DNA Replication, Recombination

							DNA Replication, Repair and Recombination
Topics: 1. An introduction to Cell Cycles 2. Semi Conservative Method of Replication: An Experimental View 3. Enzymes Involved in Replication 4. DNA Replication: Mechanism 5. DNA Recombination 6. Repair of Damaged DNA 1. An introduction to Cell Cycles
Cell division: Cell division is an important phenomenon in the life cycle of a cell. A cell should duplicate its entire DNA before it is going to divide. In higher organisms like eukaryotes, this occurs during S phase of the cell cycle. Invariably, all types of cells undergo a division cycle during their life span. Certain cells like stem cells are continually dividing through out the life. Others divide a specific number of times until cell death (apoptosis – cell suicide) occurs, and still others divide a few times before entering a terminally differentiated state. Mostly, all cells of our body fall into the latter category of cells. At the end of cell division, two daughter cells are formed. Hence during the process of cell division itself, every component inside the cell must be duplicated in order to make certain the survival of the two resulting daughter cells. DNA replication:

In order to make the cell survival accurate, an efficient and rapid duplication of the cellular genome needs to occur which is termed as ‘DNA replication’. Eukaryotic Cell Cycle: The sequence of events that occurs during the lifetime of a cell is defined as the ‘cell cycle’. The eukaryotic cell cycle is divided into 4 major stages. During each stage, a specific sequence of events occurs. The ultimate conclusion of one cell cycle is cytokinesis resulting in two individual, identical daughter cells. The 4 phases of a typical cell cycle and the events occurring during each phase are outlined: 1. Mitotic phase (or) M phase: This is the initial stage when cells prepare for and then undergo the process of During mitosis the cytokinesis. M phase is simply termed as ‘mitosis’ also. chromosomes are paired and then divided prior to cell division. The events in this stage of the cell cycle leading to cell division are prophase, metaphase, anaphase and telophase. All these four stages have their own significance. 2. G1phase: This stage corresponds to the gap in the cell cycle which follows cytokinesis. During this phase, the cells make a decision to either exit the cell cycle or become dormant or terminally differentiated or to continue dividing. Terminal differentiation is identified as a non-dividing state for a cell. Dormant and terminally differentiated cells are identified as being in G0 phase. Cells in G0 can remain in this state for extended periods of time without division. Specific stimuli may induce the G0 cell to re-enter the cell cycle at the G1 phase or alternatively may induce permanent terminal differentiation. During G1 phase, cells begin synthesizing all the cellular components needed in order to generate two identically complimented daughter cells. As a result the size of cells begins to increase during G1. 3. S phase: This is an important stage of the cell cycle during which the DNA is replicated. This is the DNA synthesis phase. Additionally, some specialized proteins like histones are also synthesized during S phase. 4. G2 phase: This is the final stage after the completion of DNA replication. During G2 phase,

(i) (ii) (iii)

Chromosomes begin to condense Nucleoli disappear Spindle pores are produced.

Typical eukaryotic cell cycles take up approximately 16 - 24 hrs when grown in culture. However, in the context of the multicellular organization of organisms the cell cycles can be as short as 6 - 8 hrs to as much greater as 100 days. The G1 phase of the cycle leads to this high variability of cell cycle durations.

2. Semi Conservative Method of Replication: An Experimental View
DNA Replication is semiconservative in nature. The semiconservative nature of DNA replication means that the newly synthesized daughter strands remain associated with their respective parental template strands. Hence the newly formed duplex will consist of an old parental strand paired with the new daughter stand copied from it.

Instead, if the two newly synthesized strands together paired themselves to for a new duplex, then it is termed to be ‘conservative’.

The process of DNA replication begins at specific sites in the chromosomes termed as the origins of replication. It requires a specific primer bearing a free 3'-OH end, proceeds particularly in the 5'----->3' direction on both strands of DNA concurrently. This continuous process results in the copying of the template strands in a semiconservative manner.

When the replication process is complete, two DNA molecules which are identical to each other and identical to the original DNA have been produced. Each strand of the original molecule has remained intact as it served as the template for the synthesis of a complementary strand.

This mode of replication is described as semi-conservative, (i.e) one-half of each new molecule of DNA in the daughter cell is an old one and the other-half is a newly synthesized one.

Watson and Crick had suggested that this was the way the DNA would be replicated. The experimental proof of the model was given by the experiments of M. Meselson and

W.F.Stahl. This classic experiment first gives a clear-cut evidence for the semiconservative mechanism of DNA replication.

Meselson and Stahl’s experiment:

The most popular bacterium Escherichia coli (E.coli) which lives in the digestive tract of humans is selected by these two eminent scientists to notice the significant changes made during DNA replication. E.coli cells were initially cultured in a selective medium of ammonium salts prepared with the isotope “heavy nitrogen” (15N) until all the cellular DNA of the bacterial cells contained this isotope. After this, the bacterial cells which contain this heavy isotope were transferred into a culture medium which contained the normal ‘light isotope’ (14N). In between, the samples are periodically from this culture. The samples subjected to density-gradient equilibrium centrifugation, showed a clear distinction of light-light (L-L), heavy-heavy (H-H) and heavy-light (H-L) DNA duplexes.

All the banding patterns resulted were reliable with the semiconservative mode of replication and inconsistent with the conservative mechanism. The analysis of the same experimentation with the higher eukaryotic DNA such as plant sells also revealed out with similar patterns. Hence, unanimously, whether prokaryotic or eukaryotic, the DNA is getting replicated in a semi conservative mechanism.

3. Enzymes Involved in Replication
DNA replication is a very complex process and involves multiple enzymatic activities. Replication of DNA is a necessary process during cell division cycles. As the genetic complement of the resultant daughter cells must be the same as the parental cell, DNA replication must possess a very high degree of fidelity. The mechanics of DNA replication was originally characterized in the bacterium, Escherichia. coli which contains 3 distinct enzymes capable of catalyzing the replication of DNA. These have been identified as DNA polymerase I, II, and III. DNA polymerase I is the most abundant replicating activity in E. coli. Also, it has the primary role in order to ensure the fidelity of replication by means of the repair of damaged and mismatched DNA. Replication of the E. coli genome is carried out by DNA Polymerase III. This enzyme is much less abundant than DNA polymerase I, even its activity is nearly 100 times that of pol I. The capability of DNA polymerases to replicate DNA requires a number of additional accessory proteins. The combination of polymerases with several of the accessory proteins yields the DNA polymerase holoenzyme. These accessory proteins are primase, processivity accessory proteins, helicases, DNA ligases, and single strand binding proteins, and topoisomerases.

A portion of the double helix is unwound by the enzyme called as helicase. A molecule of a DNA polymerase binds to one of the double strand of the DNA and begins moving along it in the 3' to 5' direction. This strand is being used as a template for assembling a leading strand of nucleotides and reforming a double helix. In eukaryotes, this molecule is called DNA polymerase delta (δ). DNA synthesis can only occur from 5' to 3', a molecule of a second type of DNA polymerase called as epsilon (ε) in eukaryotes binds to the other template strand as the double helix opens. This molecule must synthesize discontinuous segments of polynucleotides called as Okazaki fragments.

Another enzyme called as DNA ligase I then stitches these together into the lagging strand.

4. DNA Replication: Mechanism
The eukaryotic chromosomes are larger in size. Hence in order to overcome the limitation of DNA synthesis, multiple origins of replication are present in order to complete replication in a reasonable period of time.   At the replication origin the strands of DNA must dissociate and unwind in order to allow access to DNA polymerase. Unwinding of the duplex at the origin as well as along the strands as the replication process proceeds is carried out by helicases. The resultant regions of single-stranded DNA are stabilized by the binding of single-strand binding proteins. The stabilized single-stranded regions are then accessible to the activities of other enzymes required for replication to proceed. The site of the unwound template strands is termed the replication fork.

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In order for DNA polymerases to synthesize DNA they must encounter a free 3'OH which is the substrate for attachment of the 5'-phosphate of the incoming nucleotide. During replication the 3'-OH is supplied through the use of an RNA primer, synthesized by the primase activity. The primase utilizes the DNA strands as templates and synthesizes a short stretch of RNA generating a primer for DNA polymerase. Synthesis of DNA proceeds in the 5'---->3' direction through the attachment of the 5'-phosphate of an incoming deoxy nucleosidetriphosphate (dNTP) to the existing 3'-OH in the elongating DNA strands with the concomitant release of pyrophosphate. Initiation of synthesis, at origins of replication, occurs simultaneously on both strands of DNA. DNA synthesis process then proceeds bidirectionally, with one strand in each direction being copied continuously and one strand in each direction being copied discontinuously.

The experimental support for this bidirectional mode of replication in the cells of higher eukaryotes was acquired by the fiber autoradiography of labeled DNAs got from mammalian cell cultures. When the replicating DNA extracted and isolated from eukaryotic cells are examined under the electron microscope, the clear “bubble” like structures extending from multiple replication origins are clearly visible. Synthesis of leading and lagging strand: During the process of DNA polymerases incorporating dNTPs into DNA in the 5'---->3' direction they are moving in the 3'---->5' direction with respect to the template strand. In order for DNA synthesis to occur simultaneously on both template strands as well as bidirectionally one strand appears to be synthesized in the 3'---->5' direction. In real, one strand of newly synthesized DNA is produced discontinuously. The strand of DNA synthesized continuously is termed the leading strand and the discontinuous strand is termed the lagging strand. The lagging strand of DNA is composed of short stretches of RNA primer along with the newly synthesized DNA which is of approximately 100-200 bases long (the approximate distance between adjacent nucleosomes). The lagging strands of DNA are also called as Okazaki fragments. The ability of a particular polymerase to remain associated with the template strand is termed its' processivity. The longer it associates the higher the processivity of the enzyme. DNA polymerase processivity is enhanced by additional protein activities of the processivity accessory proteins. One such example is a replisome. Role of DNA polymerases: DNA polymerases exist as dimers associated with the other necessary proteins at the replication fork and this representation is identified as the replisome. The template for the lagging strand is temporarily looped through the replisome such that the DNA polymerases are moving along both strands in the 3'---->5' direction simultaneously for short distances, up to the distance of an Okazaki fragment. As the replication forks progresses along the template strands, the newly synthesized daughter strands and parental template strands reform a DNA double helix. Hence it is explicit that that only a small stretch of the template duplex is single-stranded at any given time. The progression of the replication fork requires that the DNA ahead of the fork be continuously unwound. Since the eukaryotic chromosomal DNA is attached to a protein scaffold the progressive movement of the replication fork thus intruding severe torsional stress into the duplex ahead of the fork. Role of Topoisomerases: This torsional stress is relieved by the enzymes DNA topoisomerases. Topoisomerases relieve torsional stresses in duplexes of DNA by introducing either double- (topoisomerases II) or single-stranded (topoisomerases I) breaks into the backbone of the DNA. These breaks allow unwinding of the duplex and removal of the replication-induced torsional strain. The nicks are then resealed by the topoisomerases.

Role of DNA Ligases: The RNA primers of the leading strands and Okazaki fragments are removed by the repair DNA polymerases simultaneously replacing the ribonucleotides with deoxyribonucleotides. The gaps that exist between the 3'-OH of one leading strand and the 5'-phosphate of another is repaired by the DNA ligases. The gaps between one Okazaki fragment and another are also repaired by DNA ligases thereby the process of replication is completed resulting in two individual daughter strands. Biochemical Approach of DNA Replication: In a biochemical view of, the following are the processes happening during replication.
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DNA replication begins with the "unzipping" of the double stranded parent molecule of DNA as the hydrogen bonds between the base pairs are broken. Once exposed, the sequence of bases on each of the separated strands serves as a template to guide the insertion of a complementary set of bases on the strand being synthesized. Here the specificity of base – pairing comes into effect.

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The new strands being synthesized are assembled from deoxynucleoside triphosphates Each incoming nucleotide is covalently linked to the "free" 3' carbon atom on the pentose by the formation of covalent bonds. The second and third phosphates are removed together as a molecule of pyrophosphate (PPi). The nucleotides are assembled in the order that complements the order of bases on the strand serving as the template. Thus each G on the template guides the insertion of a C on the new strand, each C a G, and so on. When the process is complete, two DNA molecules have been formed identical to each other and to the parent molecule.

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5. DNA Recombination
DNA recombination refers to the phenomenon whereby two parental strands of DNA are tied together resulting in an exchange of portions of their respective strands.

DNA recombination results in the process leads to new molecules of DNA that contain a mix of genetic information from each parental strand. There are 3 main forms of genetic recombination. (i) Homologous recombination (ii) Site-specific recombination (i) Transposition Homologous recombination: Homologous recombination is the process of genetic exchange that occurs between any two molecules of DNA that share a region (or regions) of homologous DNA sequences. This form of recombination occurs frequently while sister chromatids are paired during reduction division of meiosis.   Indeed, it is the process of homologous recombination between the maternal and paternal chromosomes that imparts genetic diversity to an organism. Homologous recombination generally involves exchange of large regions of the chromosomes when compared to the other two types of recombination.

Most of the cases of homologous recombination are initiated by the doublestrand breaks in DNA. These breaks later broaden into gaps, resulting in singlestranded recombinogenic ends. This end is much active in invading the next duplex. The repair mechanism to fill the missing regions results in the formation of an intermediate having two crossed –strand Holliday junctions. The resolution of this intermediate happens by rotation followed by the cleavage and ligation of two strands at each Holliday junction. The recombination proteins expressed in the higher eukaryotes are similar to that of the E.coli. Hence, it is believed that the process of homologous recombination follows a similar mechanism both in prokaryotes and eukaryotes. Site-specific recombination: This involves exchange between much smaller regions of DNA sequence (approximately 20 - 200 base pairs) and requires the recognition of specific sequences by the proteins involved in the recombination process. Site-specific recombination events occur primarily as a mechanism to alter the program of genes expressed at specific stages of development. The site – specific recombinases has the ability to identify, cleave, and recombine the short homologous DNA sequences present in two different DNA molecules. The most significant site-specific recombinational events in humans are the body cells. The immune responses deal with the rearrangements that take place in the immunoglobulin genes during B-cell differentiation in response to antigen presentation are an exclusive of this type. The antibody production resulting from these gene rearrangements in the immunoglobulin genes is extremely diverse.

6. Repair of Damaged DNA
DNA can be damaged by various factors both intrinsic and extrinsic. DNA repair is a major defense against environmental damage to cells. This exclusive mechanism is present in almost all organisms examined including bacteria, yeast, drosophila, fish, amphibians, rodents and humans. DNA repair process can assist the cell to avoid the risk involved in processes that minimize cell killing, mutations, replication errors, persistence of DNA damage and genomic instability to the minimum. Persistent abnormalities in these processes may result in cancer and aging.

Using the combination of the biochemical and genetic approaches, the DNA repair mechanisms are extensively studied in E.coli which gives us three broad categories of repair pathways in mammalian cells. a) Mismatch repair of single base mispairs A single step reaction which involves a direct reversal by a single enzyme like photolyase b) Single and multi-step base excision mechanisms (i.e., glycosylases) and c) Multi-step reactions involving multiple protein components. Direct reversal mechanism: An example of the single step reaction is the direct reversal that can be accomplished by the bacterial photolyase enzyme: a cyclobutane pyrimidine dimer is converted into two adjacent pyrimidines, and thereby the lesion is repaired. Base excision repair (BER) is a multi-step process that corrects non-bulky damage to bases resulting from oxidation, methylation, deamination, or spontaneous loss of the DNA base itself. These alterations, although simple in nature, are highly mutagenic and therefore represent a significant threat to genome fidelity and stability. Simple base modifications such as monofunctional alkylations can be removed by the base excision repair system. But, in case of more complex, bulky lesions, the nucleotide excision repair pathways are required. Nucleotide excision repair mechanism: The most common type of DNA damage caused by the UV light is the thymine-thymine dimers and can be repaired by this nucleotide excision repair mechanism. Hence nucleotide excision repair is considered as the most important DNA repair pathway that fixes the majority of bulky lesions in DNA. These lesions include UV induced photoproducts, and bulky adducts (the DNA regions which contain chemically modified bases) such as those derived from cisplatin and 4-nitroquinoline oxide.

Nucleotide excision repair pathways differ in different parts of the mammalian genome: separate pathways operate for the repair of active or essential genomic regions versus regions that are noncoding. NER Nucleotide excision repair (NER) is the most flexible of the DNA repair pathways considering the diversity of DNA lesions it acts upon. The most significant of these lesions are pyrimidine dimers (cyclobutane pyrimidine dimers and 6-4 photoproducts) caused by the UV component of sunlight.

Points to Remember:
    Cell division is an important phenomenon in the life cycle of a cell which occurs in higher organisms like eukaryotes during S phase of the cell cycle. Cell division results in the formation of two new daughter cells. DNA replication is an efficient and rapid duplication of the cellular genome needs to occur In order to make the cell survival accurate. The sequence of events that occurs during the lifetime of a cell is defined as the ‘cell cycle’.

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The 4 phases of a typical cell cycle are Mitotic phase (or) M phase, G1phase:, S phase, and G2 phase. Typical eukaryotic cell cycles take up approximately 16 - 24 hrs when grown in culture. DNA Replication is semiconservative in nature. Semiconservative nature of DNA replication means that the newly synthesized daughter strands remain associated with their respective parental template strands.

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In semi-conservative mode of replication, one-half of each new molecule of DNA in the daughter cell is an old one and the other-half is a newly synthesized one. The experimental proof of semi-conservative model was given by the experiments of M. Meselson and W.F.Stahl. The mechanics of DNA replication was originally characterized in the bacterium, Escherichia. coli which contains 3 distinct enzymes capable of catalyzing the replication of DNA which are identified as DNA polymerase I, II, and III.

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DNA polymerase I is the most abundant replicating activity in E. coli. Also, it has the primary role in order to ensure the fidelity of replication. Replication of the E. coli genome is carried out by DNA Polymerase III. Efficiency of DNA polymerases to replicate DNA requires a number of additional accessory proteins like primase, processivity accessory proteins, helicases, DNA ligases, and single strand binding proteins, and topoisomerases.

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Combination of polymerases with several of the accessory proteins yields the DNA polymerase holoenzyme. The newly synthesized discontinuous segments of polynucleotides called as Okazaki fragments. The site of the unwound template strands is termed the replication fork. The strand of DNA synthesized continuously is termed the leading strand and the discontinuous strand is termed the lagging strand. The lagging strands of DNA are also called as Okazaki fragments. The ability of a particular polymerase to remain associated with the template strand is termed its' processivity. Torsional stress is relieved by the enzymes DNA topoisomerases.

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Topoisomerases relieve torsional stresses in duplexes of DNA by introducing either double- (topoisomerases II) or single-stranded (topoisomerases I) breaks into the backbone of the DNA.

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The gaps that exist between the 3'-OH of one leading strand and the 5'phosphate of another is repaired by the DNA ligases. DNA recombination refers to the phenomenon whereby two parental strands of DNA are tied together resulting in an exchange of portions of their respective strands.

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DNA recombination results in the process leads to new molecules of DNA that contain a mix of genetic information from each parental strand. Three main forms of genetic recombination are Homologous recombination, Sitespecific recombination, and Transposition. DNA can be damaged by various factors both intrinsic and extrinsic. DNA repair is a major defense against environmental damage to cells and this exclusive mechanism is present in almost all organisms examined including bacteria, yeast, drosophila, fish, amphibians, rodents and humans.

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Three broad categories of repair pathways in mammalian cells .Mismatch repair of single base mispairs, Single and multi-step base excision mechanisms (i.e., glycosylases) and c) Multi-step reactions involving multiple protein components.