Based on the study of how DNA is synthesized when it is copied and the method of DNA replication answer the following 1. What is the experimental basis for concluding that DNA replicates semiconservatively in prokaryotes and Eukaryotes 2. how was it demon
Meselson Stahl experiment used E.Coli labeled radioactively and then grown in normal media as well as density labeling showed semiconservative in prokaryotes while Taylor philip and hughes used radioactive thymidine and bean roots to show it in eukaryotes 2. The DNA polA1 mutant could still synthesize active DNA leading to conclusion that at least one other enzyme responsible in vivo 3. In vitro studies by Kornberg showed nucleotides added to 3' end 4. In newly formed DNA relatively short nucleotide fragments are bonded to template strand and these short fragments accumulate in ligase deficient E.coli
Compare conservative, semiconservative, and dispersive modes of DNA replication
Conservative- original double helix remains a complete unit and new double helix is produced as single unit semiconservative- each daughter strand has one parental strand and one new DNA strand, separation of hydrogen bonds required dispersive- original dna strand broken into pieces and new strand interspersed among old pieces, requires separation of individual phosphodiester bonds
describe role of 15N in Meselson-Stahl experiment
Labeling with this heavy isotope allowed experimenters to follow the parental DNA after it went to new media by taking density measurements
What are the requirements for in vitro synthesis of DNA under direction of DNA polymerase 1
DNA template, a primer, Mg2+, and all four of the deoxyribonucleoside triphosphates dATP, dGTP, dCDP, dTTP
Kornberg showed that nucleotides are added to 3' end of each growing DNA strand. In what way does an exposed 3'OH group participate in strand elongation
Each precursor to DNA synthesis is added to the 3' end by removal of the terminal phsphates and the formation of a covalent bond. The 3' OH directly participates in the formation of that covalent bond
What was the significance of the polA1 mutation
Demonstrated that DNA polymerase one was not necessary for the formation of DNA in vivo, thus it was concluded other enzymes must be necessary
summarize and compare the properties of DNA polymerase 1, 2, and 3
Similar: None initiate but all elongate 5' to 3', all have large molecular weight and 3' to 5' exonuclease activity, 5' phosphate added to 3' end of polynucleotide DNA pol 1: 5' to 3' exonuclease activity, removal of RNA primer DNA pol 2: possibly involved in repair function DNA pol 3: essential for replication
Distinguish between unidirectional and bidirectional synthesis and continuous and discontinuous synthesis of DNA
unidirectional means only one way polymerization after synthesis while bidirectional means boths ways with some discontinuous synthesis
List the proteins that unwind DNA during in vivo DNA synthesis. How do they function
Helicase, dnaA, and single stranded binding proteins unwind open and stabilize DNa at initiation point, DNA gyrase relieves supercoiling generated by unwinding
Define and incicate the significance of Okazaki fragments DNA ligase and primer RNA during DNA replication
Okazaki framents- relatively short DNA fragments synthesized in discontinuous fashion on lagging strand during DNA replication, necessary because template DNA not available for synthesis until some degree of continuous synthesis occurs on replication fork DNA ligase- forms phosphodiester linkages in gaps that are generated when DNA polymerase 1 removes RNA primer and meets newly synthesizd DNA ahead of it primer RNA- serves as initiation point of replication provides 3' OH group necessary for DNA polymerases to begin synthesizing
Outline the current model for DNA synthesis
Opening and stabilizing of helix, priming DNA with RNA primer, movement of replication forks in both directions including elongation of RNa primers and their removal by DNA POL 1, okazaki fragments generated in the replicative process joined together by DNA ligase and DNA gyrase relieves supercoiling caused by unwinding of DNA
Why is DNA synthesis expected to be more complex in eukaryotes than in bacteria? How is DNA synthesis similar in the two types of organisms
More DNA replicated slower, multiple initiation sites for replication, telomerases needed to replicate telomeres since chromosomes are linear, similar in that both bidirectional, continuous on one strand and discontinuous on the other, requirements for synthesis DNTP's primer, template, and cation are the same, okazaki fragments 10X bigger in prokaryotes
If the analysis of DNA from two different microorganisms demonstrated very similar base compositions are the DNA sequences of the two organisms also nearly identical
Even though composition may be similar sequences can vary dramatically
Suppose E. Coli synthesizes DNA at a rate of 100,000 nucleotides per minute and takes 40 minutes to replicate its chromosome 1. How many base pairs are present in E.Coli chromosomes 2. what is the physical length of the chromosome in its helical configura
1. 4,000,000 (100,000 X 40) 2. 1.3 X 10^6 nm (4,000,000 bp times .34 nm/bp)
Several temperature sensitive mutant strains of E. coli display the following characteristics Predict what enzyme or function is being affected by each mutation 1. newly synthesized DNA contains many mismatched base pairs 2. okazaki fragments accumulate a
1. No repair from DNA POL 3 and or DNA Pol 1 2. No linking of fragments by DNA ligase 3. No primase activity 4. Only DNA pol 1 activity 5. No DNA gyrase activity
as interval between time of H-thymidine and time of centrifugation increased the proportion of short strands decreased and more labeled DNA was found in larger strands What would account for this observation
radioactive thymidine would be incorporated into new DNA, under denaturing centrifugation conditions okazaki fragments free to form small peak, as time passes okazaki fragments on lagging strand are joined by DNA ligase so larger strands are formed with a higher peak
Since inappropriate activation of telomerase can cause cancer why do you think the genes coding for this enzyme have been maintained in the human genome throughout evolution? Are there any types of human body cells where telomerase activity would be advan
Germ like tissue maintains telomere length from one generation to the next, telomeres cannot shorten indefinitely without eventually eroding coding information
The genome consists of 1.7 X 10^8 base pairs DNA synthesis occurs at a rate of 30 base pairs per second In the early embryo the entire genome is replicated in 5 minutes how many bidirectional origins of synthesis are required to accomplish this
Bidirectional synthesis one can multiply rate of synthesis by 2 to find 18,000 base pairs for 5 minutes (30 per second times 300 seconds X 2) , 1.6X10^8/ 1.8X10^4 =8800 replication sites
If an unlabeled G1 chromosome went a round of replication in 3 H thymidine and that the metaphase chromosome has both labeled which replicative models could be eliminated
conservative is eliminated because clearly there was a new strand with labeled DNa