1. What are the structural differences between DNA and RNA? a.

The nitrogenous bases of DNA are Glycine, Cytosine, Thymine and Adenine while the nitrogenous bases of RNA are the same except for Thymine which is replaced with Uracil b. DNA s sugar is deoxyribose, has a hydrogen atom at this position and contains one oxygen atom fewer overall, while RNA s sugar is ribose, sugar that has a hydroxyl group attached to the 2 -carbon atom. c. RNA is single-stranded while DNA is double-stranded with a helical secondary structure 2. Name the enzyme that adds the nucleotides during DNA replication. DNA polymerase III 3. What is the difference between a nucleotide and nucleoside? Nucleotide- consists of a sugar, a phosphate, and a nitrogenous base Nucleoside- a compound consisting of a purine or pyrimidine base linked to a sugar, especially ribose or deoxyribose 4. What are Okazaki fragments? Why are they formed? Short lengths of DNA produced on the lagging strand during DNA replication. They are rapidly joined by DNA ligase to form a continuous DNA strand. They are formed because the DNA polymerase can only polymerize the 5 -3 direction, therefore the lagging strand is first synthesized with RNA primers and then would leave an interval with an open OH for the DNA polymerase to add nucleotides 5. What is meant by semi conservative replication? In a round of replication, each of the two strands of DNA is used as a template for the formation of a complementary DNA strand. The original strands therefore remain intact through many cell generations. 6. List the functions of the DNA polymerase molecule.

7. What enzyme seals the Okazaki fragments? DNA ligase 8. What is a replication fork? The point of unwinding, where the two single nucleotide strands separate from the doublestranded DNA helix 9. What is the advantage of numerous replication forks in replicating DNA molecule? -so that if one fork is damaged, the replication can still proceed in the other fork

-speed 10. Describe the steps involved in DNA replication.

Initiation

Unwinding Elongation Termination

The circular chromosome of E. coli has a single replication origin (oriC). The minimal sequence required for oriC to function consists of 245 bp that contain several critical sites. Initiator proteins bind to oriC and cause a short section of DNA to unwind. This unwinding allows helicase and other single-strand-binding proteins to attach to the polynucleotide strand DNA helicases, SSBP, topoisomerase, primase DNA pol I,II,III, ligase

11. What are the differences in DNA replication between a prokaryote and eukaryote? a. Initiation of replication Prokaryote Replication origins are specified by specific DNA sequences that are only several hundred nucleotides pair long. They also have a single origin of replication in a circular chromosome. Eukaryote The sequences needed to specify an origin of DNA replication seem to be less well-defined, and the origin can span several thousand nucleotide parts. Eukaryotic replication takes places at S phase. The replication fork in eucaryotes moves about t0

times more slowly than the bacterial replication fork, and the much longer eucaryotic chromosomes each require many replication origins to complete their replication in an S phase, which typically lasts for 8 hours in human cells b. Termination of replication Prokaryote Bacteria have a circular DNA as chromosomes in where replication ends when two replication forks meet starting from the initiation point Eukaryote Eukaryotes have specialized nucleotide sequences at the end of the chromosomes that are incorporated into structures called telomeres *telomerase- the enzyme that replenishes the nucleotide sequences every time the cell divides Telomerase extends one of the DNA strands at the end of a chromosome by using an RNA template that is an integral part of the enzyme itself, producing a highly repeated DNA sequence that typically extends for thousands of nucleotide pairs at each chromosome end. c. Lagging strand polymerization Prokaryote 1000-2000 interval Eukaryote About 100-200 nucleotides interval

12. Describe how the following enzymes or proteins would function in DNA replication: a. Primase -synthesizes short RNA primers on the lagging strand which uses a ribonucleoside triphosphates b. Ligase -joins the 3 end of the new DNA fragment to the 5 end of the previous one to produce a continuous DNA chain from the many DNA fragments made on the lagging strand. c. DNA pol 5 -3 polymerizing activity -elongates the DNA d. DNA pol 3 -5 editing function
-3'-to-5 ' exonuclease activity attached to DNA polymerase chews back to create a base paired 3'OH end on the primer strand. DNA polymerase continues the process of adding nucleotides to the base-paired 3-OH end of the primer strand

e. DNA pol 5 -3 exonuclease activity

-Clips off any unpaired residues at the primer terminus, continuing until enough nucleotides have been removed to regenerate a correctly base-paired 3 -OH terminus that can prime DNA synthesis. A self-correcting enzyme that removes its own polymerization errors as it moves along the DNA. f. Initiator protein a) Helps unwind the origin using helicase b) Guides replication machinery to the origin c) Eukaryote: links cell cycle control to origin activation g. Helicase -facilitates the unwinding of the helix to create template strands; accomplished by ATP hydrolysis h. Single strand binding protein -binds tightly and cooperatively to expose s-s DNA w/o covering the bases made available for templating i. Sliding clamp protein - Keeps the polymerase firmly on the DNA when it is moving but releases it as soon as the polymerase runs into a double-stranded DNA Topoisomerase - Add itself covalently to a DNA backbone phosphate thus, breaking a phosphodiester bond in a DNA in a DNA strand

j.

13. Draw a replication fork and label the 5 and 3 ends. Include the leading and lagging strands in your drawing. Beneath the drawing, indicate which direction the replication fork is moving. Why is the 3 OH group on the deoxyribose ring so important for DNA synthesis?

14. What are the three major steps of polymerase chain reaction? a. Denaturation b. Annealing c. Extension/ elongation