNA

For example, ' phage adopts the lambda mode of
replication to produce several copies of circular DNA

Subsequently, these \____ serve as the template
for rolling circle synthesis of linear 'DNA molecules.

The \_______ serves as the template for the leading strand
synthesis, whereas the \______ serves as the template for the lagging strand synthesis.

Termination of DNA replication

replication complex

topoisomerases

occurs when the two replication forks moving away from the single origin of replication
meet on the opposite side of the circle in the circular chromosome of bacteria.

When the forks approach each other, the \_____disassembles and two growing strands join and this results in the interlinking of the two new
daughter DNAs. The two circular DNA molecules are separated by \__________

ter sites

unidirectional contrahelicase.

Sequences that are involved with termination
are called \_______.


contains a short, 23-bp sequence.

The termination sequences are unidirectional and
can be recognized by a \_______

Tus

helicase known as \___ in E. coli can recognize the consensus
sequences and prevent the replication fork from proceeding.

multiple points

replication
forks

topoisomerase

In eukaryotes, termination is completed at \___________. This is because the linear chromosomes have multiple
origins of replication, each of which initiate a replication bubble.

During the replication elongation, the two \_______ form different bubbles will meet in the same way as the two bacterial forks.

For the purpose of termination, the
resulting interlinked chromosomes will be unlinked by \____________