DNA Damage

Mutations arise not only from the errors in the replication but also from damage to the DNA. Some damage is caused by environmental factors, such as radiation and chemicals like mutagens, which are chemical agents that increase the rate of mutation. DNA also undergoes spontaneous damage from the action of water.

Mechanisms which causes DNA Damage include:

  1. Hydrolysis (Deamination and Depurination)
  2. Alkylation
  3. Oxidation
  4. Radiation Ractions
  5. Base analogue and intercalating agents.

1. Hydrolysis

The most frequent and important kind of hydrolytic damage is deamination of the base cytosin. Deamination is the removal of an amine group from a molecule. Enzymes that catalyse this reaction are called deaminases.

  • The deamination of cytosin to uracil happens at a significant rate in cells. Deamination can be repaired by a specific repair process which detects uracil, not normally present in DNA; otherwise the U will cause A to be inserted opposite it and cause a C:G to T:A transition when the DNA is replicated.
  • Deamination converts adenine to hypoxanthine, which hydrogen bonds to cytosine rather than to thymine; guanine is converted in to xanthine, which continues to pair with cytosin, though with only two hydrogen bonds.
  • Deamination of 5-methylcytosine to thymine also occur. 5- Methylcytosine occurs in the human genome at the sequence 5’CpG3′, which normally avoided in the coding regions of genes. if the meC is deaminated to T, there is no repair system which can recognise and remove it (becase T is normal base in DNA). This means that whatever CpG occurs in genes it is a “hotspot” for mutation.

Deamination of Bases

DNA also undergoes depurination by spontaneous hydrolysis of the N-glycosyl linkage, and this produces an abasic site (that is deoxyribose lacking a base) in the DNA.

Depurination in DNA is a chemical reaction of purine deoxyribonucleosides, deoxyadenosine and deoxyguanosine, in which the β-N-glycosidic bond is hydrolytically cleaved releasing a nucleic base, adenine or guanine

depurination

2. Alkylation

Alkylation is the transfer of an alkyl group from one molecule to another. In alkylation, methyl orethyl groups are transferred to reactive sites on the bases and to phosphates in DNA backbone.

Alkylations do not lead immediately to mispairing. However, they do make the bond between sugar and base more labile, or more apt to break. When this break occurs, it leaves an apurinic site, a sugar without its purine. This obviously cannot be replicated properly unless it is first repaired, but cells sometimes attempt to replicate apurinic DNA anyway. If they do, they frequently insert the wrong base across from an apurinic site, and this generates a mutation. Alkylation can also enhance the tendency of a base to form the rare (wrong) tautomer, which changes the base-pairing properties, leading to mutations. Moreover, all of the nitrogen and oxygen atoms involved in base-pairing are also subject to alkylation, which can directly disrupt base-pairing and lead to mutation.

fig2

Many environmental carcinogens, or cancer-causing agents, are electrophiles that act by attacking DNA and alkylating it. Many of the favorite mutagens used in the laboratory for the express purpose of creating mutations are also alkylating agents. One example is ethylmethane sulfonate (EMS), which transfers ethyl (CH3-CH2) groups to DNA. The product of this methylation, O6-rthylgaunine, often mispaires with thymine, resulting in the change of G:C base pair into an A:T base pair when the damaged DNA is repliacted.

3. Oxidation

DNA oxidation is the process of oxidative damage on Deoxyribonucleic Acid. It occurs most readily at guanine residues due to the high oxidation potential of this base relative to cytosine, thymine, and adenine. Reactive oxygen species (O2-, H2O2, and OH) are generated by ionizing radiation and by chemicals agents that generate free radicals. An important oxidation product is 8-hydroxyguanine, which mispairs with adenine, resulting in G:C to T:A transversions.

fig3

4. Radiation Ractions

Ultraviolet radiation (UV radiation) cross-links adjacent pyrimidines on the same DNA strand, forming pyrimidine dimers, usually thymine dimers. Figure shows the structure of a thymine dimer and illustrates how it interrupts base-pairing between the two DNA strands. These dimers block DNA replication because the replication machinery cannot tell which bases to insert opposite the dimer. As we will see, replication sometimes proceeds anyway, and bases are inserted at random. If these are the wrong bases, a mutation results.

fig3

Thymine dimers. (a) Ultraviolet light cross-links the two thymine bases on the top strand. This distorts the DNA so that these two bases no longer pair with their adenine partners. (b) The two bonds joining the two thymines form a four-membered cyclobutane ring (red).

Ultraviolet radiation has great biological significance; it is present in sunlight, so most forms of life are exposed to it to some extent. The mutagenicity of UV radiation explains why sunlight can cause skin cancer: Its UV component damages the DNA in skin cells, which leads to mutations that sometimes cause those cells to lose control over their division.

Damage by Gamma and X -rays:

The much more energetic gamma rays and X rays, like ultraviolet rays, can interact directly with the DNA molecule. However, they cause most of their damage by ionizing the molecules, especially water, surrounding the DNA. This forms free radicals, chemical substances with an unpaired electron. These free radicals, especially those containing oxygen, are extremely reactive, and they immediately attack neighboring molecules. When such a free radical attacks a DNA molecule, it can change a base, but it frequently causes a single- or double-stranded break. Single-stranded breaks are ordinarily not serious because they are easily repaired, just by rejoining the ends of the severed strand, but double-stranded breaks are very difficult to repair properly, so they frequently cause a lasting mutation. Because ionizing radiation can break chromosomes, it is referred to not only as a mutagen, or mutation-causing substance, but also as a clastogen, which means “breaker.”

5. Base analogue and intercalating agents.

Mutations ae al;so caused by compounds that substitue for normal bases (base analogs) or slip between the bases (intercalating agents) to cause errors in replication.

Base analogs are structurally similar to proper bases but differ in ways that make them treacherous to the cell. Thus base analogs are similar enough to the proper base to get taken by cells, converted in to nucleoside triphosphates, and incoporated into DNA during replication. But, because of the structural difference between these analogues and the proper bases, the analogues base-pair inaccurately, leading to frequent mistakes during the replication process.

one of the most mutagenic base analogs is 5-bromouracil, an analog of thymine. The presence of bromo substituent allows the base to mispair with guanine via enol tautomer.

5-bromouracil (5-BU) ia a derivative of uracil and behaves as a thymine analog, which increases the probability of a tautomeric shift from the normal keto form to the enol form, mis-pairing with guanine instead of adenine. After one round of replication, an A - T to G - C transition mutation results.

5-bromouracil (5-BU) ia a derivative of uracil and behaves as a thymine analog, which increases the probability of a tautomeric shift from the normal keto form to the enol form, mis-pairing with guanine instead of adenine. After one round of replication, an A – T to G – C transition mutation results.

Intercalating agents are flat molecules containing several polycyclic rings that bind to the equally flat purine or pyrimidine bases of DNA. Intercalating agents are compounds, such as proflavin, acridine and ethidium,  that can bind to the major and minor grooves of DNA and cause addition or deletion of bases during replication. They may result in a frameshift mutation, which can alter the codon reading frame and result in aberrant DNA transcription and replication.

mutation-intercal-2441

The Replicon Model of Replication Initiation

The initial replication fork requires the separation of the two strands of the DNA duplex to provide a template for the synthesis of both the RNA primer and new DNA. Specific genomic DNA sequence direct the initiation of DNA Replication. The specific sites at which DNA unwinding and initiation of replication occur are called Origin of Replication.

The Replicon Model of Replication Initiation:

In 963 Frabcois Jacob, Sydney Brenner and Jacques Cuzin proposed a model to explain the events controlling the initiation of replication in bacteria. It proposed two components that control the initiation of replication :

  • Replicator: Replicator is defined as the entire set of cis-acting DNA sequences that is sufficient to direct the initiation of Replication.
  • Initiator: Initiator is the protein which specifically recognizes a DNA elements in the replicator and activates the initiation of replication. The initiator protein is the only sequence specific DNA-binding protein involved in the initiation of replication.

The model proposed that all the DNA replicated from a particular origin as a replicon. In prokaryotes since there is only one origin of replication at the single chromosome, the entire chromosome is a single replicon. In contrast the presence of multiple origins of replication divides each eukaryotic chromosomes into multiple replicons.

Replicator:

The origin of replication is the site on DNA where the DNA is unwound and DNA synthesis initiates. Although the origin of replication is always part of the replicator, sometimes the origin of replication is only a fraction of the replicator.

DNA Sequences of Replicator Share two common features:

  • First, they include a binding site for the initiator protein.
  • They include a stretch of AT-rich DNA that unwinds readily but not spontaneously. Unwinding of DNA at replicators is controlled by the replication Initiation Proteins.

The single replicator required for E-Coli chromosomal replication is called oriC. There are two repeated motif that are critical for oriC function. The 9-mer motif is the binding site for the E-Coli initiator, DnaA, and is repeated four times at oriC. The 13-mer motif, repeated 3 times, is the initial site of ssDNA formation during initiation.

Initiator:

Initiator proteins typically perform 3 different functions:

  1. Binding to the replicator – Bind a specific sequence DNA sequence within the replicator.
  2. Unwind DNA – once bound to the DNA, they frequently distort or unwind a region of DNA adjacent to the site of binding.
  3. Recruiting other replication proteins – Initiator proteins interact with additional factors required for replication initiation, thus recruiting them to the replicator.

In Prokaryotes, for example, the E-coli initiator, DnaA binds the repeated 9-mer elements in oriC and is regulated by ATP. When bound to ATP, DnaA also interacts with DNA in the region of the repeated 13-mer repeats of oriC. These additional interactions result in the separation of the DNA strands over more than 20bp within the 13-mer repeat region. this unwound DNA provides an ssDNA template for additional replication proteins such ad DNA helicase to begin the RNA and DNA synthesis steps of replication.

In Eukaryotes, the initiator is known as the origin recognition complex (ORC). It is a complex formed of 6 proteins. Like DnaA in E-Coli, ORC binds and hydrolyze ATP. ATP is required for sequence-specific DNA binding at the origin. Unlike DNA binding ORC at the replicator does not separate the DNA strands. ORC is , however, required to recruit all the remaining replication proteins to the replicator before it unwinds. Unwinding the strands requires the origin to get activated. Origin activation occurs only when the cell enters S phase of cell cycle.

Unlike prokaryotic initiator, ORC of eukaryotes have two functions:

  1. Binding to the replicator
  2. Recruiting other replication proteins

once the initiator binds to the replicator, the remaining steps in the initiation of replication is largely driven by the protein-protein interactions and protein-DNA interactions that are sequence independent. The end result is the assembly of two replication fork.

Mismatch Repair System

The replication machinery achieves remarkably high degree of accuracy using a proofreading mechanism. Proofreading improves the fidelity of DNA replication by a factor about 100 . The proof reading exonuclease is not, however, foolproof. Some mis incorporated nucleotides escape detection and become a mismatch between the newly synthesized strand and the template strand.

If the misincoporated nucleotide in the first round of replication is not subsequently detected and replaced, the sequence change will become permanent in the genome in the second round of replication.  At this point the mismatch will no longer exist; instead it will have resulted in a permanent change in the DNA sequence.

Fortunately, a mechanism exists for detecting the mismatches and repairing them, known as Mismatch Repair System. This system increases the accuracy of DNA synthesis by an additional two to three orders of magnitude.

Aims of Mismatch repair system are as follows:

  • Quick response : It must scan the genome for mismatches. Because mismatches are transient (they are eliminated in the second of replication) the mismatch repair system must find and repair mismatches.
  • Accuracy: The system must correct the mismatch accurately. that is it must replace the misincoporated nucleotides in the newly synthesized strand and not the correct nuceotide in the parent strand.

Mismatch Repair Pathway in Prokaryotes:

  1. In E-Coli mismatches are detected by a dimer of the mismatch repair protein MutS. MutS scan the DNA, recognizing the mismatches from the distortion they cause in the DNA backbone.
  2. MutS embraces the the mismatch-containing DNA , inducing a kink in the DNA and a conformational change in MutS itself.
  3. This complex of MutS and mismatch containing DNA recruits MutL, a second component of mismatch repair system .
  4. MutL, in turn, activates the mutH, an enzyme that causes incision or nick on one strand near the site of the mismatch.
  5. Nicking is followed by the action of specific helicase and one of three exonucleases. The helicase unwind the DNA, starting from the incision and moving in the direction of the site of the mismatch, and exonucleases progressively digest the displaced single strand, extending to and beyond the site of the mismatched nucleotide.
  6. This action produces a single-stranded gap, which is then filled by DNA polymerase III and sealed with DNA Ligase.

  • How does the mismatch repair system know which of the two mismatched nucleotides to replace?

The E-Coli enzyme Dam methylase methylates A residue on both strands of the sequence 5′-GATC-3′. The GATC sequence is widely distributed along entire genome , and all of these sites are methylated by the Dam methylase. When replication fork passes through DNA that is methylated at GATC sites on both strands , the resulting daughter DNA duplex will have hemimethylated (that is methylated on only parent strand). Thus for few minutes, until the Dam mathylase catches up and methylates the newly synthesized strand, daughter DNA duplex will be methylated only on the strand that served as the template. Thus the newly synthesized strand lacks methyl group and are so marked and hence can be recognized as the strand for repair.

The MutH proteins binds at such hemimethylated sites, but its endonuclease  activity is normally latent. Only when it is contacted by MutL and LutS located at a nearby mismatch does MutH become activated. Once activated, MutH selectively nicks the unmethylated strand.

Mismatch repair system in Eukaryotes:

Eukaryotes cells also repair mismatches and do so using homologs to MutS (Called MSH proteins for MutS homologs) and MutL (called MLH and PMS). Indeed, eukaryotes have multiple MutS-like proteins with different specificities.

Even though eukaryotic cells have MMR, mismatch repair systems, they lack MutH and E-coli’s clever trick of using hemimethylation to tag the parent strand.

Following are the detailed view how Eukaryotic Mismatch repair system works.

All eukaryotic organisms, including yeast, mouse and human, have MutS homologs (MSHs) and MutL homologs (MLHs). The eukaryotic MMR system has been well conserved during the evolutionary process. However, in contrast to MutS and MutL in bacteria, which function as homodimers, in eukaryotes MSHs and MLHs form heterodimers with multiple proteins. Five highly conserved MSHs (MSH2 to MSH6) are present in both yeast and mammals.

MSH1, which is present in mitochondria, exists only in yeast . MSH4 and MSH5 show reproductive tissue- specific expression, and null mutations of these genes do not confer mutator phenotypes because they are involved in meiotic recombination but not postreplication repair. Genetic and biochemical studies have indicated that MSH2 is required for all mismatch correction in nuclear DNA, whereas MSH3 and MSH6 are required for the repair of some distinct and overlapping types of mismatched DNA during replication. These three MutS homologs make two heterodimers: MutSa (MSH2⁄MSH6) and MutSb (MSH2⁄ MSH3). The former plays the major role in recognition of mismatched DNA in eukaryotic MMR. That is, MutSa functions in the repair of base–base mispairs as well as a range of insertion ⁄ deletion loop mispairs, whereas MutSb primarily functions in the repair of insertion ⁄ deletion loop mispairs.

MutL homologs in eukaryotic organisms were identified as genes whose amino acid sequences showed high similarity with prokaryotic MutL proteins, or whose mutation phenotypes were increased levels of postmeiotic segregation (PMS) that resulted from a failure to repair mismatches in meiotic recombination intermediates. There are four homologs of MutL in both yeast and mammals. In a genetic analysis, defects in MLH1 and PMS1 in yeast resulted in more severe mutator phenotypes, reminiscent of those of MSH2 and MSH6, than defects in the two other MutL homologs. Also, MLH1 interacted with the other three MutL homologs in a yeast two-hybrid analysis. Overall, yeast MLH1⁄PMS1 and mammalian MLH1⁄PMS2 heterodimers (each known as MutLa) play a major role in mutation avoidance, and the other two heterodimers of MutL homologs take part in the repair of specific classes of mismatches. The biochemical activities and structure of MutL homologs are closely related to those of prokaryotic MutL proteins, especially in the N-terminal domain. The X-ray crystallographic structure of the conserved N-terminal 40-kDa fragment of human PMS2 resembles that of the ATPase fragment of E. coli MutL.

Extensive genetic studies in yeast have failed to find orthologs of MutH and UvrD in the MMR system, and there may be no homolog of these two proteins in the eukaryotic genome. Therefore, some divergence in the MMR system from strand discrimination and the nicking process might occur between prokaryotes and eukaryotes.

A recent increase in our knowledge of the eukaryotic MMR system provides some understanding of this divergence. In mammalian cell extracts, mismatches provoke initiation of excision at pre-existing nicks in exogenous DNA substrates with high efficiency and specificity. The molecular nature of eukaryotic MMR could be assessed using cell extract assays in vitro, and components of the eukaryotic MMR system have been identified with depletion and complementation assays using cell extracts. One protein, identified in this way, is proliferating cell nuclear antigen (PCNA). PCNA is known to function as a processivity factor for replicative polymerase, but some mutations in the PCNA gene result in mutator phenotypes, and its interactions with MSH2 and MLH1, and with MSH6, suggest that it functions in MMR. PCNA has biochemical activity that increases the binding of MutSa to mismatched DNA; the interactions between PCNA and MSH6 are essential for this biochemical activity, which suggests that PCNA might play a role in MMR at the mispair recognition stage. PCNAhas been proposed to function in the mismatch recognition stage of MMR by helping MutSa search for mismatched DNA or increasing the mismatchbinding specificity of MutSa. One intriguing point about the role of PCNA in eukaryotic MMR is that the requirement for PCNA depends on the direction of the nick in the in vitro MMR assay. Although PCNA is required for mismatch-provoked excision directed by a 3’ strand break in HeLa nuclear extracts, it is not essential for excision directed by a 5’ nick. Moreover, whereas 3’ nick-directed excision iscompletely abolished by the inhibition of PCNA, 5’ nick-directed excision is affected only minimally.

Finally, a mismatch-provoked 5’ to 3’ excision reactioncan be reconstituted in a purified system that comprises only MutSa, MutLa, ExoI and replication protein A (RPA), without PCNA, and the process is similar to that observed in nuclear extracts. RPA, the eukaryotic single-stranded DNA-binding protein, has been shown to enhance excision and stabilize excision intermediates in crude fractions. The activities of ExoI are described below.

Genetic studies in yeast, and biochemical studies of MMR activity in cell extracts, indicate that eukaryotes use a mechanism similar to prokaryotes, with both 3’ to 5’ and 5’ to 3’ exonuclease activities for mismatch correction. ExoI, a 5’ to 3’ exonuclease, was found to play a role in mutation avoidance and mismatch repair in yeast, and its physical interaction with MSH2 and MLH1 also support a role in MMR. Intriguingly, the mammalian ExoI was reported to be involved in both 5’- and 3’ nick-directed excision in extracts of mammalian cells, but how ExoI can have a 3’ to 5’ exonuclease activity was unclear. Recent research by the Modrich group provides a plausible answer to this question [49]. They reconstituted mismatch-provoked excision, directed by a strand break located either 3’ or 5’ to the mispair, in a defined human system using purified human proteins. In the presence of the eukaryotic clamp loader replication factor C (RFC) and PCNA, 3’ to 5’ excision was supported by MutSa, MutLa, ExoI and RPA. Moreover, RFC and PCNA act to suppress 5’ to 5’ excision when the strand break that directs hydrolysis is located 3’ to the mismatch, which suggests that the polarity of mismatch-provoked excision by ExoI is regulated by PCNA and RFC. Once the strand is excised beyond the mismatch, DNA resynthesis occurs by the activity of polymerase d in the presence of PCNA and RPA. The remaining nick is then sealed by an as-yet-unidentified ligase, completing the repair process.

Mutation

Mutation is a permanent change in the DNA sequence that makes up a gene. Mutation range in size from one DNA base to a whole chromosome change.

Gene mutation can occur in two ways:

  • Germ line Mutation or hereditary Mutation – Mutations that can be passed down (are hereditary) and are present in all cells of an individual, including the germ cells.
  • Acquired or sporadic Mutations – Mutations caused by genetic changes that happen mostly by chance and are not inherited. They are acquired sometime during a person’s lifetime. These genetic changes are called sporadic (spontaneous) or acquired mutations. Most of these mutations occur in somatic cells. Somatic cells are any cells in the body other than egg and sperm cells (germ cells). Mutations in somatic cells cannot be passed down from a parent to a child.

These changes can be caused by environment factors such as ultraviolet radiation from the sun, or can occur due to the errors that occur during cell division. Errors in replication and damage to DNA have two consequences:

  • Permanent change to DNA, which can alter the coding sequence of a gene or its regulatory sequences.
  • Some chemical alterations to the DNA prevents its use as a template for replication and transcription.

The perpetuation of the genetic material from generation to generation depends on the maintaining rates of mutation at low levels. High rates of mutation in the germ line would destroy the species (lead to species extinction), and high rates of mutation in the soma would destroy the individual (leads to development of new species along with the existing one).

Sources of Mutation:

  1. Inaccuracy in DNA replication: The enzymatic machinery for replicating DNA attempts to cope with the mismatch incorporation of incorrect nucleotides through a proofreading mechanism, but some errors escape detection.
  2. Chemical Damage: Natural and unnatural chemicals and radiations break its backbone and chemically alters its bases. Example: UV radiation, X- ray and Gamma Radiation.
  3. Transposons: It is a DNA sequence that can change its position within the genome, sometimes creating or reversing mutations and altering the cell’s genome size. It can insert large portion of base pairs (may be even thousand bp) from one place to another.

Types of Mutation:

Mutation may  be gross or large ( which contain large portion or whole chromosome), or point (mutations not visible as cytological abnormalities), which can involve change in just a single nucleotide pair in DNA.

A. Base Pair (Nucleotide Pair) Substitutions:

Base pair substitution are the simplest mutation and involve switching of one base pair for another. These are of two types:

  • Transitions (Purine to purine or pyrimidine to pyrimidine substitution)
  • Transversions (Purine to pyrimidine or pyrimidine to purine substitution)

The consequences of base substitution mutations in protein coding regions of a gene depends on the substitution and its location. They may be

Silent: not resulting in a new amino acid in the protein sequence, eg GCA or GCG codons in mRNA both mean Arginine.

Missense Mutation: A base substitutaion which result in amino acid substitution. Eg. CTC in the DNA sense strand will specify glumate residue in the protein; this is altered to CAC in the DNA or GUG in the mRNA, resulting in a Valine residue in the beta-globulin protein chain causing sickle-cell anemia.

Nonsense Mutation: Base substitutions in a protein coding region may mutate an amino acid codon to termination code or vise versa, which results in a prematurely shortened protein.

Base substitution mutation may also occur in promoters or 5′ regulatory regions of genes or in introns and may affect their transcription, translation, or splicing. Many of the beta-thalassemias are the result of these types of non-structural mutations that may affect the level of expression of the globin genes. All of the types of the mutation described above have been observed in human globin genes. Their consequences depends on what they do to the level of expression of the gene products and/or on what amino acid substitution may have occured and where it is in the protein.

B. Frameshift Mutation (Insertions, Deletions and Duplications)

These result from insertion or deletion of one or more nucleotides in the coding region of the gene. This causes an alteration of the reading frame: since codons are groups of three nucleotides, there are three possible reading frames for each gene although only one is used. A mutation of this sort changes all the amino acids downstream and is very likely to create a nonfunctional product since it may differ greatly from the normal protein.

C. Repeat Expansions

Nucleotide repeats are short DNA sequences that are repeated a number of times in a row. For example, a trinucleotide repeat is made up of 3-bp sequences. A repeat expansion is a mutation that increase the number of times that the short DNA sequence is repeated. This type of mutation can cause resulting protein to function improperly.

D. Changes in Chromosomes

Changes that affect entire chromosomes or segments of the chromosomes can cause problems with growth, development, and function of the body’s systems. These changes can affect many gene along the chromosome and alter the proteins made by these genes. Conditions caused by the change in the number or structure of chromosomes are known as chromosomal disorders. these changes can occur during the formation of productive cells or in early fetal development. Many cancer cells also have changes in their chromosome number or structure. These changes most often occur in somatic cells during a person’s lifetime.

Proneness to Mutations

DNA sequences contain regions which are more prone to mutation. These mutation-prone sequences are repeats of simple di, tri, or teranucleotide sequences, which are known as DNA microsatellites.

 

Replication in Prokaryotes

The synthesis or replication of DNA molecule can be divided into three stages

  1. Initiation (Formation of Replisome)
  2. Elongation (Initiation of synthesis and elongation)
  3. Termination

I) Initiation

The replication begins at a specific initiation point called OriC point or replicon. (Replicon: It is a unit of the genome in which DNA is replicated; it contains an origin for initiation of replication)  It is the point of DNA open up and form open complex leading to the formation of prepriming complex to initiate replication process.

The OriC site is situated at 74″ minute near the ilv gene.  The OriC site consists of 245 basepairs, of which three of 13 basepair sequence are highly conserved in many bacteria and forms the consensus sequences (GATCTNTTNTTTT).  Close to OriC site, there are four of 9 basepair sequences each (TTATCCACA).

The sequence of reactions in the initiation process is as follows:

a) Dna A protein recognizes and binds up to four 9bp repeats in OriC to form a complex of negatively supercoiled OriC DNA wrapped around a central core of Dna A protein monomers.  This process requires the presence of the histone like HU or 1 HC proteins to facility DNA bending.

 

b) Dna A protein subunits then successively melt three tandemly repeated 13bp segments in the presence of ATP at >=22*C (open complex).

c) The Dna A protein then guides a Dna B – Dna C complex into the melted region to form a so called prepriming complex.  The Dna C is subsequently released.  Dna B further unwinds open complex to form prepriming complex.

d) DNA gyrase, single stranded binding protein (SSB), Rep protein and Helicase – II are bound to prepriming complex and now complex is called as priming complex.

e) In the presence of gyrase and SSB, helicases further unwinds the DNA in both directions so as to permit entry of primase and RNA polymerase.  Then RNA polymerase forms primer for leading strand synthesis while primase in the form of primosome synthesis primer for lagging strand synthesis.

f) To the above complex, DNA polymerase – III will bind and forms replisome.

REPLISOME:  It is the multiprotein structure that assembles at the bacterial replicating fork to undertake synthesis of DNA.  It contains DNA polymerase and other enzymes.

II) ELONGATION:

Now the stage is set for the initiation of synthesis and the elongation to proceed.  But this occurs in two mechanistically different pathways in the 5′–>3′ template strand and 3′–>5′ template strand.

a) Initiation of synthesis and Elongation on the 5′–>3′ template (synthesis of leading strand) (If replication fork moves in 3′–>5′ direction)

The DNA daughter strand that is synthesized continuously on 5′–>3′ template is called leading strand.  DNA pol-III synthesizes DNA by adding 5′-P of deoxynucleotide to 3′-OH group of the already presenting fragment.  Thus chain grows in 5′–>3′ direction.  The reaction catalyzed by DNA pol-III is very fast.  The enzyme is much more active than DNA pol – I and can add 9000 nucleotides per minute at 37*C.  The RNA primer that was initially added by RNA polymerase is degraded by RNase.

b) Initiation of synthesis and Elongation on 3′–>5′ template when fork moves in 3′–>5′ direction (Synthesis of lagging strand) 

The daughter DNA strand which is synthesized in discontinuous complex fashion on the 3′–>5′ template is called lagging strand.  It occurs in the following steps:

i) Synthesis of Okazaki fragment:

To the RNA primer synthesized by primosome, 1000-2000 nucleotides are added by DNA pol-III to synthesis Okazaki fragments.

ii) Excision of RNA primer:

        When the Okazaki fragment synthesis was completed up to RNA primer, then RNA primer was removed by DNA pol – I using its 5′–>3′ exonuclease activity.

iii) Filling the gap (Nick translation)

        The gap created by the removal of primer, is filled up by DNA pol – I using the 3′-OH of nearby Okazaki fragment by its polymerizing activity.

iv) Joining of Okazaki fragment: (Nick sealing)

        Finally, the nick existing between the fragments are sealed by DNA ligase which catalyze the formation of phosphodiester bond between a 3′-OH at the end of one strand and a 5′ – phosphate at the other end of another fragment.  The enzyme requires NAD for during this reaction.

III) TERMINATION:

 

Termination occurs when the two replicating forks meet each other on the opposite side of circular E.Coli DNA.  Termination sites like A, B, C, D, E and F are found to present in DNA. Of these sites, Ter A terminates the counter clockwise moving fork while ter C terminates the clockwise moving forks.  The other sites are backup sites.   Termination at these sites are possible because, at these sites tus protein (Termination utilizing substance) will bound to Dna B protein and inhibits its helicase activity.  And Dna B protein released and termination result.

After the complete synthesis, two duplex DNA are found to be catenated (knotted).  This catenation removed by the action of topoisomerase.  Finally, from single parental duplex DNA, two progeny duplex DNA synthesized.

REGULATION OF PROKARYOTIC REPLICATION:

Especially initiation of replication is regulated.  Dna A protein when available in high concentration then ratio of DNA to cell mass is quiet high but at low Dna A concentration, the ratio found to be low.  This shows that Dna A protein regulates the initiation of replication.

The sequence most commonly methylated in E.Coli is GATC including in three of 13mer sequence.  Thus, the observation that E.Coli defective in the GATC methylation enzyme are very inefficiently replicated, suggests that the DNA replication trigger also responds to the level of OriC methylation.

Replication in Eukaryotes

Eukaryotic replication occurs during s-phase of cell cycle.  Replication usually occurs only one time in a cell.  Replication in eukaryotes occur in five stages namely,

  1. Pre-initiation
  2. Initiation
  3. Elongation
  4. Termination
  5. Telomerase function
  1. Pre-initiation:

Actually during pre-initiation stage, replicator selection occurs.  Replicator selection is the process of identifying the sequences that will direct the initiation of replication and occur in G1 phase. and occurs in Gl (prior to S phase). This process leads to the assembly of a multiprotein complex at each replicator in the genome. Origin activation only occurs after cells enter S phase and triggers the Replicator – associated protein complex to initiate DNA unwinding and DNA polymerase recruitment.  Replicator selection is mediated by the formation of pre-replicative complexes (pre-RCs). The first step in the formation of the pre-RC is the recognition of the replicator by the eukaryotic initiator, ORC (Origin recognition Complex). Once ORC is bound, it recruits two helicase loading proteins (Cdc6 and Cdtl). Together, ORC and the loading proteins recruit a protein that is thought to be the eukaryotic replication fork helicase (the Mem 2-7 complex). Formation of the pre-RC does not lead to the immediate unwinding of origin DNA or the recruitment of DNA polymerases. Instead the pre-RCs that are formed during Gl are only activated to initiate replication after cells pass from the Gl to the S phase of the cell cycle.

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  1. Initiation:

Pre-RCs are activated to initiate replication by two protein  kinases namely Cdk (Cyclin Dependant Kinase) and Ddk (Ddt4 Dependant Kinase). Kinases are proteins that  covalently attach phosphate groups to target proteins. Each of these kinases is inactive in Gl and is activated only when cells enter S phase. Once activated, these kinases target the pre-RC and other replication proteins. Phosphorylation of these pro-proteins results in the assembly of additional replication proteins at the origin and the initiation of replication.

 

These new proteins include the three eukaryotic DNA polymerases and a number of other proteins required for their recruitment. Interestingly, the polymerases assemble at the origin in a particular order. DNA Pol d and e associate first, followed by DNA Pol a/primase. This order ensures that all three DNA polymerases are present at the origin prior to the synthesis of the first RNA primer (by DNA Pol a/primase).   Once present at the origin, DNA Pol a/primase synthesizes an RNA primer and briefly extends it. Thus initiation of replication started.

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  1. Elongation:

 

The resulting primer-template junction is recognized by the eukaryotic sliding clamp loader (RF-C), which assembles a sliding clamp (PCNA) at these sites. Either DNA Pol d or e recognizes this primer and begins leading strand synthesis. After a period of DNA unwinding, DNA Pol a/primase synthesizes additional primers, which allow the initiation of lagging strand DNA synthesis by either DNA Pol d or e. In the diagram, Pol d was used for leading strand and Pol e was used for lagging strand synthesis.  DNA Pol e possess activity to remove primer and fills the gap with DNA like DNA Pol I in prokaryotes.  SSB like activity was played by replication protein A (RP A) which is denoted as accessory factors during replication.

 

  1. Termination:

 

When the replication forks meet each other, then termination occurs.  It will result in the formation of two duplex DNA.  Eventhough replication terminated, 5’ end of telomeric part of the newly synthesized DNA found to have shorter DNA strand than the template parent strand.  This shortage corrected by the action of telomerase enzyme and then only the actual replication completed.

  1. Terlomerase Function:

In Linear eukaryotic chromosome, once the first primer on each strand is remove, then it appears that there is no way to fill in the gaps, since DNA cannot be extended in the 3′–>5′ direction and there is no 3′ end upstream available as there would be in a circle DNA.  If this were actually the situation, the DNA strand would get shorter every time they replicated and genes would be lost forever.

Elizabeth Blackburn and her colleagues have provided the answer to fill up the gaps with the help of enzyme telomerase.  So, that the genes at the ends, are conserved.  Telomerase is a ribonucleoprotein (RNP) i.e. it has RNA with repetitive sequence.  Repetitive sequence varies depending upon the species example Tetrahymena thermophilia RNA has AACCCC sequence and in Oxytrica it has AAAACCCC.  Telomerase otherwise known as modified Reverse Transcriptase.  In human, the RNA template contains AAUCCC repeats.  This enzyme was also known as telomere terminal transferase.

The 3′-end of the lagging strand template basepairs with a unique region of the telomerase associated RNA.  Hybridization facilitated by the match between the sequence at the 3′-end of telomere and the sequence at the 3′-end of the RNA.  The telomerase catalytic site then adds deoxy ribonucleotides using RNA molecule as a template, this reverse transcription proceeds to position 35 of the RNA template.  Telomerase then translocates to the new 3′-end by pairing with RNA template and it continues reverse transcription.  When the G-rich strand sufficiently long, Primase can make an RNA primer, complementary to the 3′-end of the telomere’s G-rich strand.   DNA polymerase uses the newly made primer to prime synthesis of DNA to fill in the remaining gap on the progeny DNA.  The primer is removed and the nick between fragments sealed by DNA ligase.

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DNA replication in Prokaryotes

All living cells are capable of giving rise to a new generation of cells by undergoing

DNA replication and cell division. During cell division in eukaryotic cells, the replicated DNA is equally distributed between two daughter cells.

During replication process the hydrogen bonds between complimentary strands break and allow the DNA helix to unzip. Each strand of unwound DNA acts as template to build complimentary strand.

The process of DNA replication:

1. The first step in the process of DNA replication is to unwind the DNA. An enzyme known as helicase helps in breaking the hydrogen bonds and unwinding the DNA.

2. Even though the strands are separated they have a tendency of annealing back again due to complimentary nature of base pairs. Single stranded binding proteins (SSBs) bind to the exposed DNA and keep them apart by blocking the hydrogen bonding.

DNA replication

3. Another enzyme gyrase helps to release the tension in the separated strands by cutting and resealing them.

4. DNA is unwound at multiple locations forming bubbles known as replication bubbles. The junction where DNA is still attached is known as replication fork.

Replication bubble

5. DNA polymerase III enzyme helps in synthesizing the complimentary strand using template strand as a guide.

6. DNA polymerase III can function only under certain conditions

i) it synthesizes DNA in 5’ to 3’ direction

ii) it require the presence of RNA primer to initiate complimentary strand

7. RNA primer is synthesized by primase enzyme.

8. Complimentary stand can be made continuously on one of the template strand known as leading strand. Other strand is known as lagging stand.

replication showing okazaki fragments

9. On leading strand one RNA primer is attached at 3’ end and complimentary stand is made uninterrupted.

10. On lagging stand multiple RNA primers are attached to template strand and synthesize small fragments of complimentary strands. These fragments are known as Okazaki fragments.

11. RNA primers are removed by DNA polymerase I and are replaced with appropriate deoxyribonucleotides.

12. Ligase enzyme joins all Okazaki fragments together by forming bonds between them.

Replication Forks and Origins of Replication

In the cell, both strands of the DNA duplex are replicated at the same time. The first step in DNA replication is the separation of the two DNA strands that make up the helix that is to be copied to make two template DNAs. DNA Helicase untwists the helix at locations called replication origins. The junction between the newly separated template strands and the unreplicated duplex DNA is known as the replication fork.

The replication fork moves down the DNA strand, usually from an internal location to the strand’s end (unreplicated DNA). The result is that every replication fork has a twin replication fork, moving in the opposite direction from that same internal location to the strand’s opposite end.

Single-stranded binding proteins (SSB) work with helicase to keep the parental DNA helix unwound. It works by coating to stabilize the unwound strands with rigid subunits of SSB that keep the strands from snapping back together in a helix. Binding of one SSB promotes the binding of another SSB to the immediately adjacent ssDNA. This is called cooperative binding.The SSB subunits coat the single-strands of DNA in a way as not to cover the bases, allowing the DNA to remain available for base-pairing with the newly synthesized daughter strands.

As the strands of DNA are seperated at the replication fork, the duplex DNA infront of the fork becomes increasingly positively supercoiled. This accumulation of super is the result of DNA helicase eliminating the base parts between the two strands. If there is no mechanism to relieve the accumulation of these supercoils, the replicating machinery would grind to a halt in the face of mounting pressure. These super coils produced by the DNA unwinding at the replication fork is removed by Toposiomerases.

Because of the antiparallel nature of DNA and because DNA is only sythesized by elongating 3′ end, daughter strands synthesize through different methods, one adding nucleotides one by one in the direction of the replication fork, the other able to add nucleotides only in chunks. The first strand, which replicates nucleotides one by one (continuously) is called the leading strand; the other strand, which replicates in chunks (discontinuous fashion), is called the lagging strand.

Leading strand DNA polymerase can replicate its template as soon as it is exposed, synthesis of the lagging strand must wait for movement of the replication fork to expose a substantial length of new lagging strand is exposed.

The lagging strand replicates in small segments, called Okazaki fragments. These fragments are stretches of 100 to 200 nucleotides in humans (1000 to 2000 in bacteria) that are synthesized in the 5′ to 3′ direction away from the replication fork. Yet while each individual segment is replicated away from the replication fork, each subsequent Okazaki fragment is replicated more closely to the receding replication fork than the fragment before. These fragments are then stitched together by DNA ligase, creating a continuous strand. This type of replication is called discontinuous.

The initiation of New strand of DNA requires an RNA Primer with free 3’OH. DNA polymerase cannot initiate a new DNA strand them selves. To accomplish this the cell take advantage of the ability of RNA polymerase to do what DNA polymerases cannot: initiate new RNA strand. Primase is a special RNA polymerase dedicated to making short, RNA primers on an ssDNA template. These primers are subsequently extended by DNA Polymerase.

Although both the leading and lagging strands require primase to initiate DNA synthesis, the frequency of primase function on the two strands is dramatically different. Leading strand requires only a single RNA primer. In contrast, the discontinuous synthesis of the lagging strand means that new primers are needed for each Okazaki fragments.

Primase does not require a specific DNA sequence to initiate the synthesize of new RNA primer. Instead primase is activated only when it is associated with other DNA replication protein such as DNA helicase. Once activated, the primase synthesize a RNA primer using the most recently exposed lagging strand template regardless of sequence.

To complete the DNA replication, the RNA primase used for the initiation must be removed and replaced by DNA. to replace the RNA primers with DNA, an enzyme called RNase H recognize and removes most of the each RNA primer. This enzyme specifically degrades RNA that is base paired with DNA. RNase H removes all of the RNA primer except the ribonucleotide directly linked to the DNA end. This is because RNase H can only cleave bonds between tow ribonucleotides.The final ribonuceotide is removed by an a exonuclease, that degrages RNA and DNA from there 5′ end.

Removal of RNA leaves a gap in the double-stranded DNA. DNA polymerase fill the gap until every nucleotide is base-paired, leaving a DNA molecule that is complete except for the break in the backbone bvetween ythe 3’Oh and 5′ phosphate of the repaired strand. This nick in the DNA can be repaired by an enzymes called DNA ligase. Only after all RNA primase are replaced and asscociated nicks are sealed, DNA synthesis is completed.

Mechanism of DNA Polymerase

The synthesis of DNA is catalyzed by an enzyme called DNA Polymerase.  Unlike most enzymes, which have an active site dedicated to a single reaction, DNA polymerase uses a single active site  to catalyze addition of any of the four deoxynucleotide triphosphates. Polymerase shows kinetic selectivity, in which an enzyme favors catalysis using one of several possible substrates by dramatically increasing the rate of bond formation only when the correct substrate is present. Only when the correct pair is formed the catalysis occurs. Incorrect base pairing leads to dramatically lower rate of nucleotide addition due to the catalytically unfavorable alignment of these substrates. DNA polymerase show an impressive ability to discriminate between ribo- and deoxyribonucleoside phosphates. This ability is mediated by steric exclusion of rNTPs from the DNA polymerase active site, because in DNA polymerase the nucleotide binding pocket is too small to allow the presence of a 2’OH on the incoming nucleotide.

DNA Polymerase Structure

DNA Polymerase resemble a right hand that partially grips the primer: Template junction. Based on anatomy of the hand, there are three domains in the polymerase:

  • Palm: The palm domain is composed of a B Sheet and is the active site of DNA Polymerase. This region of DNA polymerase binds two divalent metal ions (Mg2+ or Zn2+) that alter the chemical environment around the correctly base-paired dNTP and the 3’OH of the primer. One metal ion reduces the affinity of the 3’OH for its hydrogen. This generates a 3’O- that is primed for the nucleophilic attack of the alpha-phosphate of the incoming dNTP. The secong metal ion coordinates the negative charges of the Beta and Gamma phosphates of the dNTP and stabilizes the pyrophosphate produced by joining the primer and the incoming nucleotide. The palm domain also monitors the accuracy of base-pairing for the most recently added dNTP.
  • Fingers: once the correct base-pair is formed, the finger domain moves to enclose the dNTP. this close form of the polymerase hand stimulates catalysis by moving the incoming nucleotide in close contact with the catalytic metal ions.
  • Thumb: Thumb interacts with the DNA that has been most recently synthesized. this serves two purpose: (1) It maintains the correct position of the primer and the active site, (2) helps to maintain a strong association between the DNA polymerase and its substrate, which contribute to the processive nature of DNA polymerase.

Features of DNA Polymerase:

  • High Processivity: Catalysis by DNA polymerase is rapid, are capable of adding as many as 1000 nucleotide per second to a primer strand. In case of DNA polymerase, the degree of processivity is defined as the number of nucleotides added each time the enzyme binds a primer:template junction.
  • Multiple substrates: In processive enzyme each time the enzyme binds a primer:template junction multiple substrates, dNTP is added to the primer. In contrast with no processive enzyme adds a single dNTP to primer and then is released from the primer:template junction.

Increased processivity is facilitated by the ability of DNA polymerase to slide along the DNA Template. DNA polymerase interacts tightly with much of the double-stranded portion of the DNA in a sequence non specific manner. These interactions include electrostatic interactions between the phosphate backbone and the thumb domain, and interaction between the minor groove of the DNA and the palm domain. Further increase in processivity are achived through the interactions between the DNA polymerase and the “Sliding clamp” protein.

  • Exonuclease Activity: Occasional flickering of the bases into the ‘wrong’ tautomeric form. This alternate form of bases allow incorrect base pairs to be correctly positioned for catalysis. Exonucleases proofread newly synthesized DNA. These type of exonuclease is called proofreading exonuclease. Theses exonucleases are capable of degrading DNA starting from the 3′ DNA end, that is the from the growing end of the new DNA strand and remove the incorrect nucleotide.

Mispaired DNA alters the geometry of the 3′ OH and the incoming nucleotide due to poor interactions with the palm region. This altered geometry reduces the rate of nucleotide addition and increase the rate of proofreading exonuclease activity. proofreading can occur without releasing the DNA from the polymerase. When a mismatched pair is detected, the primer:template junction slides away from the DNA polymerase active site and into the exonuclease site. after the incorrect nucleotide is removed the correctly paired primer:template junction slides back ingto the active site of polymerase and DNA synthesis continues.

The DNA polymerase performing synthesis and editing

  • Nucleotides are added in to both templates in replication fork at the same time.
  • Primer is required. DNA polymerase require primer to initiate DNA Polymerase.

Specialization of DNA Polymerase

DNA polymerases are specialized for different roles in the cell. The central role of DNA polymerases in the efficient and accurate replication of the genome requires that cell have multiple specialized DNA polymerases.

DNA polymerases in Prokaryotes:

1. DNA polymerase II (pol II) is a minor DNA polymerase in E. соli.

  • Function. Pol II may be involved in some DNA repair processes, but E соli mutants lacking this enzyme show no replication or growth deficiencies.
  • Structure. Pol II is a single polypeptide.
  • Other enzymatic activities. Pol II has proofreading (3′ to 5′ exonuclease) activity but lacks excision-repair (5′ to 3′ exonuclease) activity.

2. DNA polymerase III (pol III) is the primary DNA polymerase involved in cellular replication.

  • Function. Pol III catalyzes leading and lagging strand synthesis.
  • Structure. Pol III is structurally complex. The pol III core enzyme, made of three different polypeptides, was first isolated. Gentle purification schemes and careful reconstitution showed that the active cellular form of pol HI, called pol III holoenzyme, is made of 10 subunit polypeptides. The subunits are organized into an asymmetric dimeric structure with two catalytic centers. This is important in understanding how it catalyzes both leading and lagging strand syntheses at the replication fork.
  • Other enzymatic activities. Pol III holoenzyme has proofreading (3′ to 5′ exonuclease) activity but no excision-repair (5′ to 3′ exonuclease) activity. The two α subunits both contain the DNA polymerase activity, and the two ε subunits both contain the 3′ to 5′ exonuclease activity. The two subunits form a dimer that circles around the DNA. This β2 “DNA clamp” enables the pol III holoenzyme to stay bound to the DNA and facilitates its high rate of replication.

3. DNA polymerase I (pol I) was the first DNA polymerase discovered.

  • Function. Pol I functions in the replication of DNA and in the repair of damaged DNA.
  • Structure. Pol I is a single polypeptide.
  • Other enzymatic activities. Pol I has two enzymatic activities—besides DNA polymerase activity—that are important to its cellular function.
    • (1) Proofreading. Pol I does not typically add a nucleotide to the growing DNA chain that cannot properly base pair with the template strand. If a mismatched nucleotide is added, the enzyme halts polymerization. A 3′- to 5′-exonuclease activity removes the mismatched nucleotide, and polymerization resumes. This activity is called proofreading, and it assures the high-level fidelity of replication that is a desirable trait of genetic material.
    •  (2) Excision-repair. Pol I has a 5′ to 3′ exonuclease activity, called excision-repair activity, that can hydrolytically remove a segment of DNA from the 5′ end of a strand of duplex DNA.
      • (a) From 1-10 nucleotide segments of DNA can be removed simultaneously.
      • (b) This activity is essential for the removal of primers in DNA replication.
      • (c) This activity is essential for the repair of damaged DNA.
    • (3) Nick-translation. A nick is a break in a phosphodiester bond of one strand of DNA in a double helix. A nick leaves a free 3′-hydroxyl and a free 5′-phosphate. Pol I can function at nicks as an exonuclease and a polymerase at the same time. As the 5′-phosphate nucleotide is removed or displaced by pol I, it is replaced with the polymerase activity of pol I. Pol I cannot reseal the nick; therefore, the nick is moved, or translated, along the DNA in the direction of synthesis.

DNA polymerases in Eukaryotes:

1. DNA polymerase alpha /primase

  • Function. DNA polymerase alpha is almost exclusively required for the initiation of DNA replication and the priming of Okazaki fragments during elongation. In most organisms no specific repair role beyond that of checkpoint control has been assigned to this enzyme.
  • Structure. Pol alpha contain 4 subunit. (two subunit DNA Pol alpha and a two-subunit primase)

2. DNA polymerase beta

  • Function. Base excision repair. The role of DNA polymerase beta in base-excision repair is well established for mammalian systems, but in yeast, DNA polymerase delta appears to fulfill that function.
  • Structure. Pol beta contain 1 subunit.

3. DNA polymerase gamma

  • Function. Mitrochondrial DNA replication and repair.
  • Structure. contain 3 subunit.

4. DNA polymerase Delta

  • Function. DNA polymerase delta functions as a dimer and, therefore, may be responsible for both leading and lagging strand DNA replication. In addition, this enzyme is required for mismatch repair and, together with DNA polymerase zeta, for mutagenesis.
  • Structure. contain 2-3 subunits.

5. DNA polymerase Epsilon

  • Function. The function of DNA polymerase epsilon in DNA replication may be restricted to that of Okazaki fragment maturation. In contrast, either polymerase delta or epsilon suffices for the repair of UV-induced damage.
  • Structure. contain 4 subunits.

During Replication, due to relatively low processivity , DNA pol alpha/primase is replaced by the highly processive DNA polymerase Delta and Epsilon by the process called polymerase switching. The majority of other DNA polymerase in Eukaryotes are involved in DNA repair.

DNA Replication: Introduction

DNA replication is the process of producing two identical replicas from one original DNA molecule. This biological process occurs in all living organisms and is the basis for biological inheritance. DNA is made up of two strands and each strand of the original DNA molecule serves as template for the production of the complementary strand, a process referred to as semiconservative replication. Cellular proofreading and error-checking mechanisms ensure near perfect fidelity for DNA replication.

For the DNA synthesis two key substrate is required.

Substrate: The four deoxynucleoside triphosphates (dNTPs)—deoxyadenosine tri- phosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphos-phate (dCTP), and deoxythymidine triphosphate (dTTP)—are needed as substrates for DNA synthesis.

Primer:template junction: second substrate for the DNA Synthesis is a particular arrangement of ssDNA and dsDNA called primer:template junction.

  • Template. DNA replication cannot occur without a template. A template is required to direct the addition of the appropriate complementary deoxynucleotide to the newly synthesized DNA strand. In semiconservative replication, each strand of parental DNA serves as a template. Then, each template strand and its newly synthesized complementary strand serve as the DNA in daughter cells.
  • Primer. DNA synthesis cannot start without a primer, which prepares the template strand for the addition of nucleotides. The primer must also have an exposed 3’OH because new nucleotides are added to the 3′ end of a primer that is properly base paired to the template strand of DNA, new synthesis is said to occur in a 5′ to 3′ direction.

The DNA synthesis that occurs during the process of replication is catalyzed by enzymes called DNA-dependent DNA polymerases. These enzymes depend on DNA to the extent that they require a DNA template. They are more commonly called DNA polymerases.