In this article we will discuss about:- 1. Subject-Matter of Replication and Synthesis of Nucleic Acids 2. Unidirectional and Bidirectional Replications of Nucleic Acids 3. Enzymes and Proteins Involved in DNA Replication and Repair 4. Replication of Double-Stranded Circular DNA 5. RNA Primer Synthesis 6. Leading Strand and Lagging Strand and Other Details.
Contents:
- Subject-Matter of Replication and Synthesis of Nucleic Acids
- Unidirectional and Bidirectional Replications of Nucleic Acids
- Enzymes and Proteins Involved in DNA Replication and Repair
- Replication of Double-Stranded Circular DNA
- RNA Primer Synthesis
- Leading Strand and Lagging Strand
- A Generalized Model of DNA Replication
- Roiling Circle Model of DNA Replication
- Replication of Single-Stranded Circular DNA
1. Subject-Matter of Replication and Synthesis of Nucleic Acids:
DNA replication is semiconservative, i.e., complimentary copy is synthesized against an old strand of DNA so that each progeny molecule has one new and one old strand. There are specific sites or points called origin at which replication is initiated. Each bacterial chromosome, viral genome and plasmid has a single origin of DNA replication, so that the whole chromosome replicates as one unit.
The unit of replication is termed as replicon. On the contrary, DNA replication in eukaryotes occurs at many points along a single chromosome; thus a single eukaryotic chromosome contains several replicons, e.g., Drosophila genome has 3500 replicons, while Viciafaba has 35,000 replicons, (Table 3.6). Toad (Xenopuslaevis) has 15,000 replicons in its genome.
However, the total number of active origins in the genome varies considerably among the different developmental stages, e.g., during the early cleavages of Drosophila embryos, the number of active origins is many times more than that in the dividing cells of adults. A large number of active origins permits a rather rapid replication of the DNA during the early embryonic development.
2. Unidirectional and Bidirectional Replications:
Radiographic studies have shown that DNA replication may be either unidirectional (in few organisms) or bidirectional (in most of the organisms). In case of unidirectional replication, the DNA strands begin to separate from the origin point and replication proceeds in one direction only, i.e., the replication fork (the Y like configuration produced by the progressive separation of the two stands of a DNA double helix generating single-stranded regions against which the new strands are synthesized) proceeds in one direction from the origin (Fig. 3.11). Unidirectional replication is known to occur in mitochondrial DNA, and in coliphage P2.
In bidirectional replication, on the other hand, DNA strands begin to separate at the origin point, but the DNA replication proceeds in both the directions from the origin. Thus there are two active replication forks which proceed in the opposite directions (Fig. 3.11).
The evidence for bidirectional replication is available both in eukaryotes like yeast, Drosophila and mammals, and in prokaryotes, e.g., bacteria and bacteriophages. The replication of DNA double helix starts at the origin producing an “eye” or “bubble” shaped structure due to the separation of the complementary strands of the double helix; this “bubble” moves in both the directions as it increases in size.
The “eyes” can be observed either through autoradiography or by using an electron microscope. The number of “eye” or “bubbles” indicates the number of origins and hence “replicons present in DNA molecule.
When the origin is located near one end of a chromosome, which is the case in phage T7, one fork of the “eye” (-O-) will reach the chromosome end much before the other fork does; this will convert the “eye” into a “Y” structure. The remaining replication fork remains active and keeps on advancing till the chromosome is fully replicated. Both the ‘eye’ and ‘Y’ configurations of the T7 chromosome have been actually observed during replication.
3. Enzymes and Proteins Involved in DNA Replication and Repair:
A number of enzymes and proteins having specific functions are involved in the replication of DNA (Table 3.7); they constitute the “replication apparatus” of organisms. Several enzymes of replication apparatus are present in the plasma membrane of bacteria; the bacterial chromosome is also attached to the plasma membrane, especially at the point of origin and at the growing point of the replications fork.
Eukaryotic DNA polymerases differ from the prokaryotic DNA polymerases. Some of their chief characteristics are summarised in Table 3.8.
Bacterial DNA polymerase I:
DNA bacterial polymerase I molecule has the following functional sites (Fig. 3.12).
(a) Template site: it binds to the template strand of DNA.
(b) Primer site: it is the growing chain site.
(c) Primer terminus site: It is the 3’à5′ exonuclease site used for the removal of a mismatched nucleotide incorporated (due to error) at the end of the growing chain.
(d) Triphosphate site: It binds the complementary nucleotide triphosphate (A = T; G = C) to be added to the growing chain and forms the phosphodiester bond between the nucleotides at the 3′ end of the growing chain.
(e) 5’à3 exonuclease site: it removes any strand coming in the path of the growing chain.
Bacterial DNA polymerase III:
It is a hetero-multimeric enzyme with the molecular weight of 9,00,000daltons (Fig. 3.13. ; Table 3.9). Three subunits, namely a, e and 0 from the “core enzyme”. The function of these subunits are DNA synthesis (a), 3’—>5′ proof reading exonuclease activity (ε), and assembly (θ). Two units (αεθ + αεθ) of the core enzyme combine to form a dimer with the help of the subunit (T); this dimer is called Pol III’.
Addition of 7 8 complex makes it a Pol III* complex. The subunit (3 and the complex γδ have the ability to recognize the prime template. The “holoenzyme” has only one y8 complex and therefore, it is asymmetrical. It is suggested that the core with γδ complex synthesizes the lagging strand, while the core with t dimer synthesizes the leading strand.
4. Replication of Double-Stranded Circular DNA:
Bacterial chromosome is a single circular double stranded DNA molecule; similar chromosomes occur in some viruses, such as phage γ (Table 3.1); Cairns in 1963, using tritiated thymidine (3H- thymidine), demonstrated that E. coli chromosome replicated in a semiconservative manner.
He concluded that one strand of the newly produced DNA double-helix is old, while the other one is newly synthesized. The replication chromosome produced the ‘theta’ (θ) shaped intermediate before completion of replication (Fig. 3.14).
The replication of bacterial or phage chromosome beings at the “origin” and the two stands of the DNA double helix begin to separate; the replication proceeds in both the directions from the origin point (Fig. 3.14) till the entire chromosome is replicated to yield two progeny circular, double-stranded chromosomes.
In E. coli, DNA replication occurs at the rate of about 50,000 base pairs (50 kb)/minute. But in eukaryotes, the replication rate is much lower (Table 3.6); it is 2,600 bp/minutes in D. melanogaster, while only 500 bp/minute in Xenopuslaevis.
This may be due to the highly complex organization of eukaryotic chromosomes which have repeated “nucleosome” units. Therefore, during DNA replication in eukaryotes, the following events must take place;
(i) DNA has to be separated from the histones participating in the nucleosome organization;
(ii) It has to unwind;
(iii) Histones have to be synthesized during the DNA synthesis;
(iv) DNA replication has to occur, and
(v) The newly produced DNA double helices have to be assembled into nucleosome and chromatin fibres.
5. RNA Primer Synthesis:
At the beginning of DNA replication, a short RNA segment complementary to DNA strand is synthesized by the enzyme “primase”; this RNA is known as prime RNA or simply as primer. The primer RNA is required since DNA polymerase cannot initiate the synthesis of a DNA strand without “proof reading” the base at the primer terminus site.
Therefore, a primer having a free 3′-OH is essential for DNA polymerase to function. There are several findings that suggested that DNA replication has a prerequisite of RNA primer synthesis.
6. Leading Strand and Lagging Strand:
The two DNA strands are oriented in opposite polarity
, but DNA replication occurs only in the 5’—>3′ direction. Therefore, replication of the two strands of a DNA molecule has to occur in the opposite directions. Thus the replication of the strand oriented in the 3’—>5′ direction begins first and is continuous as the new chain being synthesized in the 5’—>3′ direction; hence this strand is called the leading strand.
However, the replication of the other strand (oriented in the 5’—>3′ direction) begins somewhat later, and is discontinuous; therefore, this strand is called the lagging strand (Fig. 3.11, 3.15). In the case of bidirectional replication, however, the same strand acts as the leading strand at the replication fork on one side of the origin, while it acts as the lagging strand at the fork on the other side of the origin (Fig. 311).
7. A Generalized Model of DNA Replication:
DNA replication is unidirectional in some organisms, while in the vast majority, it is bidirectional. In both the cases, replication of one of the two DNA strands is continuous, while that of the other occurs in small fragments called Okazaki fragments (after their discoverer Reiji Okazaki).
The length of Okazaki fragments varies from 1000 to 2000 nucleotides in prokaryotes, and from 100 to 200 nucleotides in eukaryotes. These fragments are ultimately joined together by the polynucleotide ligase.
A generalized scheme of DNA replication is depicted in the Fig. 3.15, and is briefly described below:
(1) There are unique and fixed sites on the chromosomes at which DNA replication is initiated. These sites are called origin.
(2) The enzyme gyrase relaxes the super coiling caused by unwinding of the two strands of the DNA double helix.
(3) The enzyme helicase unwinds the two strands of DNA molecule to generate single stranded regions. The DNA molecule rotates at the growing fork at the rate of about 100 revolutions/second during the unwinding process.
(4) The single-stranded binding proteins (SSB) bind to the single-stranded regions of the DNA produced by unwinding; this prevents enzymatic degradation of the single-stranded regions as well as the formation of hydrogen bonds between the two complementary strands (that were originally separated).
(5) Pre-priming proteins recognize the initiation site for primer synthesis. The enzyme primase now synthesizes a short RNA sequence complementary to the DNA template strand oriented in the 3’—>5′ direction (the leading strand). This RNA sequence serves as the “primer”.
(6) The enzyme DNA polymerase III elongates the RNA primer by adding deoxyribonucleotides at the 3′-OH end of the primer ; as a result, the polynucleotide chain grown in the 5’—>3′ direction, and at the same time, is complementary to the template DNA strand.
(7) Another enzyme DNA polymerase I (also called Kornberg’s enzyme) removes the RNA primer by its exonuclease activity. This enzyme also adds deoxyribonucleotides in place of the ribonucleotides by its polymerase activity and thus fills the gap produced due to the degradation of the RNA primer.
(8) In prokaryotes, the replication forks are associated with plasma lemma, while in eukaryotes, they are associated with the nuclear membrane.
(9) Replication of the second strand which is oriented in the 5’—>3′ direction (lagging strand) begins somewhat later than that of the leading strand (3’—>5′). Replication of the leading strand is continuous, while that of lagging strand is discontinuous and occurs in fragments called “Okazaki fragments”.
(10) On the lagging strand, an RNA primer is synthesized near the replication fork. DNA polymerase keeps on adding nucleotides to this primer so that the polynucleotide chain grows in the 5’—>3′ direction, i.e., from the fork toward the origin.
It may be emphasized that the directions of the chain growth on the leading (from origin toward the replication fork) and the lagging (from fork toward origin) strands are opposite to each other.
(11) The Okazaki fragments are joined together by the enzyme polynucleotide ligase. But before this, the RNA primers are removed and the gaps so created are filled by the DNA polymerase I (in prokaryotes). DNA ligase enzyme joins the 3′-OH end of one fragment with the 5′-P end of the other segment.
8. Roiling Circle Model of DNA Replication:
This mode of DNA replication occurs in circular double-stranded DNAs of certain prokaryotes and eukaryotes, which are listed below.
(i) Transfer of E. coli chromosome from donor into the recipient cell during conjugation.
(ii) Late stages of growth of phage lambda (a).
(iii) Replication of the replicative forms (RF) of phage φ X 174 chromosome.
(iv) Replication of the circular double-stranded DNA segments produced in the amphibians Xenopus for rRNA synthesis.
At the start of replication, a nick is produced in one strand of the DNA double helix; this generates one 5′- and one 3′-end (Fig. 3.16). The 5′-end of the strand becomes attached to a membrane, while DNA synthesis proceeds at the 3′-end (in 5′-»3′ direction) using the intact strand as a template.
The intact circular strand “rolls” on and the 5′-end attached to the membrane progressively separates to keep on generating single-stranded regions of the intact strand, which are used as template for the DNA synthesis proceeding in the 5’—>3′ direction.
In this mode of DNA replication, RNA primer is not required, since the 3′-end of the nicked strand provides the 3′-OH essential for DNA polymerase action. Replication also begins on the single-stranded 5′-end of the nicked strand, but this synthesis requires RNA primer and is discontinuous. The enzyme polynucleotide ligase joins the Okazaki fragments (Fig. 3.16).
9. Replication of Single-Stranded Circular DNA:
The phage φ X174 contains single-stranded circular DNA molecule consisting of 5386 nucleotides. This strand is designated as the plus (+) strand. Following infection of E. coli, the (+) strand of phage φX174 synthesizes its complementary minus (-) strand.
The synthesis of (-) strand is discontinuous, the RNA primers being synthesized by the enzyme primase in conjunction with at least 6 priming proteins; this complex is called “primosome”. DNA polymerase III-elongates the RNA primer at 3′-OH end by adding deoxyribonucleotides.
Subsequently, DNA polymerase I degrades the primers and tills the gaps so created, while the polynucleotide ligase joins the ends of the DNA fragments.
The double-stranded circular DNA having the (+) and (-) strands is called the replicative form (RF). Each parental RF molecule replicates to produce about 60 copies of RF molecules These RF molecules produce about 500 (+) strands through the “rolling circle” mechanism of replication.
A nick is produced in the origin of the (+) strand of RF by the gene A protein which remains attached to the 5′-end so produced. The 5′-end of the (+) strand progressively unwinds and separates from the (-) strand. DNA polymerase III progressively adds nucleotides to the 3′- OH end of (+) strand, using the (-) strand as the template; this synthesis is continuous.
The intact (-) strand rolls about and its replication continues till the origin point is reached. The gene A protein now cleaves the strand in the origin and joins its 3′-end to the 5′-end. The RecA protein frees itself from the circularized (+) strand and binds to the 5′-end generating the plus (+) strand due to its cleavage.
The DNA polymerase III keeps adding nucleotides to the 3′-end of the (-) strand, while its 5′-end goes on unwinding till the origin is reached within the RecA again cleaves the (+) strand. In this manner, rolling circle replication goes on generating circular single-stranded φX174 chromosome (+ strands). The free circularized (+) strands become enclosed into the head of the phage φX174.