Bio340 DNA

Pyrimidines

(cytosine, thymine, and uracil) have a single six-membered ring

As synthesis proceeds, the RNA primers are replaced by DNA. The primers are removed by the exonuclease activity of DNA pol I, and the gaps are filled in by deoxyribonucleotides. The nicks that remain between the newly synthesized DNA (that replaced the RNA primer) and the previously synthesized DNA are sealed by the enzyme DNA ligase that catalyzes the formation of the phosphodiester linkage between the 3'-OH end of one nucleotide and the 5' phosphate end of the other fragment

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The carbon atoms of the five-carbon sugar are numbered

1', 2', 3', 4', and 5'

The two polynucleotide chains of a double helix form a stable structure that follows two rules

1. 1. The bases of one strand are complementary to the bases in the corresponding strand (A pairs with T and G pairs with C) 2.The two strands are antiparallel with respect to their 5' and 3' ends

The process of DNA replication can be summarized as follows:

1. DNA unwinds at the origin of replication. 2.Helicase opens up the DNA-forming replication forks; these are extended bidirectionally. 3. Single-strand binding proteins coat the DNA around the replication fork to prevent rewinding of the DNA. 4.Topoisomerase binds at the region ahead of the replication fork to prevent supercoiling. 5. Primase synthesizes RNA primers complementary to the DNA strand. 6. DNA polymerase III starts adding nucleotides to the 3'-OH end of the primer. 7. Elongation of both the lagging and the leading strand continues. 8. RNA primers are removed by exonuclease activity. 9. Gaps are filled by DNA polymerase I by adding dNTPs. 10. The gap between the two DNA fragments is sealed by DNA ligase, which helps in the formation of phosphodiester bonds. The following table summarizes the enzymes involved in prokaryotic DNA replication and the functions of each.

The haploid human genome is 3 Gbp long (3x10^9 bp). There are approximately 50 trillion cells in the human body. How long would all of the human genomic DNA in your body be if it were stretch out end to end?

100 billion Km

Purines

Adenine and Guanine have 2 rings together

In prokaryotes, three main types of polymerases are known:.

DNA pol I, DNA pol II, and DNA pol III. It is now known that DNA pol III is the enzyme required for DNA synthesis; DNA pol I and DNA pol II are primarily required for repair

can now extend this RNA primer, adding nucleotides one by one that are complementary to the template strand.

DNA polymerase

can only extend in the 5' to 3' direction, which poses a slight problem at the replication fork.

DNA polymerase

These nucleotides are identified collectively as

Deoxynucleoside monophosphates ( N can refer to any 4 nucleotide base. ) (dNMP)

Basis of complementary bonding

Hydrogen bond donor: hydrogen on nitrogen or oxygen Hydrogen bond acceptor: free pair of electrons on nitrogen or oxygen

The base pairs are stabilized by hydrogen bonds; adenine and thymine form two hydrogen bonds and cytosine and guanine form three hydrogen bonds.

Hydrogen bonds are non-covalent bonds that form between the partial charges that are associated with the hydrogen, oxygen, and nitrogen atoms of nucleotide bases.

The antiparallel arrangement of the two strands of the double helix is essential for forming stable H bonds. It brings the partial charges of complementary nucleotides into alignment

If two strands were to align in parallel, the charges of complementary nucleotides would repel each other

reported the first evidence of bacterial origins of replication in 1963. Cairn's work showed expansion around the origin of replication, forming a replication bubble, once replication gets under way in bacteria. At each end of the replication, bubble is a replication fork; replication is complete when the replication forks meet.

John Cairns

The other strand, complementary to the 5' to 3' parental DNA, is extended away from the replication fork, in small fragments known as ________ each requiring a primer to start the synthesis. Okazaki fragments are named after the Japanese scientist who first discovered them. The strand with the Okazaki fragments is known as the _______

Okazaki fragments ,lagging strand.

synthesizes an RNA primer that is about five to ten nucleotides long and complementary to the DNA.

RNA primase,

is a method used to make a clone or an identical copy of an entire multicellular organism.

Reproductive cloning

coat the single strands of DNA near the replication fork to prevent the single-stranded DNA from winding back into a double helix. DNA polymerase is able to add nucleotides only in the 5' to 3' direction (a new DNA strand can be only extended in this direction). It also requires a free 3'-OH group to which it can add nucleotides by forming a phosphodiester bond between the 3'-OH end and the 5' phosphate of the next nucleotide.

Single-strand binding proteins

The diameter of the molecule is 20Å, where 1Å is 0.1 nm

The diameter results from the fact that each complementary base pair (A and T or G and C) is 20Å wide

prevents the over-winding of the DNA double helix ahead of the replication fork as the DNA is opening up; it does so by causing temporary nicks in the DNA helix and then resealing it.

Topoisomerase

During DNA replication, each of the two strands that make up the double helix serves as a template from which new strands are copied. The new strand will be complementary to the parental or "old" strand.

When two daughter DNA copies are formed, they have the same sequence and are divided equally into the two daughter cells.

A hydrogen bond is formed when

a weakly positive hydrogen atom already bonded to one electronegative atom (for example, the oxygen in the water molecule) is attracted to another electronegative atom from another molecule.

The two strands are _________in orientation; that is, the 3' end of one strand faces the 5' end of the other strand. I

antiparallel

Several experiments have demonstrated that DNA replication is

bidirectional

Following the transformation protocol, bacterial cells are plated onto an antibiotic-containing medium to inhibit the growth of the many host cells that were not transformed by the plasmid conferring antibiotic resistance. A technique called___________is then used for lacZ-encoding plasmid vectors such as pUC19.

blue-white screening

The chemical basis of the pairing is the formation of stable hydrogen (H)

bonds between the bases on the antiparallel strands

"mating" with other cells

conjuction

the parental DNA remains together, and the newly formed daughter strands are together

conservative replication

DNA nucleotides that are not part of a polynucleotide chain carry a string of three phosphate groups at the 5' carbon and are identified as dATP, dGTP, dCTP, and dTTP.

deoxynucleotide tryiphosphates (dNTP)

both copies of DNA have double-stranded segments of parental DNA and newly synthesized DNA interspersed.

dispersive model

If the of an egg cell is replaced with a diploid nucleus from the cell of any individual of the same species (called a donor), it will become a zygote that is genetically identical to the donor. The resulting zygote forms a blastocyst, the first step of embryonic development.

haploid nucleus

Sexual reproduction requires two cells; when the ________ egg and sperm cells fuse, a ________ zygote results

haploid, diploid

unwinds the DNA by breaking the hydrogen bonds between the nitrogenous base pairs

helicase

using the energy from ATP hydrolysis opens up the DNA helix. Replication forks are formed at each replication origin as the DNA unwinds. The opening of the double helix causes over-winding, or supercoiling, in the DNA ahead of the replication fork.

helicase

conjugation, transduction and transformation are examples of

horizontal gene transfer.

Blue colonies have a functional beta-galactosidase enzyme because the _____________ is uninterrupted, with no foreign DNA inserted into the polylinker site

lacZ gene

This continuously synthesized strand is known as the

leading strand.

Two of the three phosphates of a deoxynucleotide triphosphate (dNTP) are removed (as a pyrophosphate group) during phosphodiester bond formation,

leaving the nucleotides of a polynucleotide chain in their monophosphate form.

The important components of the nucleotide

nitrogenous base, deoxyribose (5-carbon sugar), and a phosphate group

progressing in both directions from particular positions in chromosomes called _________ where specialized initiator proteins bind to DNA to start the process of replication.

origins of replication

These are resolved by the action of topoisomerases. Primers are formed by a complex between primase, and the low-processivity enzyme pol α. After the RNA primer has been synthesized, pol α synthesizes DNA for a short distance and it is soon replaced by the main DNA polymerase.

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DNA nucleotides that are part of a polynucleotide chain have one phosphate residue attached to the hydroxyl group of the 5' carbon of one sugar of one nucleotide and the hydroxyl group of the 3' carbon of the sugar of the next nucleotide, thereby forming a 5'-3'

phosphodiester bond

For bases as part of nucleotides, all extra-cyclic primary oxygens are in the keto (not enol) form, all extra-cyclic primary amines are in the amino (not imino) form

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The 5' and 3' designations of the phosphate and hydroxyl at the ends of the DNA strands establish polarity; the two strands are antiparallel

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major grove and minor groove

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• Complementary base pairing combines one purine with one pyrimidine

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white colonies lack a functional beta-galactosidase enzyme, indicating the insertion of foreign DNA within the ________ of the plasmid vector, thus disrupting the lacZ gene.

polylinker site

Base pairing takes place between a __________ namely, A pairs with T and G pairs with C. Adenine and thymine are complementary base pairs, and cytosine and guanine are also complementary base pairs.

purine and pyrimidine;

In nature, free DNA typically comes from other lysed bacterial cells; in the laboratory, free DNA in the form of _________is introduced to the cell's surroundings.

recombinant plasmids

ATP hydrolysis is required for this process. As the DNA opens up, Y-shaped structures called __________are formed. Two replication forks are formed at the origin of replication and these get extended bi- directionally as replication proceeds.

replication forks

Multiple origins are necessary in eukaryotes as they have much larger genomes than bacteria and eukaryotic __________move slower than bacterial replication forks.

replication forks

An overview of three postulated types of DNA replication

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DNA has (a) a double helix structure and (b) phosphodiester bonds. The (c) major and minor grooves are binding sites for DNA binding proteins during processes such as transcription (the copying of RNA from DNA) and replication.

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DNA polymerase catalyzes the formation of a new phosphodiester bond attaching guanosine monophosphate to the 3' end of the new strand. Image modified from DNA chemical structure by Madeleine Price Ball

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Dolly the sheep was the first mammal to be cloned. To create Dolly, the nucleus was removed from a donor egg cell. The nucleus from a second sheep was then introduced into the cell, which was allowed to divide to the blastocyst stage before being implanted in a surrogate mother.

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Each nucleotide is made up of a sugar, a phosphate group, and a nitrogenous base. The sugar is deoxyribose in DNA and ribose in RNA.

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In a reaction catalyzed by DNA polymerase, and using cytosine on the template strand (right), the activated 3' OH of the deoxyguanosine in the growing strand (left) attacks the triphosphate group of the incoming dGTP. Image modified from DNA chemical structure by Madeleine Price Ball

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Notice that the bonds occur between the weakly positive charge on the hydrogen atoms and the weakly negative charge on the oxygen atoms. Hydrogen bonds are relatively weak and therefore are indicated with a dotted (rather than a solid) line.

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Recombinant DNA technology is the artificial recombination of DNA from two organisms. In this example, the human insulin gene is inserted into a bacterial plasmid. This recombinant plasmid can then be used to transform bacteria, which gain the ability to produce the insulin protein

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The steps involved in molecular cloning using bacterial transformation are outlined in this graphic flowchart.

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method suggests that each of the two parental DNA strands acts as a template for new DNA to be synthesized; after replication, each double-stranded DNA includes one parental or "old" strand and one "new" strand

semi conservative

white colonies resulting from this blue-white screening contain plasmids with an insert and can be further screened to characterize the foreign DNA. To be sure the correct DNA was incorporated into the plasmid, the DNA insert can then be

sequenced

A protein called the _______ holds the DNA polymerase in place as it continues to add nucleotides.

sliding clamp

Recent advances in biotechnology have made it possible to artificially induce asexual reproduction of mammals in the laboratory. It is achieved through a process called

somatic cell nuclear transfer.

Meselson and Stahl experimented with E. coli grown first in heavy nitrogen (15N) then in 14N. DNA grown in 15N (red band) is heavier than DNA grown in 14N (orange band), and sediments to a lower level in cesium chloride solution in an ultracentrifuge. When DNA grown in 15N is switched to media containing 14N, after one round of cell division the DNA sediments halfway between the 15N and 14N levels, indicating that it now contains fifty percent 14N. In subsequent cell divisions, an increasing amount of DNA contains 14N only. This data supports the semi-conservative replication model.

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Each polynucleotide chain has a _________________consisting of alternating sugar and phosphate groups throughout its length.

sugar-phosphate backbone

The addition of nucleotides requires energy; this energy is obtained from the nucleotides that have three phosphates attached to them, similar to ATP which has three phosphate groups attached. When the bond between the phosphates is broken,

the energy released is used to form the phosphodiester bond between the incoming nucleotide and the growing chain.

process of transferring a diploid nucleus from a somatic cell (usually a skin cell) into an enucleated egg.

therapeutic cloning

Two H bonds form between A and T;

three H bonds form between G and C

viral infrection

transduction

The most commonly used mechanism for introducing engineered plasmids into a bacterial cell is

transformation

a process in which bacteria take up free DNA from their surroundings.

transformation

taking up DNA from the en

transformation

Individual nucleotides are assembled into a polynucleotide chain by the enzyme DNA polymerase,

which catalyzes the formation of a phosphodiester bond between the 3' hydroxyl group of one nucleotide and the 5' phosphate group of an adjacent nucleotide.

The________ nucleus contains the genetic information to produce a new individual. However, early embryonic development requires the cytoplasmic material contained in the egg cell.

zygote

The haploid human genome is 3 Gbp long (3x10^9 bp). How long would the genomic DNA in a single diploid cell in your body be if it were stretched out end to end

~2m (Remember that 1 Angstrom = 0.1 nm, and that each base pair is separated from the other base pair by a distance of 3.4 Angstroms.)