Mamalapat, Mohamidin K 10/1/2023.

Chem 2065 AY2023-24

10 Biosynthesis of Nucleic Acids: Replication

1. Provide an overview of the central dogma of biology (replication, transcription,

translation)

The central dogma of biology describes the genetic information’s flow from the
DNA to RNA to protein. It focuses on those genes that play a role in specifying the
sequence of the mRNA molecules which specify the protein sequence. The cell makes sure
that the DNA is protected and copies it in the form of RNA since the information inside
the DNA is vital for cellular function.

● Replication – process of duplication of DNA

● Transcription – production of complementary RNA copy of a DNA sequence;

● Translation – mRNA is decoded and translated to a polypeptide sequence

2. Provide the requirements of RNA

viruses and retroviruses (reverse transcriptase, RNA replication)

In retroviruses, instead of the DNA, the RNA serves as the genetic material. Here,
the RNA directs its own synthesis like the DNA. The enzyme that catalyzes this process is
the reverse transcriptase. However, not all viruses with RNA as genetic material are a
retrovirus, but all the retroviruses have the reverse transcriptase. In fact, the term retrovirus
is coined based on that, referring to the reverse of the usual situation with transcription.

3. State the scope and limitation of studies on prokaryotic replication (E. coli, double stranded
DNA or dsDNA versus single stranded DNA or ssDNA, challenges: separation of the two
strands and the chemical reality that polynucleotide synthesis occurs only in the 5' to
3' direction

Duplication of the double-helical DNA molecule to produce two such double-

stranded molecules is a complex process. This allows for a high-degree of fine-tuning that
ensures fidelity in replication. In this process, the cell undergoes three challenges.

(a) separating the two DNA strands – the two strands of the DNA are wound around each
other in a manner wherein they must be unwound if they are to be separated. To be able to
achieve continuous unwinding of the helices, the unwound portions must be protected from
the action of the nucleases which attack single-stranded DNA.
 (b) synthesizing of DNA from the 5’ to the 3’ end – two parallel strands must be
synthesized in the same direction on antiparallel templates. So, if the template has one 5’
> 3’ strand and one 3’ > 5’ strand.

(c) guarding against errors in replication – the correct base should be added to the growing
polynucleotide chain

4. Explain how the semiconservative replication was deduced by the Mesehlson and Stahl
experiments (density-gradient centrifugation)

They grew E. coli bacteria with 15NH4Cl as the sole nitrogen source with 15N as
a heavy isotope of nitrogen. In this medium, all newly formed nitrogen compounds were
labeled with 15N. The labeled ones were higher in density compared to the unlabeled ones,
which contains the usual isotope, 14N. The 15N-labeled cells were transferred to a medium
which only contains 14N and then allowed the cells to continue to grow. Through density-
gradient centrifugation, a sample of DNA was extracted and analyzed with every new
generation of growth. The heavy 15N DNA forms a band at the bottom of the tube while
the light 14N DNA appears on top of the tube. DNA containing a 50-50 mixture of both
appears at a halfway between two bands. In the said experiment, the 50-50 hybrid DNA
was observed after one generation, a result to be expected with semiconservative
replication. After two generations in the lighter medium, half of the DNA in the cells should
be the 50-50 hybrid and half should be the lighter 14N DNA.

5. Explain how replication is bidirectional while polynucleotide synthesis occurs only in

the 5' to 3' direction (origin of replication oriC, plasmids, replication fork, theta
structure, bubble, eye, daughter DNA)

At the origin of replication (OriC in E. coli), the DNA double helix unwinds during
replication. Two possibilities exist for the growth of the new strands: synthesis may take
place in both directions from the origin of the replication or in one direction only. There
are two points for each origin of replication called the replication forks where new
polynucleotide chains are formed. A “’bubble” or an “eye” of the new synthesized DNA
between regions of the original DNA is a manifestation of the advance of the two
replication forks in opposite directions. These bubbles grow larger and then merge
eventually which gives rise to two complete daughter DNAs. This bidirectional growth of
the new polynucleotide chains represents net chain growth. Both chains are also
synthesized in the 5’ to 3’ direction.

6. Write the mechanism of nucleophilic attack of the 3'-hydroxyl group from an exisiting
polynucleotide to the phosphorus of a nucleosidetrisphosphate (NTP) in the formation of
a new phosphodiester bond

Nucleotide chain synthesis occurs in the 5’ to 3’ direction from the perspective of

the chain being synthesized due to the nature of the reaction of DNA synthesis. The last
nucleotide added to a growing chain has a 3’-hydoxyl on the sugar while the incoming
nucleotide has a 5’-triphosphate. The 3’-hydroxyl group at the end of the growing chain is
 a nucleophile which attacks the phosphorus adjacent to the sugar in the nucleotide to be
added to the rowing chain, which leads to the elimination of the pyrophosphate and the
formation of a new phosphodiester bond.

7. Explain how replication can proceed under the mechanism stated in the previous objective
considering that the polynucleotides in DNA are antiparallel (leading strand, lagging
strand, continuous and semi-discontinuous replication, Okazaki fragments, DNA
ligase

It can proceed by different modes of polymerization for the two growing strands.
One newly formed strand, the leading strand, is formed continuously from the 5’ end to
the 3’ end at the replication fork on the exposed 3’ to 5’ template strand. On the other hand,
the lagging strand is formed discontinuously in small fragments which is sometimes called
the Okazaki fragments. The 5’ end of these fragments is closer to the replication fork than
the 3’ end. Through the enzyme DNA ligase, the fragments of the lagging strand are linked
together.

8. Describe the properties and functions of the five DNA polymerases isolated form E.
coli ( DNA polymerase, Polymerase III, Pol III, main polymerizing enzyme, DNA
ligase, proofreading, repair enzyme, processivity)

• DNA polymerase catalyzes the successive addition of each new nucleotide to the
growing chain. The first DNA polymerase discovered was found in the E. coli.
• DNA polymerase I (Pol I) – a single polypeptide chain that was discovered first;
functions in the replication-repairing, and patching of DNA
• DNA polymerase II (Pol II) – a multisubunit protein that shares common subunits with
Pol III; not required for replication but is strictly a repair enzyme
• DNA polymerase III (Pol III) – multisubunit protein that shares common subunits with
Pol II; consists of a core enzyme responsible for the polymerization and 3’ exonuclease
activity; main polymerizing enzyme
• DNA polymerase IV and V – repair enzymes and are involved in the SPS response]

→ There are two important considerations on the polymerases: the speed of the synthetic
reaction (turnover number) and the processivity or the number of nucleotides joined
before the enzyme dissociates from the template
→ Proofreading – incorrect nucleotides are removed from the polynucleotide so that the
correct ones can be incorporated. The 3’ to 5’ exonuclease activity is part of the
proofreading function; done one nucleotide at a time.

9. Explain the need for the presence of a primer in replication

DNA polymerase cannot catalyze de novo synthesis. All three enzymes require the
primer, a short oligonucleotide strand to which the growing polynucleotide chain is
attached covalently in early stages of replication. The DNA polymerase must have a
nucleotide with free 3’-hydroxyl in place to add the first nucleotide as part of the growing
chain. RNA is the primer in natural replication and it is hydrogen bonded to the template
 (DNA); the primer provides a stable framework where the nascent chain can start to grow.
The newly synthesized DNA strand begins to grow by forming covalent linkage to the free
3’-hydroxyl group of the primer.

10. List the required biomolecules and ions involved in replication

dTTP, dATP, dGTP, and dCTP are the four deoxyribonucleoside triphosphates
required in the DNA polymerase reaction. The Mg2+ and a DNA template are also
necessary. Due to the requirement for an RNA primer, ATP, UTP, GTP, the four
ribonucleoside triphosphates are also needed which are incorporated into the primer. DNA
replication is carried out by replisome, a multiprotein complex.

11. Explain the roles of the different proteins involved in prokaryotic replication (DNA gyrase,
helicase, single-strand binding protein SSB, primase, ligase)

• DNA gyrase – enzyme that catalyzes the conversion of relaxed, circular DNA with a
nick in one strand to the supercoiled form with the nick sealed; a slight unwinding of
the helix before the nick is sealed introduces supercoiling; fights the positive supercoils
by putting negative supercoils ahead the replication fork
• Helicase – helix-destabilizing protein that promotes the unwinding by binding at the
replication fork; dnaB protein and rep protein are some known helicases
• Single-strand binding protein (SSB) – protein that stabilizes the single-stranded regions
of the DNA by binding tightly to these portions of the molecule
• Primase – enzyme that copies a short stretch of the DNA template strand to produce
the RNA primer sequence; first primase was discovered in E. coli
• Ligase – enzyme that links the separate stretches of DNA

12. Use Figure 10.10 in your textbook to summarize the process of DNA replication in
prokaryotes (primosome, replisome)

Through the DNA gyrase and helicase, the DNA duplex is unwound, and the single
strands are coated with the ssDNA-binding protein. The primase periodically primes
synthesis on the lagging strand. Each half of the dimeric replicative polymerase is a
holoenzyme bound to its template strand by a beta-subunit sliding clamp. DNA polymerase
I and DNA ligase act downstream on the lagging strand to remove RNA primers, replace
them with DNA, and ligate the Okazaki fragments.

13. Describe the inefficiency of Pol III is corrected by Pol I during the proofreading stage in
replication

Incorporation of an incorrect nucleotide into a growing DNA chain once every 104
to 105 base pairs happens because of the errors in the hydrogen bonding. Pol I has two
major fragments: the (1) Klenow fragment, which contains the polymerase activity and
proofreading activity, and (2) the 5’ to 3’ repair activity. DNA polymerase I uses its 3’
exonuclease activity to remove the incorrect nucleotide.
14. Describe how 'nick translation', mismatch repair, base-excision repair, nucleotide-
excision repair mechanisms remove errors in replication or in damaged DNA to prevent
or minimize mutation

In the nick translation, the polymerase I uses its 5’ to 3’ exonuclease activity to

remove the RNA primers or the damaged nucleotides as it moves along the DNA. It then
fills in behind it with its polymerase activity.

• Mismatch repair – in this mechanism the enzyme recognizes that two bases are
incorrectly paired. The area that contains the mismatch is removed and the DNA
polymerases replicate the area again. However, this will only be possible if the
repair system identifies which of the strands is correct by modifying the bases with
added methyl groups before the replication process
• Base-excision repair – when a base is damaged by oxidation or chemical
modification it is removed by the DNA glycosylase leaving an AP site. The AP
endonuclease removes the sugar and phosphate from the nucleotide and then an
excision exonuclease removes more bases. Then, the DNA polymerase I fills the
gap and the DNA ligase seals the phosphodiester backbone
• Nucleotide-excision repair – when there are DNA lesions, the section of the DNA
with the lesion is removed by the ABC exonuclease then the DNA polymerase I
and ligase work to fill the gap.

15. Use the disease xeroderma pigmentosum to highlight the importance of repair mechanisms

In xeroderma pigmentosum, affected individuals develop numerous skin cancers at

an early age because they do not have the repair system to correct damages caused by UV
light. The endonuclease that nicks the damaged portion of DNA is probably the missing
enzyme. XPA protein is the repair enzyme that recognizes the lesion. These cancerous
lesions spread throughout the body which causes death.

16. Explain the advantage of life having evolved such that DNA contains thymine but not
uracil

Thymine helps guarantee replication fidelity. The determination of cytosine to

uracil is one of the most common spontaneous mutations of bases. So, when this happens
during the replication process, it would base-pair to adenine instead of guanine. Since uracil
is an unnatural base in DNA, DNA polymerases can recognize it as a mistake and can
replace it, thus the incorporation of thymine into DNA. Even if this costs energy, it helps
ensure that DNA is replicated faithfully.

17. [Optional] Describe how DNA Recombination allow genetic information to form new
associations

New associations are formed when genetic information is rearranged. Example,

progeny may have new combinations of traits due to recombination. At the molecular level,
 it is the exchange of one DNA sequence to another or the incorporation of a DNA sequence
into another. Recombination occurs by the breakage and reunion of DNA strands so that
physical change of DNA parts takes place.

18. Describe how eukaryotic replication works. Highlight only the major differences
between prokaryotic and eukaryotic replication

Eukaryotic replication is more complicated than prokaryotic replication in three basic

ways: (a) multiple origins of replication, (b) timing must be controlled to that of cell
divisions, (c) more proteins and enzymes involved.

• Cell growth and division are divided into three phases - M, G1, S, and G2. During
a few hours in the S phase, DNA replication happens, and pathways exist to make
sure that the replication of DNA happens only once per cycle. The process of
replication can only be initiated in cells that have reached the G1 phase. Throughout
this cycle, the origin recognition complex or the ORC forms and remains bound to
the DNA which serves as the attachment site for proteins that plays a role in the
control of replication.
• Replication starts and requires the binding of the replication activator protein to the
ORC then the replication licensing factors bind. Some of these factors are cytosolic
which means that they access the chromosome only when the nuclear membrane
dissolves during mitosis. The DNA then is now ready for replication.
• Cyclins are the proteins that are produced in one part of the cell cycle and degraded
in another thus they are present only in some cycles. They combine with the
cyclindependent protein kinases. The activation of this initiates the DNA
replication. It also phosphorylates the RAP and RLFs. This targets these proteins
for degradation thus preventing the formation of another pre-RC. In the G2 phase,
DNA has been replicated and is separated into two daughter cells in the M phase.

19. Describe the roles of telomeres in replication and their relationship to cancer and in aging

Telomerase provides a mechanism for synthesis of the telomeres. It is an enzyme

that is a ribonuclear protein which contains a section of the RNA that is complement to the
telomere. In humans, this sequence is 5’CCCUAA3’. The telomerase then binds to the 5’
strand at the chromosome end and uses a reverse transcriptase activity to synthesize DNA
on the 3’ strand using its own RNA as the template. Telomerase is not active in most of the
adult tissues thus cell division does not preserve the chromosome ends leading to DNA
loss and cell death. It is also found to be reactivated in cancer cells thus explaining their
immortality and their ability to divide rapidly