29. DNA Structure and Replication

Prokaryotic and eukaryotic DNA polymerases elongate a new DNA strand by adding deoxyribonucleotides, one at a time, to the 3'-end of the growing chain

DNA chain elongation is catalyzed by DNA polymerase III. Using the 3′-hydroxyl group of the RNA primer as the acceptor of the first deoxyribonucleotide, DNA polymerase III begins to add nucleotides along the single-stranded template that specifies the sequence of bases in the newly synthesized chain. DNA polymerase III is a highly "processive" enzyme—that is, it remains bound to the template strand as it moves along, and does not have to diffuse away and rebind before adding each new nucleotide The nucleotide substrates are 5′-deoxyribonucleoside triphosphates The new strand grows in the 5′→3′ direction, antiparallel to the parental strand a total of two high-energy bonds are used to drive the addition of each deoxynucleotide. All four deoxyribonucleoside triphosphates (dATP, dTTP, dCTP, and dGTP) must be present for DNA elongation to occur. If one of the four is in short supply, DNA synthesis stops when that nucleotide is depleted. To ensure replication fidelity, DNA polymerase III has, in addition to its 5′→3′ polymerase activity, a "proofreading" activity (3′→5′ exonuclease). The enzyme requires an improperly base-paired 3′-hydroxy terminus and, therefore, does not degrade correctly paired nucleotide sequences.]

Each chromosome in the nucleus of a eukaryote contains one long linear molecule of double-stranded DNA, which is bound to a complex mixture of proteins to form chromatin

Eukaryotes have closed circular DNA molecules in their mitochondria, as do plant chloroplasts. A prokaryotic organism contains a single, double-stranded, supercoiled, circular chromosome. Each prokaryotic chromosome is associated with histone like protein and RNA that can condense the DNA to form a nucleoid. Plasmids may carry genes that convey antibiotic resistance to the host bacterium, and may facilitate the transfer of genetic information from one bacterium to another

DNA polymerase III continues to synthesize DNA on the lagging strand until it is blocked by proximity to an RNA primer. When this occurs, the RNA is excised and the gap filled by DNA polymerase I.

In addition to having the 5′→3′ polymerase activity that synthesizes DNA, and the 3′→5′ exonuclease activity that proofreads the newly synthesized DNA chain like DNA polymerase III, DNA polymerase I also has a 5′→3′ exonuclease activity that is able to hydrolytically remove the RNA primer Differences between 5′→3′ and 3′→5′ exonucleases: The 5′→3′ exonuclease activity of DNA polymerase I differs from the 3′→5′ exonuclease used by both DNA polymerase I and III in two important ways. First, 5′→3′ exonuclease can remove one nucleotide at a time from a region of DNA that is properly base-paired. The nucleotides it removes can be either ribonucleotides or deoxyribonucleotides. Second, 5′→3′ exonuclease can also remove groups of altered nucleotides in the 5′→3′ direction, removing from one to ten nucleotides at a time. This ability is important in the repair of some types of damaged DNA.

In prokaryotic organisms, DNA replication begins at a single, unique nucleotide site called the origin of replication

In eukaryotes, replication begins at multiple sites along the DNA helix. These sites include a short sequence composed almost exclusively of AT base pairs.This is referred to as a consensus sequence, because the order of nucleotides is essentially the same at each site. Having multiple origins of replication provides a mechanism for rapidly replicating the great length of the eukaryotic DNA molecules.

DNA is a polydeoxyribonucleotide that contains many mono-ribonucleotides covalently linked by 3-5 phosphodiester bonds.

In eukaryotic cells, DNA is found associated with various types of proteins (known collectively as nucleoprotein) present in the nucleus, whereas in prokaryotes, the protein-DNA complex is present in the nucleoid.

The DNA polymerases responsible for copying the DNA templates are only able to "read" the parental nucleotide sequences in the 3′→5′ direction, and they synthesize the new DNA strands in the 5′→3′ (antiparallel) direction.

Leading strand: The strand that is being copied in the direction of the advancing replication fork is called the leading strand and is synthesized continuously. Lagging strand: The strand that is being copied in the direction away from the replication fork is synthesized discontinuously, with small fragments of DNA being copied near the replication fork. These short stretches of discontinuous DNA, termed Okazaki fragments, are eventually joined to become a single, continuous strand. The new strand of DNA produced by this mechanism is termed the lagging strand.

Phosphodiester linkages between nucleotides (in DNA or RNA) can be cleaved hydrolytically by chemicals, or hydrolyzed by a family of nucleases:

Nucleases that cleave the nucleotide chain at positions in the interior of the chain are called endonucleases. Those that cleave the chain only by removing individual nucleotides from one of the two ends are called exonucleases

Eukaryotic DNA polymerases At least five key eukaryotic DNA polymerases have been identified and categorized on the basis of molecular weight, cellular location, sensitivity to inhibitors, and the templates or substrates on which they act

Pol α and pol Δ: Pol α is a multisubunit enzyme. One subunit has primase activity, which initiates strand synthesis on the leading strand and at the beginning of each Okazaki fragment on the lagging strand. The primase subunit synthesizes a short RNA primer that is extended by the pol α 5′→3′ polymerase activity, which adds a short piece of DNA. Pol Δ is then recruited to complete DNA synthesis on the leading strand and elongate each Okazaki fragment, using 3′→5′ exonuclease activity to proofread the newly synthesized DNA. Note: DNA polymerase Δ associates with the protein, proliferating cell nuclear antigen, which serves as a sliding DNA clamp in much the same way the β subunit of DNA polymerase III does in E. coli, thus ensuring high processivity. Pol β and pol ε are involved in DNA repair Pol γ replicates mitochondrial DNA.

This flow of information from DNA to RNA to protein is termed the "central dogma of molecular biology", and is descriptive of all organisms, with the exception of some viruses that have RNA as the repository of their genetic information.

Prokaryotic cells, which lack nuclei, have a single chromosome, but may also contain extrachromosomal DNA in the form of plasmids

DNA polymerases cannot initiate synthesis of a complementary strand of DNA on a totally single-stranded template. In de novo DNA synthesis, that free 3'-hydroxyl is provided by the short stretch of RNA, rather than DNA.

Rather, they require an RNA primer—that is, a short, double-stranded region consisting of RNA base-paired to the DNA template, with a free hydroxyl group on the 3′-end of the RNA strand This hydroxyl group serves as the first acceptor of a nucleotide by action of DNA polymerase. [Note: Recall that glycogen synthase also requires a primer

As the two strands unwind and separate they form a "V" where active synthesis occurs. This region is called the replication fork.

Replication of double-stranded DNA is bidirectional that is, the replication forks move in both directions away from the origin.

There are three major structural forms of DNA: the B form, described by Watson and Crick in 1953, the A form, and the Z form Q. Which structural form of DNA is not present? a. A form b. B form c. D form d. Z form Q. A scientist was studying the denaturation pattern of DNA strands. DNA were heated to temperatures at which one half of the helical structure is lost called as the melting temperature. Which of the following statment regarding melting temperature is true a. DNA that contains high concentrations of A and T denatures at a lower temperature than G- and C-rich DNA. b. DNA that contains high concentrations of G and C denatures at a lower temperature than A and T rich DNA. c. DNA that contains equal concentration of A and G denature at a lower temperature. d. None of the above

The B form is a right-handed helix with ten residues per 360° turn of the helix, and with the planes of the bases perpendicular to the helical axis. Chromosomal DNA is thought to consist primarily of B-DNA. The A form is produced by moderately dehydrating the B form. It is also a right-handed helix, but there are eleven base pairs per turn, and the planes of the base pairs are tilted 20° away from the perpendicular to the helical axis. The conformation found in DNA-RNA hybrids or RNA-RNA double-stranded regions is probably very close to the A form. Z-DNA is a left-handed helix that contains about twelve base pairs per turn [Note: The deoxyribose-phosphate backbone "zigzags," hence, the name"Z"-DNA.] Stretches of Z-DNA can occur naturally in regions of DNA that have a sequence of alternating purines and pyrimidines. For example polyg GC Transition between helical forms of DNA may play an important role in regulating gene expression.

As the two strands of the double helix are separated, a problem is encountered, namely, the appearance of positive supercoils (also called supertwists) in the region of DNA ahead of the replication fork

The accumulating positive supercoils interfere with further unwinding of the double helix. To solve this problem, there is a group of enzymes called DNA which are responsible for removing supercoils in the helix. Type DNA topoisomerases reversibly cut a single strand of the double helix. They have both nuclease (strand-cutting) and ligase (strand-resealing) activities. They do not require ATP, but rather appear to store the energy from the ester bond they cleave, reusing the energy to reseal the strand. Type I topoisomerease relax negative super coils (that is, those that contain fewer turns of the helix than relaxed DNA) in E. coli and both negative and positive super coils (that is, those that contain fewer or more turns of the helix than relaxed DNA) in eukaryotic cells.

Base pairing: The bases of one strand of DNA are paired with the bases of the second strand, so that an adenine is always paired with a thymine and a cytosine is always paired with a guanine. [Note: The base pairs are perpendicular to the axis of the helix]. One polynucleotide chain of the DNA double helix is always the complement of the other.

The amount of guanine equals the amount of cytosine, and the total amount of purines equals the total amount of pyrimidines. The base pairs are held together by hydrogen bonds: two between A and T and three between G and C These hydrogen bonds, plus the hydrophobic interactions between the stacked bases, stabilize the structure of the double helix

The process of DNA replication is called semi-conservative approach. Initiation of DNA replication commits the cell to continue the process until the entire genome has been replicated

The enzymes involved in the DNA replication process are template-directed polymerases that can synthesize the complementary sequence of each strand with extraordinary fidelity.

3'->5'-Phosphodiester bonds Phosphodiester bonds join the 5'-hydroxyl group of the deoxypentose of one nucleotide to the 3'-hydroxyl group of the deoxypentose of an adjacent nucleotide through a phosphate group.

The resulting long, unbranched chain has polarity, with both a 5'-end (the end with the free phosphate) and a 3'-end (the end with the free hydroxyl) that are not attached to other nucleotides

The chains are paired in an anti parallel manner, that is, the 5'-end of one strand is paired with the 3'-end of the other strand. The spatial relationship between the two strands in the helix creates a major (wide) groove and a minor (narrow) groove.

These grooves provide access for the binding of regulatory proteins to their specificcrecognition sequences along the DNA chain. [Note: Certain anti-cancer drugs, such as Dactinomycin, exert their cytotoxic effect by intercalating into the narrow groove of the DNA double helix, thus interfering with RNA and DNA synthesis.

Initiation of DNA replication requires the recognition of the origin of replication and/or the replication fork by a group of proteins that form the prepriming complex.

These proteins are responsible for maintaining the separation of the parental strands, and for unwinding the double helix ahead of the advancing replication fork. a. DNAa protein: Twenty to fifty monomers of dnaA protein bind to specific nucleotide sequences at the origin of replication, which is particularly rich in AT base pairs b. Single-stranded DNA-binding (SSB) proteins: Also called helix-destabilizing proteins, these bind only to single-stranded DNA. They bind cooperatively; that is, the binding of one molecule of SSB protein makes it easier for additional molecules of SSB protein to bind tightly to the DNA strand. The SSB proteins are not enzymesThese proteins not only keep the two strands of DNA separated in the area of the replication origin, thus providing the single-stranded template required by polymerase but also protect the DNA from nucleases that cleave single-stranded DNA. DNA helicase: These enzymes bind to single-stranded DNA near the replication fork, and then move into the neighboring double-stranded region, forcing the strands effect, unwinding the double helix. Helicases require energy provided by ATP. When the strands separate, SSB proteins bind, preventing reformation of the double helix

The cell cycle is controlled at a series of "checkpoints" that prevent entry into the next phase of the cycle until the preceding phase has been completed.

Two key classes of proteins that control the progress of a cell through the cell cycle are the cyclins and cyclin-dependent kinases (Cdk).]

Type II DNA topoisomerases bind tightly to the DNA double helix and make transient breaks in both strands

Type II DNA topoisomerases are also required in both prokaryotes and eukaryotes for the separation of inter locked molecules of DNA following chromosomal replication. Anticancer agent Etoposide target Topoisomerase II. DNA gyrase, a Type II topoisomerase found in bacteria and plants, has the unusual property of being able to introduce negative supercoils into relaxed circular DNA using energy from the hydrolysis of ATP. This facilitates the future replication of DNA because the negative supercoils neutralize the positive supercoils introduced during opening of the double helix. It also aids in the transient strand separation required during transcription. Bacterial DNA gyrase is the unique target of a group of antimicrobial agents called quinolones, for example, ciprofloxacin.

The two strands of the double helix separate when hydrogen bond between the paired bases are disrupted. Disruption can occur by a. Change in pH of the solution b. Heating Phosphodiester bonds are not broken.

When DNA is heated, the temperature at which one half of the helical structure is lost is defined as the melting temperature. The loss of helical structure in DNA, called denaturation, can be monitored by measuring its absorbance at 260 nm. ss DNA has a higher relative absorbance at this wavelength than does double-stranded DNA. DNA that contains high concentrations of A and T denatures at a lower temperature than G- and C-rich DNA. Under appropriate conditions, complementary DNA strands can reform the double helix by the process called renaturation (or reannealing) Q. The loss of helical structure of DNA is called denaturation. It can be monitored by measuring the absorbance of light at-- a. 235nm b. 260nm c. 325 nm d. 560 nm

Telomerase Eukaryotic cells face a special problem in replicating the ends of their linear DNA molecules. Following removal of the RNA primer from the extreme 5′-end of the lagging strand, there is no way to fill in the remaining gap with DNA. To solve this problem, and to protect the ends of the chromosomes from attack by nucleases, a complex of noncoding DNA plus proteins is found at these ends

a complex of noncoding DNA plus proteins is found at these ends called Telomeres telomeric DNA consists of several thousand tandem repeats of a noncoding hexameric sequence, AG3T2, base-paired to a complementary region of As and Cs. The TG strand is longer than its AC complement, leaving ssDNA a few hundred nucleotides in length at the 3′-end. In most normal human somatic cells, telomeres shorten with each successive cell division. Once telomeres are shortened beyond some critical length, the cell is no longer able to divide and is said to be senescent. In germ cells and other stem cells, as well as in cancer cells, telomeres do not shorten and the cells do not senesce. This is a result of the presence of a ribonucleoprotein, telomerase, which maintains telomeric length in these cells. Telomerase contains a protein that acts as a reverse transcriptase, and a short piece of RNA that acts as a template

Primase: A specific RNA polymerase, called primase, synthesizes the short stretches of RNA (approximately 10 nucleotides long) that are complementary and antiparallel to the DNA template

these short RNA sequences are constantly being synthesized at the replication fork on the lagging strand, but only one RNA sequence at the origin of replication is required on the leading strand Primosome: The addition of primase converts the prepriming complex of proteins required for DNA strand separation to form a primosome. The primosome makes the RNA primer required for leading strand synthesis, and initiates Okazaki fragment formation in lagging strand synthesis. As with DNA synthesis, the direction of synthesis of the primer is 5′→3′ (antiparallel to the template strand).