Given the structures of ddATP and DATP: NH2 NH2 N. HO-P- OH OH OH OH OH OH ddATP DATP A) What would be the effect of adding ddATP (5'-dideoxyadenosine triphosphate) to a DNA replication reaction with DTTP, DCTP, and dGTP instead of DATP (5'-deoxyadenosine triphosphate)? B) What if 33% of the adenosine triphosphate added was ddATP (and the other 67% was DATP)?
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- In the following sequence, a cytosine was deaminated and is now a uracil (underlined). 5’-GGTAUTAAGC-3’ a. Which repair pathway(s) could restore this uracil to cytosine? b. If the uracil is not removed before a DNA replication fork passes through, what will be the sequences of the two resulting double helices? Provide the sequences of both strands of both helices. Label the old and new strands and underline the mutation(s). c. Could the mismatch repair pathway fix the mutations you’ve indicated in part b? d. If the cell undergoes mitosis, and the replicated DNAs are distributed into the two daughter cells. Will 0, 1, or 2 daughter cells have a mutation in this sequence?Although DNA polymerases require both a template and a primer, the following single-stranded polynucleotide was found to serve as a substrate for DNA polymerase in the absence of any additional DNA.3′ HO-ATGGGCTCATAGCCGGAGCCCTAACCGTAGACCACGAATAGCATTAGG-p 5′What is the structure of the product of this reaction?A solution containing single stranded DNA with the sequence 5’ATGGTGCACCTGACTCCTGAGGAGAAGTCTNNNNN’3 undergoes DNA replication in vitro in the presence of all four nucleotides plus an amount of dideoxyadnosine triphosphate sufficient to compete for incorporation with deoxyadenosine triphosphate. 1. How many and what DNA fragments are expected?
- Deoxyadenylate residues in DNA undergo deamination fairly readily, as do deoxycytidylate residues. (a) What is the product of dAMP deamination? (b) The deamination product is known to base-pair with A, C, or T. What would be the genetic consequences if this deaminated site in DNA were not repaired and if it paired with C on the next round of replication?A mixture of four a-[32P]–labeled ribonucleoside triphosphates was added to permeabilized bacterial cells undergoing DNA replication in the presence of an RNA polymerase inhibitor, and incorporation into high-molecular-weight material was followed over time, as shown in the accompanying graph. After 10 minutes of incubation, a 1000-fold excess of unlabeled ribonucleoside triphosphates was added, with the results shown in the graph. (a) Why was the excess of unlabeled rNTPs added?(b) How could you tell that radioactivity is being incorporated as ribonucleotides rather than as an alternative such as reduction to deoxyribonucleotides, followed by incorporation?(c) What does this experiment tell you about the process of DNA replication?A mixture of four a-[32P]–labeled ribonucleoside triphosphates was added to permeabilized bacterial cells undergoing DNA replication in the presence of an RNA polymerase inhibitor, and incorporation into high-molecular-weight material was followed over time, as shown in the accompanying graph. After 10 minutes of incubation, a 1000-fold excess of unlabeled ribonucleoside triphosphates was added, with the results shown in the graph. Incorporation of radioactivity, cpm Excess (a) Why was the excess of unlabeled rNTPs added?(b) How could you tell that radioactivity is being incorporated as ribonucleotides rather than as an alternative such as reduction to deoxyribonucleotides, followed by incorporation?(c) What does this experiment tell you about the process of DNA replication?
- All known DNA polymerases catalyze synthesis only in the 5' → 3' direction. Nevertheless, during semiconservative DNA replication in the cell, they are able to catalyze the synthesis of both daughter chains, which would appear to require synthesis in the 3' → 5' direction on one strand. Explain the process that occurs in the cell that allows for synthesis of both daughter chains by DNA polymeraseDNA polymerase I, DNA ligase, and topoisomerase I catalyze the formation of phosphodiester bonds. What is the activated intermediate in the linkage reaction catalyzed by each of these enzymes? What is the leaving group?In a DNA double helix, why doesn't an A or T form two hydrogen bonds (out of the three possible ) with G or C? Explain how DNA replication is bidirectional and discontinuous in nature?
- Suppose that 28% of the nucleotides in a DNA moleculeare deoxythymidine 5′- monophosphate, and that duringDNA replication the percentage amounts of availablenucleotide bases are 22% A, 22% C, 28% G, and 28% T.Which base would be depleted first in the replicationprocess?Consider the following sequence of DNA: 3'-TTA CGG-5'What dipeptide is formed from this DNA after transcription and translation? b. If a mutation converts CGG to CGT in DNA, what dipeptide is formed? c. If a mutation converts CGG to CCG in DNA, what dipeptide is formed? d. If a mutation converts CGG to AGG in DNA, what dipeptide is formed?The 3′ → 5′ exonuclease activity of Pol I excises only unpaired 3′-terminal nucleotides from DNA, whereas this enzyme’s pyrophosphorolysis activity removes only properly paired 3′-terminal nucleotides. Discuss the mechanistic signifi cance of this phenomenon in terms of the polymerase reaction.