Module 6 DNA and Genetics Outline

1. Brief history a. Mendel - 1866 b. Watson and Crick 1953 c. Human Genome Project - current 2. DNA Structure a. Nucleic Acids i. Nuclides 1. A-T 2. C-G ii. Bonds b. Carbohydrate Component c. Phosphate Component d. Primary structure e. Secondary Structure i. Single strand ii. Double strand f. ~3 billion base pairs g. Chromosome 3. DNA Functions a. Genetic Code i. Gene ii. Codon b. RNA/protein synthesis 4. DNA recombination and repair a. Replication b. Faulty repair c. Errors 5. Genome a. Stability b. Instability tumorgenesis c. Epigenetics

Reading:
Genomic Stability and Instability: A Working Paradigm Cheng and Loeb, 1997.

References:
Cells Lewin et al., 2007. Principles of Genetics Snustad and Simmons, 2009. Cell Biology Pollard and Earnshaw, 2002.

Introduction
DNA is the blueprint for life. A blueprint contains all the information needed for construction of a building. One difference between a true blueprint and DNA is the recipe needed for making each component. DNA not only holds instructions for building a cell, but also contains the recipe for making the plasma membrane, for instance. As previously discussed, the cell contains all the processes required for life and is considered the smallest unit of life. In this module, the structure and function of DNA will be addressed in more detail. DNA structure begins with simple molecules and ends with a series of complex bonds. As for function, DNA carries the basic information of life, but transfer of that information undergoes the lengthy process of transcription and translation. Each of these processes will be discussed and their importance revealed.

1. History
Although the study of genetics developed during the twentieth century, the roots of study began in the nineteenth century with Gregor Mendel. Mendels research, observation of pea plants in the monastery garden, was performed in relative obscurity. He studied the plants and followed which physical traits were carried from one generation to the next. Eventually, Mendel began to interbreed pea plants with differing characteristics to see which would be passed to the next generation. With further observation and interbreeding, Mendel began to propose that each gene, or hereditary trait, was composed of two parts, known now as alleles. Mendels first breeding of pea plants was a cross between a tall pea plant, growing two meters in height, and a short plant, growing only half a meter. The next generation of pea plants was tall, indicating two forms of alleles. As the second generation of pea plants were bred and grown, the

result was a mixture of tall and short plants. Figure 6.1 shows Mendels pea plant experiment. These results confirmed Mendels theory of hereditary factors existing in two forms. In 1866, Mendel published his discoveries but the article was not much noticed and he went on to do other things. Sixteen years after Mendels death, in 1886, his paper was revisited. The study of genetics, as a science, was born and Mendels research technique was applied to many organisms.

Figure 6.1 Mendels pea plant experiment (Snustad and Simmons, Figure 3.1). As Mendels paper became better known, a plethora of study began on inheritance in microorganisms, plants and animals. Mendel had demonstrated that physical traits, such as height, are passed from one generation to the next via genes. Now, the big question was What is a gene? In 1953, James Watson and Francis Crick tried to answer that question. Watson and Crick had studied DNA and knew nucleotides were connected together. These linkages were the product of chemical bonds between phosphate and sugar molecules located in the nucleotide itself. By linking the nucleotides together, a chain is formed and contains a particular sequence

unique to that chain. This sequence is what differentiates each chain. Having this knowledge in hand, Watson and Crick proposed that DNA molecules consist of two chains of nucleotides and these chains were held together with weak chemical bonds. The chemical bonds are needed to create double stranded DNA. In addition to proposing double stranded DNA, Watson and Crick discovered the two strands of DNA were wound around each other in a helical configuration. Figure 6.2 shows a representation of DNA structure, both with and without helical arrangement. Although the structure of DNA was determined, the idea of separate genes that encode traits was still being investigated.

FIGURE 6.2 Basic structure of DNA (a) displaying hydrogen bonds and (b) showing helical form (Snustad and Simmons, Figure 1.4). In the early 1900s, geneticists were working at identifying what genes were made of. After Watson and Crick discovered the structure of DNA, geneticists began to work on ways to determine the sequence of bases in DNA molecules. By obtaining the sequence of bases, or sequencing the DNA, all the information necessary to analyze the organisms genes should be present. The collection of DNA molecules that is characteristic to an organism is referred to as its genome. Genome sequencing began with bacteria and was first successful in sequencing the bacteriophage !"174. Following this success, the Human Genome Project began in 1990 and was a worldwide effort to sequence the approximately 3 billion nucleotide pairs in human DNA. The Human Genome Project initially began as a collaboration of researchers in several different countries and was funded by each government. However, a privately funded project was initiated and soon developed alongside the publicly funded project. In 2001, efforts from both projects led to several lengthy articles about the human genome. The articles indicated 2.7 billion nucleotide pairs had been sequenced and the human genome was estimated to have 30,000 to 40,000 genes. Upon further sequencing and completion of the Human Genome Project in 2003, the human gene number has been revised to a lower number of 20,000 to 25,000 genes. These genes have been

catalogued by location, structure, and potential function. Now, efforts have shifted to the discovery of how genes influence characteristics of the human being.

2. DNA Structure
DNA, or deoxyribonucleic acid, is composed of a series of repeating units called nucleotides. Nucleotides consist of carbon, oxygen, hydrogen, nitrogen and phosphorus. From these molecules, three basic elements are formed and combined to make a single nucleotide. The elements are a carbohydrate unit, a phosphorus unit and a nucleobase. The nucleobase is made from a combination of nitrogen and carbon atoms that form either five- or six-member rings. Nucleobases are involved in pairing throughout DNA and RNA polymers, which is known as base pairing. There are five nucleobases. The five major bases are adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U). Three of the nucleobases are found in both DNA and RNA; however, DNA and RNA each have one unique base. The basic structure of each nuclide is shown in Figure 6.3. Adenine, guanine and cytosine are the common nucleobases, while thymine is found only in DNA and uracil replaces thymine in RNA.

Figure 6.3 Nucleobase structure. The nucleobases above can be classified into two types: pyrimidines, sixmembered rings, and purines, which are fused five- and six-membered compounds. Pyrimidines are heterocyclic aromatic rings. The rings consist of two nitrogen and four carbon molecules, the base for aromatic rings. Thymine, cytosine and uracil are placed into the pyrimidine category. Although uracil is classified as a pyrimidine, it lacks a methyl group on its ring. In the case of purines, adenine and guanine are classified here. A purine is most simply described as a pyrimidine ring fused with an imidazole ring. An imidazole ring consists of five members, two nitrogen and three carbon molecules. In the DNA double helix, pyrimidines from one strand interact with purines from the other strand. This interaction is called complementary base pairing. In Figure 6.4, complementary base pairs are shown and hydrogen bonds are indicated as dashed lines. Bases are bonded together with hydrogen bonds. The hydrogen bonds are not covalent and can be broken and rejoined with relative ease. Base pairing only occurs

as indicated in Figure 6.4. Adenine will only pair with thymine (or uracil) and cytosine will only pair with guanine. This specific interaction is critical for all the functions of DNA. It helps maintain the sequence of DNA throughout replication and allows reversible interactions between the bases. As will be seen later, DNA replication depends on the separation of its complementary strands.

Figure 6.4 Complementary base pairs. Each type of base pair forms with a different number of hydrogen bonds. G-C forms with three bonds, whereas A-T forms with only two. As one can guess, three hydrogen bonds are more stable than two bonds. However, the assumption that DNA with high G-C content is more stable than that with low G-C content is misleading. The stability of DNA does not depend on inter-strand base interactions, but on intra-strand base interactions. Intra-strand base interactions are more stable in DNA with high G-C content due to base stacking interactions. Base stacking interactions are due to dispersion attraction, exchange repulsion and electrostatic interactions. GC stacking tends to be more favorable with adjacent bases than CG stacking. The effects of base stacking are important in the secondary and tertiary structure of RNA. All nucleobases are chemically linked to a carbohydrate unit. In both DNA and RNA, the carbohydrate is a pentose (five carbon) sugar. DNA contains 2-deoxyribose sugar and RNA contains ribose sugar. The ribonucleic acids contain hydroxyl groups connected to each carbon of the pentose ring. However, the deoxyribose sugar contains only four hydroxyl groups. Seen in Figure 6.5, the 2 carbon contains a

hydroxyl group for RNA, but not DNA. This is one of the distinguishing characteristics of RNA. Once the nucleobase becomes linked to a sugar, it is then referred to as a nucleoside.

(a)

(b)

Figure 6.5 Pentose sugars of DNA and RNA (a) 2-deoxyribose (b) ribose Nucleosides contain one of the nucleobases and either a deoxyribose or ribose sugar. The sugar is bonded to the nucleobase via ester bonds. Ester bonds are flexible and allow the DNA strands to move and bend. The bond is generally located between a nitrogen molecule of the nucleobase and the 1 carbon of the sugar. Figure 6.6 shows the pairing of sugar and nucleobase molecules for RNA. DNA pairing works in the same fashion but uses deoxyribose sugar.

Figure 6.6 Nucleosides of RNA. After the nucleoside is formed, one or more phosphate groups are joined to the sugar through phosphodiester bonds. These asymmetric bonds form between the third and fifth carbon atoms of adjacent pentose sugars. Due to the bonding nature of the phosphate groups, DNA and RNA have a direction. The phosphate groups are simply a single phosphorous atom surrounded by four oxygen atoms, see Figure 6.7, and are the same in both DNA and RNA. As the phosphate binds to the pentose sugar, a nucleotide is created. Nucleotides are the building blocks of DNA and RNA. In DNA, the nucleotides form long polymers that are linked together through phosphodiester bonds between the deoxyribose sugar and phosphate groups.

Figure 6.7 Phosphate group The backbone of DNA is a series of alternating carbohydrate and phosphate groups. Nucleobases are attached to the sugars and phosphates connect the nucleosides. This rope-like structure is called a DNA polymer, and, as mentioned above, is held together via phosphodiester bonds. Phosphodiester bonds are strong covalent bonds that connect the 3 carbon of one sugar to the 5 carbon of the next. Due to the asymmetry of linkage between each sugar, DNA and RNA have a direction. The direction is determined by the terminal end of the DNA strand. If a phosphate group is terminal, the end of the strand is said to be the 5 end. However, if a hydroxyl group from the sugar is located at the DNA terminus, it is called the 3 end. Shown in Figure 6.8 (a) is a picture of DNA showing the 5 and 3 ends.

(a)

(b) Figure 6.8 Single (a) and helical (b) DNA polymer with 5 and 3 ends (www.blc.arizona.edu/Molecular_Graphics/DNA_Structure/DNA_Tutorial.HTML).

The secondary structure of DNA is similar to a ladder. The sides of the ladder are the alternating carbohydrate and phosphate groups that make up the backbone of two DNA polymers. The polymers align, in opposite directions, with each other and the

nucleobases begin to form bonds. As appropriate hydrogen bonds are formed between the bases, a right-handed double helix is formed, see Figure 6.8 (b). Each base pair, either G-C or A-T, makes the rungs of the ladder. Essential features of a DNA double helix are two strands of DNA with the nucleobases bonded together. The sugarphosphate backbone of each DNA polymer is on the outside of the helix, while the bases are on the inside. Each base pair is stacked 0.34 nm from the next. The bases are added in a nearly perpendicular fashion to the long axis of the DNA polymer. As the helix is formed, each complete turn, approximately 10.5 base pairs, fills a 3.4 nm length. The spacing of each nucleotide and turn within the double helix are shown in Figure 6.9. This is considered the regular structure of DNA and is referred to as B-DNA. B-DNA is the conformation that DNA takes on under normal physiological conditions, such as those found within the nucleus.

Figure 6.9 Spacing of nucleotides within a DNA helix (Snustad and Simmons, Figure 9.9). Beyond the organization of the nucleotides into DNA polymers and then a double helix, the DNA molecules still undergo further packaging. Chromosomal DNA molecules are much longer than the diameter of the nucleus itself and must be highly compacted. To begin the process, DNA is coiled around a series of histones. Histones are typically found as a set of eight proteins. This octamer consists of a central tetramer flanked on each side by a heterodimer. DNA winds around the surface of the histone octamer in a helical path. The histone complex contains, on average, two turns of DNA, which consists of approximately 150 base pairs. Figure 6.10 give a representation of DNA wrapping itself around a histone octamer.

Figure 6.10 DNA organization around a histone complex, forming a nucleosome (nicerweb.com). After DNA has successfully wrapped around the histone octamer, the DNA and histone complex is referred to as a nucleosome. Nucleosome structure is often referred to as a string of beads. The string is DNA between the nucleosomes and the beads are histone complexes that wrap the DNA. Both an electrograph and drawing of a nucleosome substructure are shown in Figure 6.11. As can be seen, formation of nucleosomes reduces the accessibility of DNA to transcription and protein regulatory factors. Both strands of DNA must be free for the binding of proteins. A comparison of DNA not bound to histones and DNA bound shows that unbound DNA binds 10- to 104fold better to protein factors than nucleosomal DNA.

Figure 6.11 Electron micrograph (a) and illustration (b) of nucleosome substructure (Snustad and Simmons, Figure 9.21).

Chromatin structure beyond the nucleosome is poorly understood. However, a 30-nm filament has been shown to further condense the nucleosomes. The disk shaped nucleosomes are thought to arrange themselves along the long-axis of the filament. However, discrepancies have arisen about the method of nucleosome packing. There are currently four possible models, shown in Figure 6.11, that deal with nucleosome packing. The first is classic winding, or solenoid, model. Classical winding is similar to the DNA wrapping of histones. A single strand of nucleosomes are linked together in a spherical manner along the central axis of the fiber. This formation can be compared to a circular staircase. The second is a cross-linked formation. Cross-linking is thought to occur between nucleosomes on opposite sides of the long axis. This method of packaging is similar to the solenoid model, but is a two strand winding, similar to the double helix of DNA. The third method of stacking is a random stacking of nucleosomes. In this fiber, no obvious structure is seen. A final zigzag stacking is possible. As the name implies, the nucleosomes are linked in a zigzag pattern around a central axis. Although there are theories about the construction of the 30-nm fiber, a definitive answer is still in the future. Structural studies of chromatin fibers are difficult due to the fragile nature of the fibers and higher levels of chromatin packing, at the moment, can only be theorized.

Figure 6.11 Four models of nucleosome packing into the 30-nm filament. Panels E and F are electron micrographs of chromatin without and with histone H1, respective (Pollard and Earnshaw, Figure 13.6).

As a summary, Figure 6.12 shows the organization of DNA. The packaging begins with nucleosome formation and ends with the chromosome. The total length of DNA in any nucleus is approximately 2 meters. These 2 meters must be condensed to fit within a 6 m diameter nulceus. Therefore, chromatin must be condensed about 104fold in length. This condensation is similar to trying to fit 100 elephants into the back of a VW Beetle.

Figure 6.12 Packaging of DNA from a double helix to a chromosome (biology200.gsu.edu)

3. DNA Functions
The primary function of DNA is to carry the genetic instructions used in development and function of organisms. These instructions are coded in the DNA by a set of by rules referred to as the genetic code. In essence, the genetic code is responsible for defining how the DNA sequences are interpreted. As mentioned above, DNA is a long polymer of nucleotides connected by phosphodiester bonds. The sequence of these nucleotides are translated and used for protein synthesis. The rules of the genetic code are defined as a set of three nucleotides to be translated at one time. This sequence is called a codon. Each codon specifies a single amino acid for protein synthesis. The DNA codon table is shown in Figure 6.13. As can be seen, not every amino acid is classified by only one DNA codon. Serine, for example, is coded for by six varying codons. Four of those codons begin with the same two nucleotides and

vary in the third. This repetition of coding is called degeneracy. Degeneracy is defined as the redundancy of the genetic code. Although the genetic code has redundancy, it is not ambiguous. Each codon is specific for one particular amino acid. However, as seen with serine, the codons can vary in any of the three nucleotide positions. RNA has a similar codon table, but uracil replaces thymine.

Figure 6.13 DNA codon table. Degeneracy occurs due to a need to code for 20 amino acids and a single stop codon. If only two bases were used per codon, only 16 amino acids would contain unique codes (42=16). Two nucleotides do not give enough variance for all 21 required codes (20 amino acids plus one stop). When the nucleotide number is increased to three, there are 64 possible codes (43=64). This is the cause of genomic degeneracy, as seen in Serine. A benefit to the redundancy in the genetic code is the fault-tolerance for point mutations. Point mutations are a single base substitution that causes one nucleotide to be replaced with another. Taking serine as an example again, a point mutation in the third codon position would not alter the translation of the DNA sequence. If the initial genetic code for serine was TCG and a point mutation at position three caused the codon to read TCA, serine would still be added to the protein. So, these mutations would most likely be silent and not affect protein synthesis. Since DNA contains all genetic information required for life in an organism, there must be a way to move that information out of the nucleus and into the cell for use. This

process is called transcription. Transcription is defined as the process of creating a complementary RNA copy of a specific sequence of DNA. Similar to DNA replication, a single DNA strand is used to make an RNA strand. The main difference is only one strand of DNA is copied, not both as in DNA replication. Also, RNA is formed as a single strand, not two complementary strands. RNA synthesis occurs in a 5 3 direction, like DNA, and occurs within five simple steps. The first step is the unwinding of DNA. Proteins attach to the DNA helix and unzip the strands to allow access to the nucleotides. The hydrogen bonds are broken which allows for RNA synthesis. Step two pairs RNA nucleotides to the DNA nucleotides. This is similar to DNA replication. However, thymine is replaced by uracil, as mentioned previously. RNA polymerase, shown in Figure 6.14, binds DNA to assist in unwinding and RNA synthesis. Once the strand of RNA nucleotides begins to form, a backbone of alternating ribose sugar and phosphate molecules are added, completing step three. During step four, hydrogen bonds formed between DNA and RNA nucleotides are broken. This frees the newly synthesized RNA from the DNA helix. When the RNA is freed, it undergoes further processing to protect the 5 and 3 ends and completes step five by exiting the nucleus through a nuclear pore.

Figure 6.14 RNA synthesis from an unwound portion of DNA (Snustad and Simmons, Figure 11.7). Transcription ends with five biologically active RNAs. They are messenger (mRNA), transfer (tRNA), ribosomal (rRNA), small nuclear (snRNA) and micro (miRNA) RNAs. Only mRNA, tRNA and rRNA will be discussed here. mRNA carries information copied from DNA to the ribosome. As discussed previously, ribosomes are sites of protein synthesis. When an mRNA reaches and binds to a ribosome, protein synthesis, or translation begins. The sequence of mRNA determines the amino acid sequence of the protein to be produced. The second type of RNA is tRNA. tRNA is a small RNA chain, only about 80 nucleotides, that transfers a specific amino acid to a growing polypeptide. The amino acids are linked to tRNA by peptide bonds and act as linkers between amino acids and the codons in mRNA during translation. The final type of

RNA is rRNA. Implied from the name, rRNAs are structural and catalytic components that make ribosomes. Eukaryotic ribosomes contain four differing rRNA molecules. Three of these are synthesized in the nucleolus, discussed previously, and the fourth is synthesized elsewhere. rRNA combines with protein in the cytosol and forms the ribosome. The ribosome then has the ability to bind mRNA and carry out protein synthesis. Protein synthesis is the process that cells use to build proteins. It begins in the nucleus with RNA transcription. mRNA, as discussed above, leaves the nucleus to enter the process of translation. Translation is defined as the decoding of mRNA by the ribosome to produce a specific amino acid chain. As in transcription, translation proceeds in distinct phases. These are activation, initiation, elongation and termination. Activation attaches the correct amino acid to the correct tRNA. tRNA carries the amino acid to the mRNA and attaches to the correct codon. Initiation involves the small subunit of the ribosome, shown in Figure 6.15, binding to the 5 end of the mRNA. This initiates translation of the mRNA sequence. Elongation is just what it sounds like. Amino acids are added to the growing chain. The additions occur until the end of the mRNA is reached. At this point, termination occurs. tRNA does not recognize the stop codon sequence and the ribosome/mRNA complex disassembles. At this point, the amino acid chain is transported to the internal space of the ER and folded into an active protein. Figure 6.15 shows translation of mRNA by a ribosome.

Figure 6.15 Translation of mRNA, ribosome shown as gray circle surrounding the mRNA.

4. DNA Replication and Repair

Several methods of DNA replication have been postulated. They are conservative, dispersive and semiconservative. Conservative replication maintains the two original template DNA strands as a single helix while the newly synthesized strands form a second helix. In this model, one daughter cell receives the original template DNA while the other daughter receives the copied DNA. The dispersive method produces two copies of DNA, but neither strand is completely composed of template or new DNA. DNA within the chromosome is composed of a combination of both original or both new strands. The DNA polymers become mixed and the end results are similar to crossover events that occur during meiosis. The semiconservative method, confirmed by the Meselson-Stahl experiment in 1958, conserves one strand of the original DNA in each replicated helix. That is, as the original helix is broken for replication, the complimentary nucleotides that form the new DNA strand become attached via hydrogen bonds. The final two double strand DNA helices each consist of one original and one newly synthesized strand of DNA. A comparison of the three replication methods is shown in Figure 6.15.

Figure 6.15 Replication methods Now with the basic understanding of semiconservative replication, DNA replication can be described in more detail. DNA replication is the process by which all living organisms copy their DNA. In a sense, it is the basis for inheritance between cell and organism generations. In a similar fashion to RNA and protein synthesis, DNA replication occurs in three steps which are initiation, elongation and termination.

Initiation of DNA replication occurs at specific sites within the DNA. These sites are called replication origins, or origins of bidirectional replication. As the name implies, replication occurs in opposing directions. Two sets of DNA replication machinery head out from the origin in opposite directions. Once the replication machinery has been established, new strands of DNA are synthesized at a rate of about 3000 nucleotide additions per minute. Even with this speed, multiple origins are needed to allow complete replication within the time allotted for S phase. If each chromosome contained only one replication origin, approximately 2000 hours would be needed replicate the entire genome. Clearly, 2000 hours greatly exceeds the time reserved to complete the S phase of the cell cycle. DNA at the origin contains specific sequences that allow replication proteins to attach to the DNA. The initiator proteins recruit other proteins, such as DNA helicase, to separate the DNA. A family of DNA helicases are responsible for breaking hydrogen bonds between nucleotides and unwinding the DNA helix. At this point, a replication bubble has formed. DNA replication moves from initiation into elongation. During elongation, DNA synthesis extends new DNA polymers in a 5 to 3 direction. The purpose of the directionality is the need to attach new nucleotides to the 3 hydroxyl on the primer strand. DNA polymerases responsible for elongation do not have 3 to 5 synthesis activity, so they do not recognize 5 phosphate groups. DNA polymerases are the key players in elongation. DNA polymerases are a family of enzymes that carry out DNA replication. However, they do not attach directly to DNA templates and require an existing DNA strand paired with the template. Small strands of RNA, called primers, are created and attached to the DNA template. DNA polymerases are then able to synthesize the new strand of DNA. The new DNA strand is extended in the 3 direction with the addition of complementary nucleotides. Elongation of DNA requires a special set of proteins referred to as replication machinery. These proteins include DNA topoisomerases and single strand binding proteins. DNA topoisomerases are responsible for nicking a single strand of DNA which allows the strands the ability to swivel around each other. Strand nicking removes the build-up of DNA twists during replication. In addition to nicking a single strand, topoisomerases cut both backbones, a double strand cut, that enables one strand of DNA to pass through another. The double cut removes knots and entanglements that can form during replication. The role of the single strand binding proteins (SSBP) is just as their name implies. They bind to single stranded DNA until the second strand is synthesized. By attaching to single strand DNA, the SSBP prevents secondary structure formation within the DNA. When the second strand is complete, the SSBP releases the DNA and hydrogen bonds are formed to hold the DNA helix together. Once the replication proteins and enzymes have gathered together and attached to DNA, they are called a replication fork. The replication fork forms in the nucleus and

only during DNA replication. It is responsible for breaking the hydrogen bonds that hold the two DNA strands together. The replication fork moves along the chromosome in a 3 to 5 direction. Seen in Figure 6.16 is the basic structure of the replication fork.

Figure 6.16 Replication fork (Pollard and Earnshaw, Figure 45.1). As briefly mentioned, the replication fork moves in a 3 to 5 direction during replication. The movement is along the leading strand of DNA. The leading strand is defined as the DNA template that is synthesized in a 5 to 3 direction. Synthesis of the new strand is complementary to the movement of the fork and DNA polymerase activity. In a sense, DNA polymerase is able to read the template strand and add nucleotides to the new strand continuously. However, there is the problem on the second strand of the DNA helix. The replication machinery reads it in a 5 to 3 direction, opposite to the activity of DNA polymerase. As the template DNA is being unwound in a 5 to 3 direction, the second strand of DNA becomes synthesized in short, non-continuous segments. This strand is called the lagging strand. The lagging strand is characterized by growth in the opposite direction to the movement of the replication fork. On the lagging strand, DNA polymerase reads the DNA in short, separated segments. The RNA primer is placed at the beginning of each segment, unlike the leading strand which needs only one primer. As each segment is synthesized, another primer is placed at the replication fork to enable synthesis of another segment of DNA. Some of the discontinuities are caused by the replication fork itself. Replication machinery takes up room and the RNA primer is not able to attach to the DNA. Once the replication fork opens another section of DNA, the primer binds the template and synthesis continues. The short DNA fragments formed on the lagging strand are called Okazaki fragments. The Okazaki fragments are joined together by DNA ligase. DNA ligase is an enzyme that repairs single stranded discontinuities. Shown briefly in Figure 6.17 are leading and lagging strands of synthesized DNA, however, only one lagging strand is shown. For a computer animated video of DNA replication, please go to http://dnalc.org/resources/3d/04mechanism-of-replication-advanced.html.

Figure 6.17 Continuous vs discontinuous replication (Snustad and Simmons, Figure 10.1). DNA can be damaged by any number of factors, including normal metabolic activities and environmental radiation. This damage can consist of individual base damage or DNA structural damage. In any given day, DNA in human cells can be exposed to 1 million molecular lesions. These lesions can cause structural damage to the DNA which can interfere with transcription of genes within the genome. Two basic types of damage that will be discussed here are single-strand breaks (SSB) and doublestrand breaks (DSB). In the case of SSB, one strand of the DNA helix is severed; however, the two DNA strands have not separated from each other. DSB cleaves both DNA strands and results in two free ends of DNA. DSB is the most hazardous to the cell due to an increased possibility of genomic rearrangements. DNA damage is shown in Figure 6.18 as base damage. This can easily be translated to a break within the DNA strand.

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(b)

Figure 6.18 (a) Single-strand and (b) double-strand damage Repair of DNA damage begins with the identification of damage. As previously discussed, there are two DNA checkpoints within the cell cycle. These checkpoints are used to pause the cell cycle and allow repair of DNA. The primary repair method of a SSB uses the same proteins used by the base excision repair (BER) mechanism. BER, briefly, is the repair of a single damaged base. The base can be damaged by oxidation or hydrolysis, among others. BER removes the damaged base and replaces the missing nucleotide with the assistance of DNA polymerase. The DNA polymerase activity is similar to that used during DNA synthesis. DNA ligase acts on the new base to seal the nick in the DNA strand. For repair of a single-strand break, BER skips to the final step of ligation. The broken phosphodiester bonds are reformed and the DNA strand break is repaired. In addition to single-strand breaks, DNA can be damaged with double-strand breaks. Double-strand breaks severe both DNA strands at the same location resulting in two free ends of DNA. The DNA ends at the DSB typically have short single-stranded sequences that serve as microhomologies within the break. In order to repair damage, the cell has a choice of two pathways. They are non-homologous end joining (NHEJ) and homologous recombination (HR). NHEJ is called non-homologous because it does not require a homologous template for repair. The broken ends of DNA are ligated together using the microhomologies found in the DNA overhangs for alignment. NHEJ repair is mostly accurate, but can have some imprecision that leads to the loss of nucleotides. However, this is only seen with DNA overhangs that are not compatible. The other method of repair is homologous recombination (HR). HR does not rely on short microhomologies. Instead, the repair proteins search out homologous DNA or the sister chromosome. The damaged DNA is resected. Resection is the process by which DNA surrounding the damage is removed from the 5 end of the break. Once resection is complete, strand invasion occurs. Strand invasion takes the 3 end of the broken DNA and invades the homologous DNA. The lost DNA is then synthesized and the

chromosomes separate when repair of the DSB has been completed. The first steps of HR are shown in Figure 6.19

Figure 6.19 First steps of Homologous Recombination In addition to DNA damage, replication has its own set of inherent errors. Replication slippage is the most common cause of error. During replication, DNA polymerase is responsible for coping DNA. DNA polymerase moves at a speed comparable to the replication fork. However, on the lagging strand, DNA polymerase pauses between Okazaki fragments. The pause in replication can cause the polymerase to dissociate from the DNA and lead to replication slippage. Replication slippage occurs in regions of DNA that have short, repeated sequence. The dissociated polymerase leads to two types of error. The first is deletion of DNA. A genetic deletion is defined as a mutation in which part of the DNA sequence is missing. The deletion can range from a single base to an entire piece of a chromosome. When DNA strands become misaligned, replication can skip over a section of DNA. In the cause of p53, a deletion of part of the gene results in the development of Li-Fraumeni syndrome. In addition to deletions, replication slippage can result in insertions. An insertion is the addition of one or more nucleotides into the DNA sequence. One way in which insertions are created is multiple replications of the same DNA. Insertions can cause frameshift mutations within the DNA sequence, if the number of nucleotides is not divisible by three. Frameshift mutations alter the normal reading frame of a gene and the amino acids encoded for by the gene.

5. Genome
Maintaining genome integrity is just as important as maintaining genetic accuracy during replication. The genome, on average, is bombarded with up to a million, or 106, DNA breaks and lesions per day. Repair, as previously discussed, ensures the damage is fixed and genomic integrity remains intact. In addition to repair of damage, a series of checkpoints are in the cell cycle as quality control mechanisms. Mentioned previously, the two DNA checkpoints within the cell cycle are located in the G1 and G2 phases. The G1 checkpoint looks at the DNA to locate lesions and, more importantly, breaks. If a single-stranded nick were to be replicated, the DNA nick would become a full-fledged double-strand break. The break then causes instability within the genome. By pausing the cell cycle, the nick is repaired and replication occurs without further damaging the DNA. In the case of the G2 checkpoint, DNA is reviewed for mismatched nucleotides and unreplicated DNA. The cell cycle is, again, paused and repairs are made. Without these vital checkpoints, integrity of the genome is compromised and mutations are more likely to occur. Genome instability is a process of chromosomal alterations that can lead to a wide variety of problems. Instability leads to deletion or insertion of DNA, mentioned above, or a change in chromosome number, among other changes. During cell separation, more specifically meiosis, unequal separation of chromosomes can occur. When one daughter cell receives more than one copy of a chromosome, the cell is said to have aneuploidy. Aneuploidy is defined as an abnormal number of chromosomes in a cell. The chromosomes themselves have not been altered, but the number is either higher or lower than expected. A common occurance of aneuploidy in humans involves chromosome 21. The normal copy number for any chromosome in a human is two. When three copies of chromosome 21 appear in a developing fetus, the physical manifestation is Down Syndrome. In addition to aneuploidy, chromosomal structure can be affected by instability. Instability in the chromosomal structure can lead to rearrangements and duplications. Rearrangements most commonly occur between non-homologous chromosomes. One of the key characteristics of chromosome rearrangement is the fusion of two genes. Two separate genes cannot function properly when joined together and are common in cancer cells. Duplication of DNA is just that, a second unneeded copy. As discussed above, DNA duplication, not related to replication, can cause insertions that affect the structure of proteins. A separate type of DNA alteration does not affect the structure or chromosomal identity of the DNA itself. The DNA sequence can be altered in a superficial manner that does not affect gene expression to the same extent of DNA deletion or gene fusion. Epigenetics is the study of gene expression alterations caused by mechanisms other

than the underlying DNA sequence. The Greek prefix epi- of epigenetics refers to a feature that is on top of or in addition to genetics. These features cause changes that affect the phenotype of a cell without altering the genotype. A cell phenotype is an observable trait that has some influence over the development of an organism. These traits include characteristics such as morphology and behavior. The phenotype of a cell, or organism, depends on the genes that are expressed. The genotype of a cell is the genetic code. The genome of an organism contains the information used for gene expression. Epigenetic events alter the expression of genes within the genome. When this occurs, suppressed genes can be activated or expressed genes can be silenced. These epigenetic changes, however, do not represent changes in the genetic information. In Figure 6.21, a brief explanation of epigenetics and possible outcome is shown. Epigenetics can be split into two general classes based on mechanism:

Figure 6.21 Some epigenetic mechanisms and its consequences. DNA modification by covalent attachment of a protein or establishing a self-perpetuating protein state. DNA modification most commonly affects only one of the two alleles. As mentioned in Figure 6. , DNA methylation can activate or repress genes. The addition of a methyl group causes the genes to become less accessible for transcription leading to suppression of the gene. However, the methyl group can be removed during chromatin remodeling. Chromatin remodeling involves moving the nucleosomes to

ensure proper winding of DNA. The second class of epigenetics involves establishing a recurrent protein state. This might involve maintaining an alternative protein conformation throughout the life of the cell or modifying a specific protein. However, self-perpetuation of an altered protein becomes difficult during replication. The protein complex could divide equally between sister chromatin or remain completely on one chromatin. In the first case, there is no reason for the protein complex to split and then reconstruct itself. The second case would require complete assembly of a new protein complex. The existence of epigenetic effects leads to the belief that proteins responsible for such modifications have some sort of self-templating or self-assembling capacity.

Before taking the quiz, you should be able to answer the following
1. Give a brief description of the beginning of the study of genetics 2. Explain the differences between nuclides in DNA and RNA 3. Be able to label the general structure of DNA, including hydrogen bonds between pyrimidines and purines 4. # of base pairs in humans 5. Describe the differences between genes and codons and how they are related to each other 6. Differentiate between the three methods of DNA repair 7. Discuss potential ramifications of faulty repair 8. Describe the principle behind DNA recombination 9. Explain the importance of genomic stability and why genomic instability can potentially lead to tumorgenesis 10. Have a general understanding of epigenetics and its ramifications