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Applications of Molecular genetics in
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Chapter: DNA Replication
Edited by: Korkut Ulucan
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DNA Replication
Vasiliki E Kalodimou*
Lab Supervisor of Flow Cytometry – Research & Regenerative
Medicine Department, IASO-Maternity Hospital, MedStem ServicesCryobanks International Kifisias Ave.37-39, 151 25, Marousi, Athens,
Greece
*Corresponding author: Vasiliki E Kalodimou, Lab Supervisor of Flow
Cytometry – Research & Regenerative Medicine Department, IASOMaternity Hospital, MedStem Services-Cryobanks International Kifisias
Ave.37-39, 151 25, Marousi, Athens, Greece, Tel: 00306956205250;
E-mail: [email protected].
Abstract
When all cells go through prior to any type of cell division a process called DNA replication takes place. During DNA replication
the cells replicate their DNA in two identical copies from the original one. As a result of this process the daughter cells generated
from cell division will have a complete copy of the DNA necessary for their survival.
The two strands of DNA will be separated during the process of DNA replication and each one will be the template for a new
strand synthesis. Remember that each new forming strand will be built only one nucleotide at the time.
DNA Replication
When Watson and Crick discovered the double helix structure of DNA, there was one more remarkable aspect that they
recognized immediately. The structure explained how DNA could be copied, or replicated. Replication Occurs in S phase of cell
cycle [1] (Figure 1).
Figure 1: The DNA in the cell cycle. M = Mitosis Phase; G1 = Gap 1 Phase; G2 = Gap 2 Phase; S = Synthesis Phase.
The mechanism of base pairing take place when each strand of the DNA has all the information needed to reconstruct the other
half and the strands referred to as complementary strands (refer to chapter 2.1.1). Important note is that the two strands could be
separated because of the base pairing rules and the result is the reconstruction of the base sequence of the other strand.
The starting point during DNA replication is a single point of the chromosome and then continues until chromosome
replication in two directions and will be stopped when the chromosome is completely copied. Important point to remember is that
in larger chromosomes the DNA replication takes place at hundreds of places [2,3].Replication forks are called the points where the
replication and separation of the chromosome is occurred (Figure 2).
The transmission of hereditary information depends on accurate replication of the genetic material [4].
During DNA replication, the DNA molecule separates into two strands, and then produces two new complementary strands
following the rules of base pairing [5]. Each strand of the double helix of DNA serves as a template for the new strand and each
molecule will have one original strand and one new strand (Figure 3).
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A process is called replication when a cell duplicates its DNA before divide its self and with this process we ensure that we will
get complete set of DNA molecules in each cell.
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Figure 2: DNA replication. When the two strands are separated we get the formation of the two forks and new bases are added to each
of them (base pairing).
Figure 3: DNA Replication.
The DNA molecule is unzipped by enzymes that carried out the of DNA replication process when the hydrogen bonds between the
base pairs are broken and the two strands of the molecule unwind. Important to remember is that each strand is the template for the
attachment of complementary bases.
Watson and Crick described three different stages of DNA replication: semiconservative, conservative, and dispersive (Figure 4). In
the figure the lines represent the sugar-phosphate backbones with a random base pair sequence. As we mention above each strand, by
unzipping, will get a single base and the two single strands will be acting as a template and begin to reform an identical double helix as
the unzipped one [4].
In semiconservative replication, each daughter duplex contains one parental and one newly synthesized strand whereas in conservative
replication one daughter duplex consists of two newly synthesized strands, and the parent duplex is conserved. At the end, the dispersive
replication results in daughter duplexes that consist of strands containing only segments of parental DNA and newly synthesized DNA.
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The main enzyme involved called PAHL-ih-mur-ayz and its role is to produce a DNA molecule by join individual nucleotides.
Because of its unique role the PAHL-ih-mur-ayz enzyme is also a polymer.
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Figure 4: The three alternative patterns for DNA replication. In here all newly added nucleotides in the cell are coming from a free
nucleotide pool and each daughter molecule contain one newly synthesized nucleotide chain and one parental.
Matthew Meselson and Franklin Stahl in 1958 they performed the following experiment:
They used a heavy isotope of nitrogen 15N medium in order to grow E-coli cells by inserting it into the nitrogen bases. After many 15N
divisions the cell DNA was labeled and they removed the cells from 15N medium and put them into a 14N medium for two more divisions
before they collect them. A Cesium Chloride (CsCl) solution was used for all samples after DNA extraction. The result of this experiment
was the formation of a single band of intermediate density between the densities of the heavy and light controls while the formation of
two bands was the result of two generations in 14N (Figure 5).
Figure 5: Matthew Meselson and Franklin Stahl experiment.
Figure 6: In semiconservative stage the results are like the result in the first generation of a single intermediate band while in the second generation
are one intermediate and one light band.
According to Watson - Crick during replication of the DNA a replication fork will be found. This hypothesis was tested by John
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This result would be expected to from the semiconservative model of replication. The result is compatible with only this mode if the
experiment begins with chromosomes composed of individual double helices (Figure 6).
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Cairns in 1963. His experiment was to allow DNA replication in bacteria with titrated thymidine. By his theory each newly synthesized
daughter molecule expected to contain one hot radioactive strand and one cold nonradioactive strand. Cairns extracted and examine
the cell’s DNA after many intervals and numbers of replication cycles in hot medium.
After one cycle of replication in [3H] thymidine he observed rings of dots in the autoradiograph (Figure 7).
Figure 7: John Cairns experiment. Remember that one of the two strands should be radioactive according to the semiconservative stage of replication.
In the second replication cycle, the forks predicted by the model were also could be seen. The structures called theta (θ) structures
(Figure 8).
Figure 8: The newly replicated double helix consist of two radioactive strands in theta (θ) structure.
The replication of some circular molecules such as plasmids and certain viruses, occurring by a mechanism called rolling-circle
replication: a nuclease cut provides a free 3’-OH end where nucleotides are added. As the synthesis proceeds the other end of the
template strand is displaced from the double-stranded circle and copied. There is no termination point and the synthesis often continues
beyond a single circle unit, producing a series of linked chains of several circle lengths called concatamers, and then by recombination
processed to yield normal-length circles [6] (Figure 9).
Figure 9: Rolling-circle replication.
The DNA replication is very complex mechanism and requires the participation of many different components.
The reaction works only with the triphosphate forms of the nucleotides. The total amount of DNA at the end of the reaction can be as
much as 20 times the amount of original input DNA, therefore most of the DNA present at the end must be progeny DNA (Figure 10).
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DNA polymerase was first successfully identified in 1950s by Arthur Kornberg. He was the first to purify the DNA polymerase in
E-coli, an enzyme that catalyzes the replication reaction.
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Figure 10: Chain-elongation reaction catalyzed by DNA polymerase.
We have three DNA polymerases in E-coli:
DNA polymerase I (pol I): Pol I has a polymerase activity (5’ → 3’) that catalyzes chain growth, exonuclease activity (3’ → 5’) that
removes mismatch bases and an exonuclease activity (5’ → 3’ ) that degrades double-stranded DNA.
DNA polymerase II (pol II): which could repair damage DNA and DNA polymerase III (pol III) which has an important role in E-coli
DNA replication along with pol I.
The most important polypeptide, from at least 20, subunits in Pol III are: alpha (α), epsilon (ε), and theta (θ). The epsilon must
always be bound to alpha. In the absence of epsilon in a strain the result is a functional epsilon with higher mutation rate. The Pol III will
complete the replication with a short segment of duplex that is already present called primer.
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At a prokaryotic stage replication in E-coli starts at a fixed point and proceeds with bidirectional ending to a site named terminus.
The unique origin is termed oriC, is 245 bp long and has several components. There is a side-by-side set of 13-bp sequences, which are
nearly identical and also there is a set of binding sites of a protein called DNA A protein. The consequences of bidirectional replication
can be seen in Figure 11.
Figure 11: Bidirectional replication of a circular DNA molecule with a larger view of DNA replication.
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In eukaryote stage the process of replication proceeds from multiple points and not at a fixed point as mentioned before. We could
better understand that by an experiment when a eukaryotic cell is exposed to [3H] thymidine for a short time (pulse exposure step) and
then provide an excess of unlabeled thymidine (chase step).
DNA polymerases can extend a chain but cannot start a chain in priming DNA synthesis, therefore must first be initiated with a
primer or a short oligonucleotide that generates a segment of duplex DNA.
The primase (30bp long short RNA stretch) enzyme synthesizes the RNA primers. The RNA primers could also be synthesized by
RNA polymerase. Notice that here the chain, (because of its short stretch of RNA), will be extended with DNA by DNA polymerase.
The complex termed primosome, (at E-coli), is the result of a complex formation between primase and DNA template with additional
proteins.
Only in the 5’ → 3’ direction and moving in a 3’ → 5’ direction the DNA polymerases synthesize new chains, (Figure 10), resulting in
a new strand called leading strand. This strand is synthesized continuously while the lagging strand synthesized in short, discontinuous
segments. The nucleotides along the template for the lagging strand are added toward the template’s 5’ end; therefore, the new strand
must grow in an opposite direction of the replication fork movement. A new lagging-strand fragment is begun and proceeds away from
the fork as the fork movement exposes a new section of the template and the process is stopped by the preceding fragment.
DNA polymerase III (Pol III) carries out most of the DNA synthesis on both strands while pol I fill in the gaps left in the lagging
strand. DNA ligase enzyme seals the process and also joins broken DNA pieces by catalyzing the formation of a phosphodiester bond (5’
phosphate end of a hydrogen-bonded nucleotide → 3’ OH group)
In step ‘a’ the primers are synthesized by primase for the discontinuous synthesis on the lagging strand while in step ‘b’ are extended
by DNA polymerase to yield DNA fragments the Okazaki fragments. In step ‘c’ the 5’ → 3’ exonuclease activity in pol I remove the primers
and fills in the gaps with DNA and in step d are sealed by DNA ligase.
During the process of replication we come across with a problem from the ends of chromosomes at the leading strand. This is
happening because the polynucleotide it is automatically addition primed from behind and always extended to the end. When the lagging
strand reaches a point where its system of RNA priming cannot work the result is a short chromosome with an unpolymerized section.
To solve this problem we get adjacent repeats of simple DNA sequences by the tips of chromosomes, called telomeres [7].
The enzymes in a duple helix that disrupt the hydrogen bonds that hold the two DNA strands together called Helicases. The ATP
hydrolysis drives the reaction.
The DNAB protein and the Rep protein are among E-coli helicases. The Rep protein helps to unwind the double helix ahead of the
polymerase.
The helicases action during DNA replication generates twists in the circular DNA. This twist needed to be removed in order to allow
replication to continue. Those circular DNA can be twisted and coiled.
Topoisomerases are called the enzymes which help the supercoiling to relaxed or created and also these enzymes in a chain can
remove or induce knots and also linked [7,8].
Summary
• Replication is achieved with the aid of several enzymes, including DNA polymerase, gyrase, and helicase.
• Replication starts at special regions of the DNA called origins of replication and proceeds down the DNA in both directions.
• Copying genetic information for transmission to the next generation.
• Occurs in S phase of cell cycle.
• Process of DNA duplicating itself.
• Begins with the unwinding of the double helix to expose the bases in each strand of DNA.
• Each unpaired nucleotide will attract a complementary nucleotide from the medium will form base pairing via hydrogen bonding.
• Enzymes link the aligned nucleotides by phosphodiester bonds to form a continuous strand.
References
1. Kalodimou EV (2013) Basic Principles in Flow Cytometry. AABB Press, Bethesda Md, USA.
2. Rhind N, Russell P (1998) Curr Opin Cell Biol 10: 749-758.
3. Woynarowski JM, Beerman TA (1997) Effects of bizelesin (U-77,779), a bifunctional alkylating minor groove binder, on replication of genomic and simian
virus 40 DNA in BSC-1 cells. Biochim Biophys Acta 1353: 50-60.
4. Weinberger M, Trabold PA, Lu M, Sharma K, Huberman JA, et al. (1999) Induction by adozelesin and hydroxyurea of origin recognition complex-dependent
DNA damage and DNA replication checkpoints in Saccharomyces cerevisiae. J Biol Chem 274: 35975-35984.
5. Chen FM, Sha F, Chin KH, Chou SH (2003) Unique actinomycin D binding to self-complementary d(CXYGGCCY’X’G) sequences: duplex disruption and
binding to a nominally base-paired hairpin. Nucleic Acids Res 31: 4238-4246.
7. Hsiang YH, Liu LF (1988) Identification of mammalian DNA topoisomerase I as an intracellular target of the anticancer drug camptothecin. Cancer Res 48:
1722-1726.
8. Berger JM, Gamblin SJ, Harrison SC, Wang JC (1996) Structure and mechanism of DNA topoisomerase II. Nature 379: 225-232.
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6. Adams A, Guss JM, Collyer CA, Denny WA, Wakelin LP (2000) A novel form of intercalation involving four DNA duplexes in an acridine-4-carboxamide
complex of d(CGTACG)(2).Nucleic Acids Res 28: 4244-4253.
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