A research topic submitted by some students of the first year in Al-Azhar Pharmacy in Assiut in 2020 in the subject of cell biology under the supervision of Dr. Omar Mohafez holds a PhD in biochemistry and is a professor at the same college.

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Azhar University of Assiut
Faculty of pharmacy
Biochemistry department
Cell Biology (PBC121)
First year, second semester
DNA replication and importance of its inhibition
Students Cods Contribution
Hatem Wael Mohamed 1044 Abstract and introduction
Hossam Mohamed Rashad 1045 PCR, Importance of DNA inhibition
Hussein Ali Altohamy 1046 DNA replication
Khaled Ashraf Mohamed 1047 DNA structure and polymerase
Rabie Mohamed Awad 1048 Replication fork and enzymes
Under supervision of
Prof. Omar Mohafez
2019-2020

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Abstract:
DNA structural unit is nucleotide which consists of a nitrogenous base, a sugar
group and a phosphate group. And it is in the form of compact chromosomes stacked
inside the nucleus and for this, the chromosome must be decomposed into two
separate strips where cloning bronzes can double the genetic dye material.
As the DNA consists of two complementary parallel bars, the ends of each bar
are numbered using atoms or atomic peripheral groups as a guide: the hydroxyl group
which is located on the third carbon atom takes its number and then the fifth carbon
atom is numbered to be the other end of the compound and the tape together.
DNA has several steps of replication. These are initiation, elongation and
termination. Elongation also has replication fork, leading strand, lagging strand, DNA
replication proteins, and replication machinery. And within eukaryotes, DNA
replication is controlled within the context of the cell cycle. As the cell grows and
divides, it progresses through stages in the cell cycle; DNA replication takes place
during the S phase (synthesis phase). The progress of the eukaryotic cell through the
cycle is controlled by cell cycle checkpoints. Progression through checkpoints is
controlled through complex interactions between various proteins, including cyclins
and cyclin-dependent kinases.

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Introduction
Nearly every cell in a person’s body has the same DNA. Most DNA is located in
the cell nucleus (where it is called nuclear DNA), but a small amount of DNA can
also be found in the mitochondria (where it is called mitochondrial DNA or mtDNA).
The Swiss scientist Friedrich Miescher discovered DNA near the end of the
nineteenth century. After Miescher, other scientists tried to identify the chemical
composition of sperm, reasoning that sperm must carry the genetic material to the
next generation. These scientists also reasoned that sperm cells have very little excess
cellular material other than the hereditary material found in the sperm head.
In fact, DNA constitutes over 60 percent of the sperm head; the remainder is
mostly protein. For a long time after Miescher’s discovery, DNA was thought to be a
simple molecule, consisting of nucleotides strung together like beads on a string.By
the late 1940s biochemists knew that DNA was a very long polymer made up of
millions of nucleotides. Each nucleotide is composed of nitrogen containing
nucleobase cytosine (C), guanine (G), adenine (A), and thymine (T) as well as a
monosaccharide sugar called deoxyribose and a phosphate group. The nucleotides are
joined to one another in a chain by covalent bonds between the sugar of one
nucleotide and the phosphate of the next, resulting in an alternating sugar-phosphate
backbone. According to base pairing rules (A with T, and C with G), hydrogen bonds
bind the nitrogenous bases of the two separate polynucleotide strands to make
double-stranded DNA.
Nucleic acids composed of the three major macromolecules (sugar, nitrogen
base and phosphate groups) are essential for all known forms of life. Most DNA
molecules consist of two biopolymer strands coiled around each other to form a
double helix. The two DNA strands are known as polynucleotide1
.

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DNA structure
DNA exists as a double-stranded structure, with both strands coiled together to
form the characteristic double-helix. Each single strand of DNA is a chain of four
types of nucleotides. Nucleotides in DNA contain a deoxyribose sugar, a phosphate,
and a nucleobase. The four types of nucleotide correspond to the four nucleobases
adenine, cytosine, guanine, and thymine, commonly abbreviated as A, C, G and T.
Adenine and guanine are purine bases, while cytosine and thymine are pyrimidines.
These nucleotides form phosphodiester bonds, creating the phosphate-deoxyribose
backbone of the DNA double helix with the nucleobases pointing inward (i.e., toward
the opposing strand). Nucleobases are matched between strands through hydrogen
bonds to form base pairs. Adenine pairs with thymine (two hydrogen bonds), and
guanine pairs with cytosine (three hydrogen bonds)2
.
DNA strands have a directionality, and the different ends of a single strand are
called the "3′ (three-prime) end" and the "5′ (five-prime) end". By convention, if the
base sequence of a single strand of DNA is given, the left end of the sequence is the
5′ end, while the right end of the sequence is the 3′ end. The strands of the double
helix are anti-parallel with one being 5′ to 3′, and the opposite strand 3′ to 5′. These
terms refer to the carbon atom in deoxyribose to which the next phosphate in the
chain attaches. Directionality has consequences in DNA synthesis, because DNA
polymerase can synthesize DNA in only one direction by adding nucleotides to the 3′
end of a DNA strand.
The pairing of complementary bases in DNA (through hydrogen bonding)
means that the information contained within each strand is redundant. Phosphodiester
(intra-strand) bonds are stronger than hydrogen (inter-strand) bonds. This allows the
strands to be separated from one another. The nucleotides on a single strand can
therefore be used to reconstruct nucleotides on a newly synthesized partner strand3
.

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DNA polymerase
DNA polymerase I (Pol I) of Escherichia coli, the first DNA polymerase to be
discovered, has long served as a simple model system for studying the enzymology of
DNA synthesis. ~ The original studies of Pol I relied on purification of the enzyme
from E. coli extracts without genetic manipulation, yielding around 10 mg of purified
enzyme per kilogram of cell paste.
2 Cloning of polA, the structural gene for Pol I, in a variety of phage A vectors
increased the level of expression about 100-fold. 3"4 Sequence analysis of the cloned
polA gene 5 allowed construction of a plasmid-derived expression system for the
Klenow fragment portion of Pol I, 6 comprising the C-terminal two-thirds of the
protein and having the polymerase and 3' ~ 5' (proofreading) exonuclease functions
of the parent molecule, but lacking the 5'Y-exonuclease that is used in nick-
translation.
(Earlier attempts to express whole Pol I on a plasmid vector were unsuccessful
because of the lethality of wild-type polA in multiple copies, 3 and indicated the need
for more sophisticated vectors giving tight control of the level of expression.) The
ability to purify large quantities of Klenow fragment paved the way for the
determination of its structure by X-ray crystallography] In addition to their
importance as experimental systems in their own right, both Pol I and Klenow
fragment have found extensive use as biochemical reagents in a variety of cloning,
sequencing, and labeling procedures4
.

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DNA replication
DNA polymerase has 5’-3’ activity. All known DNA replication systems require
a free 3’ hydroxyl group before synthesis can be initiated (Important note: DNA is
read in 3’ to 5’ direction where as a new strand is synthesized in the 5’ to 3’
direction—this is often confused). Four distinct mechanisms for initiation of
synthesis are recognized. These are
i.All cellular life forms and many DNA viruses, phages and plasmids use a
primase to synthesize a short RNA primer with a free 3’ OH group which is
subsequently elongated by a DNA polymerase.
ii. The retro elements (including retroviruses) employ a transfer RNA that
primes DNA replication by providing a free 3′ OH that is used for elongation by the
reverse transcriptase.
iii. In the adenoviruses and the φ29 family of bacteriophages, the 3’ OH
group is provided by the side chain of an amino acid of the genome attached protein
(the terminal protein) to which nucleotides are added by the DNA polymerase to form
a new strand.
iv. In the single stranded DNA viruses- a group that includes the circo
viruses, the geminiviruses, the parvoviruses and others and the many phages and
plasmids that use the rolling circle replication (RCR) mechanism, the RCR
endonuclease creates a nick in the genome strand (single stranded viruses) or one of
the DNA strands (plasmids). The 5′ end of the nicked strand is transferred to a
tyrosine residue on the nuclease and the free 3′ OH group is then used by the DNA
polymerase to synthesize the new strand5
.

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Replication Fork
The replication fork is a structure that forms within the nucleus during DNA
replication. It is created by helicases, which break the hydrogen bonds holding the
two DNA strands together. The resulting structure has two branching “prongs”, each
one made up of a single strand of DNA. These two strands serve as the template for
the leading and lagging strands, which will be created as DNA polymerase matches
complementary nucleotides to the templates; the templates may be properly referred
to as the leading strand template and the lagging strand template. DNA is always
synthesized in the 5’ to 3’ direction. Since the leading and lagging strand templates
are oriented in opposite directions at the replication fork, a major issue is how to
achieve synthesis of nascent (new) lagging strand DNA, whose direction of synthesis
is opposite to the direction of the growing replication fork6
.
Lagging Strand
The lagging strand is the strand of nascent DNA whose direction of synthesis is
opposite to the direction of the growing replication fork. Because of its orientation,
replication of the lagging strand is more complicated as compared to that of the
leading strand. The lagging strand is synthesized in short, separated segments. On the
lagging strand template, a primase “reads” the template DNA and initiates synthesis
of a short complementary RNA primer. A DNA polymerase extends the primed
segments, forming Okazaki fragments. The RNA primers are then removed and
replaced with DNA, and the fragments of DNA are joined together by DNA ligase.
DNA polymerase III (in prokaryotes) or Pol δ (in eukaryotes) is responsible for
extension of the primers added during replication of the lagging strand. Primer
removal is performed by DNA polymerase I (in prokaryotes) and Pol δ (in
eukaryotes). Eukaryotic primase is intrinsic to Pol α. In eukaryotes, pol ε helps with
repair during DNA replication.
Clamp proteins form a sliding clamp around DNA, helping the DNA polymerase
maintain contact with its template, thereby assisting with processivity. The inner face
of the clamp enables DNA to be threaded through it. Once the polymerase reaches the
end of the template or detects double-stranded DNA, the sliding clamp undergoes a
conformational change that releases the DNA polymerase. Clamp-loading proteins
are used to initially load the clamp, recognizing the junction between template and
RNA primers7
.

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Enzymes with their likely roles at the eukaryotic replication fork
Enzyme Role
DNA helicase unwinding of DNA
DNA primase synthesis of primers
DNA polymerases synthesis of the DNA strands
Ribonuclease H
removal of primers
5’ : 3’ exonuclease
DNA ligase ligation of DNA pieces
3’ : 5’ exonuclcase proof-reading for the DNA pol ymerases
DNA topoisomcrasc 1 conversion of topological DNA isomers
DNA topoisomcrase 2 segregation of DNA strands8
PCR
PCR is a simple and widely used process in which minute amounts of DNA can
be amplified into multiple copies. In addition to working rapidly, it is able to
quantitatively demonstrate how much of a particular sequence is present. As
with all methods, the validity of the results should be compared with the
specificity associated with the method. The future of PCR is promising, combining
various assays and approaches to produce greater insight into various gene
combinations.
For example, in a study to link distinct taxa within the microbial community to
specific metabolic processes, stable isotope probing was combined with qPCR
(Postollec et al., 2011; Smith and Osborn, 2009). Microarray experiments can be
validated by qPCR approaches because of the method’s rapidity.
DNA Damaging Agents
Another major strategy in chemotherapy is to use DNA damaging agents to
inhibit processive DNA polymerases. Since DNA damaging agents are very
electrophilic, they effectively react with nucleophilic moieties on DNA to
significantly modify the hydrogen-bonding potential and structure of nucleic acid. In
most instances, the formed DNA lesion acts as a physical barrier and hinders the
movement of a DNA polymerase to inhibit DNA synthesis. In other cases, the change
in hydrogen-bonding information on DNA tends to increase the frequency of
misincorporation events to subsequently enhance the occurrence of pro-mutagenic
DNA synthesis. The mismatches that are formed become excellent substrates for
enzymes involved in various DNA repair pathways which can either correct the
damaged DNA or cause cell death. The cellular effects of temozolomide (TMZ), a

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monofunctional alkylating agent, represent an excellent example of this phenomenon.
TMZ produces cytostatic and cytotoxic effects primarily through the non-enzymatic
methylation of DNA. Specifically, TMZ creates a number of DNA lesions including
N3-methyladenine, O6-methylguanine, and N7-methylguanine, the most commonly
formed DNA adduct (Gates et al., 2004). Methylation at the N7 position of guanine
produces a more toxic DNA lesion, termed an abasic site, which forms by the
spontaneous depurination of the methylated base (Friedman et al., 2000). Since
abasic sites lack Watson-Crick coding information, they are classified as non-
instructional DNA lesions and typically inhibit the synthetic activity of most high-
fidelity DNA polymerases (Shcherbakova et al., 2003). In contrast, alkylation of the
O6 position of guanine changes its hydrogen-bonding potential which increases the
frequency of misincorporation events (Woodside and Guengerich, 2002). The
resulting mispair that results from the misincorporation of dTMP opposite O6-
methylguanine activates the MMR pathway to ultimately induce apoptosis (Koç et
al., 1996)9
.
Combination Therapies
There is substantial clinical evidence supporting a strategy for combining
nucleoside analogs with DNA damaging agents. As indicated earlier, gemcitabine is
frequently combined with platinum drugs such as cisplatin and oxaliplatin to treat
ovarian and pancreatic cancer (Hoff and Fuchs, 2003; Ozols, 2005; Sehouli, 2005;
Chua and Cunningham, 2006). Several pre-clinical studies have examined the
underlying mechanism for how gemcitabine synergizes the cytotoxic effects of
platinum-based drugs. Using the ovarian cancer cell line, A2780, as a model, Jensen
et al. showed that gemcitabine combined with cisplatin caused an increase in the
amount of platinum-DNA adducts compared to cisplatin treatment alone (Jensen et
al., 1997). The higher number of DNA adducts appeared to result from a decrease in
DNA repair that was caused by the inhibition of cellular exonucleases such as
excision repair cross-complementation group 1 (ERCC1). However, other models
such as the inhibition of specialized DNA polymerases by gemcitabine have also
been invoked (Chen et al., 2008). This model is based on evidence showing that pol
η-deficient cells are more sensitive to the combination of gemcitabine and cisplatin
compared to normal fibroblast that are pol η-proficient. In addition, pol η-deficient
cells are ~10-fold more sensitive to the combined treatment of gemcitabine and
cisplatin compared to treatment with cisplatin alone10
.

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References
1
Tariku Simion, DNA Replication, Southern Agricultural Research Institute,
Arbaminch Agricultural Research Center, Arba Minch, Ethiopia, Published:
September 18, 2018 at research gate.
2
Watson JD, Crick FH. Molecular structure of nucleic acids; a structure for
deoxyribose nucleic acid. Nature. 1953;171(4356):737–738.
3
Alberts, B., et al., Molecular Biology of the Cell, Garland Science, 4th ed., 2002
4
(Methods in Enzymology 262) Campbell J. (ed.), Abelson J. (ed.), Simon M.I. (ed.) -
DNA Replication-Elsevier, Academic Press (1995).
5
Tariku Simion, DNA Replication, Southern Agricultural Research Institute,
Arbaminch Agricultural Research Center, Arba Minch, Ethiopia, Published:
September 18, 2018 at research gate.
6
Sabatinos, S. A. (2010) Replication Fork Stalling and the Fork Protection Complex.
Nature Education 3(9):40
7
Elizabeth R Barry, Stephen D Bell (2006) DNA Replication in the Archaea. Microbiol
Mol Biol Rev 70(4): 876-887.
8
Pia THOMMES and Ulrich HUBSCHER, Eukaryotic DNA replication page 700, FEBS
Journal, John Wiley and Sons, 1990.
9
Polymerase Chain Reaction , Lilit Garibyan1 and Nidhi Avashia, Journal of
Investigative Dermatology (2013) 133, e6. doi:10.1038/jid.2013.1
10
Anthony J. Berdis, Inhibiting DNA Polymerases as a Therapeutic Intervention
against Cancer, REVIEW ARTICLE, Front. Mol. Biosci., 21 November 2017.