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DNA Structure

The building blocks of nucleic acids are nucleotides, each composed of:

– a 5-carbon sugar called deoxyribose

– a phosphate group (PO4)

– a nitrogenous base

• adenine, thymine, cytosine, guanine, uracil

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Nucleosides• Nucleosides: nitrogenous base linked to specific sugar

– RNA: adenosine, guanosine, cytidine, uridine– DNA: deoxyadenosine, deoxyguanosine, deoxycytidine,

(deoxy)thymidine

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DNA nucleoside RNA nucleoside

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Nucleotides

The nucleotide structure consists of

– the nitrogenous base attached to the 1’ carbon of deoxyribose

– the phosphate group attached to the 5’ carbon of deoxyribose

– a free hydroxyl group (-OH) at the 3’ carbon of deoxyribose

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Nucleotides

• Subunits of DNA and RNA– Nucleosides

linked to phosphate group via ester bond

– “dNTP’s”: DNA– “rNTP’s”: RNA

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DNA Structure

Nucleotides are connected to each other to form a long chain

phosphodiester bond: bond between adjacent nucleotides– formed between the phosphate group of

one nucleotide and the 3’ –OH of the next nucleotide

The chain of nucleotides has a 5’ to 3’ orientation.

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DNA structure determination

Chargaff's Rules

– Erwin Chargaff determined that

• amount of adenine = amount of thymine

• amount of cytosine = amount of guanine

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DNA StructureThe double helix consists of:

– 2 sugar-phosphate backbones– nitrogenous bases toward the interior of the

molecule– bases form hydrogen bonds with

complementary bases on the opposite sugar-phosphate backbone• Adenine pairs with Thymine (2 H bonds)• Cytosine pairs with Guanine (3 H Bonds)

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DNA Structure

The two strands of nucleotides are antiparallel to each other

– one is oriented 5’ to 3’, the other 3’ to 5’

The two strands wrap around each other to create the helical shape of the molecule.

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Types of DNA Structures

• Three forms of DNA– A form: right handed

helix– B form: the most

likely biological conformation, right handed helix

– Z form: form a left handed helix;

http://www.tulane.edu/~biochem/nolan/lectures/rna/images/Image1.gif

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Chemical Properties of DNA

• Factors that affect DNA structure

– Temperature: denaturation (can be reversible)

– pH: high pH can denature DNA

– Salt concentration: lowering salt concentration can denature DNA

– Chemicals: sodium hydroxide, formamide can also denature DNA

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DNA Replication

Matthew Meselson & Franklin Stahl, 1958

investigated the process of DNA replication

considered 3 possible mechanisms:

– conservative model

– semiconservative model

– dispersive model

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At the 0 time point: all the DNA had heavy 15N nitrogen

After 1 round: the DNA was a hybrid molecule, with an intermediate location

After 2 rounds: two molecules were seen:one that was hybrid, and one that was the lighter 14N DNA molecule.

Conclusion: Semiconservative replication

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DNA Replication

Meselson and Stahl concluded that the mechanism of DNA replication is the semiconservative model.

Each strand of DNA acts as a template for the synthesis of a new strand.

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DNA Replication

DNA replication includes:

– initiation – replication begins at an origin of replication

– elongation – new strands of DNA are synthesized by DNA polymerase

– termination – replication is terminated differently in prokaryotes and eukaryotes

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Prokaryotic DNA Replication

The chromosome of a prokaryote is a circular molecule of DNA.

Replication begins at one origin of replication and proceeds in both directions around the chromosome.

--origins of replications usually are rich in Adenine and Thymine

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Enzymes of Prokaryotic DNA Replication

• The double helix is unwound by the enzymes helicase , DNA topoisomerase,and DNA gyrase– SSBP (single stranded binding protein) helps

keep strands separated• DNA polymerase III (pol III) is responsible for most

of DNA synthesis– adds nucleotides to the 3’ end of the daughter

strand of DNA; DNA synthesis is from 5' to 3'– Requires RNA primers as a guide for synthesis

• RNA primers are made by the enzyme primase

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Enzymes of Prokaryotic DNA Replication

• DNA polymerase I: involved in proofreading and DNA repair

• DNA ligase: involved in connected ends of replicated DNA together

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Prokaryotic DNA Replication

leading strand is synthesized continuously (in the same direction as the replication fork)

lagging strand is synthesized discontinuously creating Okazaki fragments

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Eukaryotic DNA Replication

The larger size and complex packaging of eukaryotic chromosomes means they must be replicated from multiple origins of replication.

The enzymes of eukaryotic DNA replication are more complex than those of prokaryotic cells.

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Eukaryotic DNA Replication

Synthesizing the ends of the chromosomes is difficult because of the lack of a primer.

With each round of DNA replication, the linear eukaryotic chromosome becomes shorter.

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Eukaryotic DNA Replication

telomeres – repeated DNA sequence on the ends of eukaryotic chromosomes

– produced by telomerase

telomerase contains an RNA region that is used as a template

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DNA organization in cells

• Prokaryotes– DNA is circular– DNA not usually associated with proteins– Some have plasmids: small circular molecules of

DNA outside of the main genomic DNA• Eukaryotes

– Three locations for DNA: nucleus, mitochondria, chloroplasts

– Nuclear DNA is linear, associated with protein– Organelle DNA is circular, not associated with

proteins

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Eukaryotic Nuclear DNA organization

• Nucleosome: DNA associated with histone protein

• Chromatin: collection of nucleosome and linker DNA

• Chromosome: condensed chromatin– Ends of chromosomes

are called telomeres (very repetitive sequences)

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What is a genome?

• Genome: the entire collection of DNA for a given organism and/or organelle

– Bacterial genomes: sum total of all DNA (not including plasmids)

– Nuclear genomes: sum total of all DNA in nucleus

– Mitochondrial, chloroplast genome

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Organization of DNA

• DNA reassociation kinetics– Allow DNA for a given species to

denature (usually by heat)– Time how long it takes for the DNA to

renature– Repetitive sequences renature faster than

nonrepetitive(unique) sequences– Complexity: more complex genomes have

more unique sequences

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Types of DNA in genomes

• Classification based on reassociation kinetics• Three classes

– Highly repetitive:  About 10-15% of mammalian DNA

– Moderately repetitive:  Roughly 25-40% of mammalian DNA.

– Single copy (or very low copy number):  This class accounts for 50-60% of mammalian DNA (thought to be regions that encode mRNA and/or protein—genes)