Project Report on DNA Replication

An exclusive project report on DNA Replication. This project report will help you to learn about: 1. Introduction to DNA Replication 2. Evidences for Semiconservative Method of Replication 3. Mechanism of DNA Replication 4. Bacterial DNA Replication.

Project Report # 1. Introduction to DNA Replication:

This is the process by which a cell copies its DNA. Replication is necessary so that the gene­tic information present in cells can be passed on to daughter cells following cell division. Delbruck suggested that Watson-Crick models of DNA could theoretically replicate by three modes – conservative, semiconservative and dispersive (Fig. 4.10).

i. According to the conservative mode the two double helices formed, one would be entire­ly of old material and the other entirely of new material.

ii. According to the semiconservative mode, each strand of the two double-helices formed would have one old and one new strand.

iii. According to the dispersive mode of replication,. DNA double-helix would break at several points forming many pieces. Each piece would replicate, then the pieces would recon­nect at random. Thus the two double helices formed would have a patchwork of old and new pieces.

However, as the semiconservative mode of replication is evidenced to be the general pattern of replication, this method has been discussed below.

Double helix model of DNA as prepared by Watson and Crick is endowed with the property of replication. The two strands of the double helix which is held together by bonding between the two bases A-T, G-C, unwind and separate into two single strands. The two strands of helix are wound anti-parallel to each other.

With the onset of unwinding, the synthesis of new strands or duplication starts with the help of the enzyme DNA polymerase, discovered by Kornberg (Fig. 4.11).

As the two strands are com­plementary, the complementary bases are attached in the sister strands against the parental one (Fig. 4.12). Of the two strands in the newly synthesized double helix, one is parental and the other is the newly synthesized one. As such, the replication is termed as semi-conservative.

If a single strand is available, one can produce the composition of sister strand on the basis of com­plementarity. The parental strand serves as a mould or template for the synthesis of new strands.

For combined replication, the same pro­cess is repeated where both the strands of the double helix serve as template for the synthesis of new double helices. The entire process is comparable to the production of print from a negative in photography, where the darker areas in a negative appear as lighter area in posi­tive.

The fundamental difference between photo­graphic processing and DNA replication is that, in the photography, negative remains as nega­tive and innumerable prints can be taken from the same negative, and prints cannot serve as a negative as in replication of DNA strands.

In DNA replication, though the two strands of the newly synthesized double helix which theoreti­cally contains a negative and a positive, can both serve as negative in next cycle of repli­cation.

Project Report # 2. Evidences for Semiconservative Method of Replication:

There is sufficient evidence to prove that double-stranded DNA replicates by semiconser­vative method.

Meselson and Stahl’s Experiment:

Meselson and Stahl in 1958 reported the results of an experiment which was designed to test whether double stranded DNA replicates in a semicon­servative manner.

The following two basic principles were involved:

(i) DNA was labelled with heavy nitrogen (N¹⁵) and was then allowed to replicate in a medium containing N¹⁵. If replication is semi-conservative then after first generation of repli­cation, one of the two strands would have nor­mal nitrogen (N¹⁴).

The resulting molecule would have a density which is intermediate between N¹⁴ DNA and. This density will gradu­ally fall in the replication of following cycles and would approach that of N¹⁴ DNA.

(ii) Another principle utilized was the prepa­ration of cesium chloride density gradient. This density gradient is prepared by gradual dilution of a heavy salt solution. When this is subjected to ultracentrifugation with a substance having density within the range of this gradient, this substance will find place at its own level of density. This enables detection of very slight differences in density.

Meselson and Stahl allowed Escherichia coli cells to grow on N¹⁵ culture medium for about 14 cell generations, so that almost all nitrogen (N¹⁴) in DNA is replaced by N¹⁴. Then the cells were abruptly transferred to N¹⁴ culture medium.

Since the time required for one cell generation was determined to be about 30 minutes, it was possible to remove cells after one or more known number of generations of replication. Their DNA could then be analysed and the following results were obtained.

DNA sample obtained one cell generation after the transfer to N¹⁴ culture medium showed only one density band as observed through ultra­violet absorption pattern. This band indicated uniform homogeneous density of DNA after one cell generation.

The band was exactly between the bands formed by N¹⁴ DNA and N¹⁵ DNA, indicating that all DNA found after first gene­ration have intermediate density (Fig. 4.13). This is what one would expect if DNA replicates in a semiconservative manner.

After two generations, when DNA was ana­lysed, two bands were observed, as one would expect according to semiconservative method of replication. These two bands were of equal intensity after the second generation. In the sub­sequent generations, although the same two bands appeared, the intensity of hybrid density band gradually decreased, while the intensity of light density band gradually increased.

Meselson and Stahl’s experiment is, thus, a classical experiment which was the first to show that DNA replicates in a semiconservative manner (Fig 4.14).

Taylor’s Experiment:

In an experiment on Vicia faba, conducted by Taylor et al. (1957), plant cells were first grown in a medium con­taining radioactive thymidine and later trans­ferred to a normal medium. Nuclei in the S phase incorporated thymidine in DNA while those which had completed DNA replication were not tagged. After metaphase arrest with colchicine, in the X₁ division, both chromatids appeared labelled, as studied through autoradiography. In the next cell generation X₁, in normal growth medium, one strand of each chromosome was labelled while the other was not.

It was concluded that prior to replication each chromosome behaves as if it has two units of DNA along its length. During S phase, two new labelled units are built along the original DNA threads following semiconservative method. Thus each chromatid includes an original non-labelled strand and a new labelled strand complementary to it.

In the second S phase, since the medium contains no isotopes, the labelled and un-labelled strands separate into labelled and non-labelled sister chromatids (Fig. 4.15).

Project Report # 3. Mechanism of DNA Replication:

The mechanism of DNA replication is very similar in most organisms. Differences exist only with respect to the enzymes and proteins involved (Table 4.3). In prokaryotes such as E. coli, the enzymes, DNA polymerases I and III, are responsible for DNA synthesis, while DNA polymerase II is involved in repair replication.

In eukaryotes DNA is replicated by different DNA polymerases (α, β, γ, δ, ԑ, к, etc.). Replication needs to be very accurate because even a small error would result in the loss of important gene­tic information after just a few cell divisions.

Accuracy is ensured by the ability of the DNA polymerases to check that the correct bases have been inserted in the newly synthesized strand. This is achieved through the reverse (3′ > 5′) exonuclease activity of the enzymes which allows them to remove incorrectly inserted bases from newly synthesized DNA and replace them with the correct base. This is referred to as proof­reading ability.

The Replication Fork:

During DNA replica­tion the entire DNA double helix is progressively unwound producing segments of single-stranded DNA which can be copied by DNA polymera­ses. Unwinding of the double helix begins at a distinct position called the replication origin and gradually progresses along the molecule, usually in both directions (bidirectional) or may be uni­directional (Fig. 4.16A).

In unidirectional mode, one replication fork leaves the origin and pro­ceeds along the DNA, whereas in bidirectional mode, two replication forks are formed and pro­ceed away from the origin in opposite directions (Fig. 4.16B).

Replication origins usually contain sequences rich in weak A-T base pairs. The region where the helix unwinds and new DNA is synthesized is called the replication fork. At the replication fork a number of distinct events occur under the influence of different enzymes and proteins (Fig. 4.17):

(i) Separation of the double helix:

This is achieved by the action of a helicase enzyme. Following separation of the strands, single-strand binding (SSB) protein attaches to the DNA and prevents the double helix from reforming.

(ii) Synthesis of leading and lagging strands:

Synthesis of DNA by DNA polymerases occurs only in the 5′ → 3′ direction. As the two strands of the double helix run in opposite direc­tions (one strand runs 5′ → 3′ and the other 3′ → 5′), slightly different mechanisms are required to replicate each. One strand, called the leading strand, is copied in the same direction as the unwinding helix and so can be synthesized con­tinuously (Fig. 4.18A).

The other strand, known as the lagging strand, is synthesized in the oppo­site direction and must be copied discontinuously. The lagging strand is synthesized as a series of segments known as Okazaki fragments (Fig. 4.18A).

(iii) Priming:

DNA polymerases require a short double-strand region to initiate or prime DNA synthesis. This is produced by an RNA polymerase, called primase, which is able to initiate synthesis on single-stranded DNA. The primase synthesizes a short RNA primer sequence on the DNA template creating a short double-stranded region.

In E. coli, DNA poly­merase III then synthesizes DNA beginning at the RNA primer. On the lagging strand, synthesis ends when the next RNA primer is encountered. At this point DNA polymerase I takes over and removes the RNA primer replacing it with DNA (Fig. 4.18B). In eukaryotes, the situation is different.

DNA polymerase α which has integral primase activity is responsible for initiating DNA synthesis. DNA is replicated by DNA polymerases α and δ with a synthesizing the lagging strand and δ synthesizing the leading strand. The other polymerases have ancillary roles. DNA polymerase e is involved in DNA repair and DNA polymerase γ replicates mitochondrial DNA.

(iv) Ligation:

The final step required to com­plete synthesis of the lagging strand is for the Okazaki fragments to be joined together by phosphodiester bonds. This is carried out by a DNA ligase enzyme (Fig. 4.18B).

Project Report # 4. Bacterial DNA Replication:

Although the mechanism of DNA replication is similar in all organisms, the overall process varies depending on the nature of the DNA molecule being copied. Different strategies are required for repli­cation of the circular DNA molecules which occur in bacteria and for the linear chromosomal DNA molecules present in eukaryotes.

The sim­plest and most common form of replication for circular DNA involves a single origin of replica­tion from which two replication forks progress in opposite directions. This results in an intermedi­ate d form (Fig. 4.19). The replication forks even­tually meet and fuse, and replication terminates.

The replication of DNA molecules requires unwinding of the DNA double helix. This causes the helix ahead of the replication fork to rotate. For circular DNA molecules that do not have free ends, this produces supercoiling of the DNA pre­venting the replication fork from progressing.

This problem is overcome by the action of enzymes called gyrases or topoisomerases. DNA topoisomerase I produces a transient break in the polynucleotide backbone of one of the DNA strands, a short distance ahead of the replication fork, allowing the DNA to rotate freely around the other intact strand removing the supercoiling.

The enzyme then rejoins the ends of the broken strand. When replication of a bacterial chromo­some is completed, two circular daughter molecules that are interlocked are produced.

These are separated by the action of DNA topoi­somerase II which acts by causing transient breaks in both strands of one of the DNA molecules allowing the other DNA molecule to pass through, thus separating the two daughter molecules. The topoisomerase II enzyme then rejoins the broken strands.

DNA Replication in Eukaryotes:

Due to the extreme length of eukaryotic chromosomes, DNA replication must be initiated at multiple origins to ensure that the process is completed within a reasonable time span. Replication forks proceed in either direction from each replication origin forming replication bubbles, which even­tually meet and merge.

DNA replicated from a single origin is called a replicon. Not all the DNA is replicated at once. Clusters of about 50 replicons initiate simultaneously at defined points during S phase. Areas containing trans­criptionally active genes are replicated first with non-active regions replicated later.

The DNA in eukaryotic chromosomes is packaged as DNA-protein complexes called nucleosomes. As the replication fork progresses, DNA must unwind from the nucleosome for replication to occur. This slows the progress of the replication forks and may explain the short length of the Okazaki fragments on the lagging strand in eukaryotes (100-200 bases) compared with prokaryotes (1000-2000 bases).

After the replication fork has passed, the nucleosomes reform. Replication of linear eukaryotic chromo­somes poses a problem not encountered with circular bacterial chromosomes. The extreme 5′ end of the lagging strand in linear DNA cannot be replicated because there is no room for an RNA primer to initiate replication.

This creates the potential for chromosomes to shorten after each round of replication leading to a loss of genetic information.

The problem is overcome by a specialized structure at the end of the chro­mosome known as the telomere which contains tandem repeats of a simple non-coding sequence. In addition, the 3′ end of the leading strand extends beyond the 5′ end of the lagging strand.

The enzyme telomerase contains an RNA molecule which partly overlaps with and binds to the repeat sequence on the leading strand. The enzyme then extends the leading strand using the RNA as a template.

The telomerase then disso­ciates and binds to the new telomere end, so that the leading strand is extended again. This pro­cess of extension may occur hundreds of times before the telomerase finally dissociates. The newly extended leading strand then acts as a template for replication of the 5′ end of the lagging strand (Fig. 4.20).

The two processes whereby the DNA is shortened during normal replication and lengthened by the action of the telomerase are roughly balanced; so the overall length of the chromosome remains approxi­mately the same.