Genetics Chapter 11
Describe 3 functions of DnaA protein
. It recognizes the origin of replication. 2. It initiates the formation of a replication bubble. 3. It recruits helicase to the region.
Describe three factors that account for the high fidelity of DNA replication. Discuss the quantitative contributions of each of the three,
1. AT and GC pairs are more likely to form compared to other types of base pairs. Limits mistakes to 1/1000. 2. Induced fitby DNA polymerase prevents covalent bond formation unless the proper nucleotides are in place. This increases fidelity another 100 to 1000 fold, from 1/100k to 1/1m 3. Exonuclease proofreading increases fidelity another 100 to 1000 fold to 1 error/100m
Match the enzyme with its appropriate role during DNA replication. Options: DNA Pol I, DNA Pol III, single strand binding protein, topoisomerase, helicase, ligase, primase 1. Covalently attaches adjacent Okazaki fragments. 2. Synthesizes an RNA primer. 3. Unwinds the two strands of DNA to create a single stranded region. 4. Removes positive supercoiling ahead of the replication fork. 5. Removes RNA primers and fills in the region with DNA. 6. Extends DNA from the RNA primer, synthesizes the leading strand and the lagging strand. 7. Binds to single stranded DNA to keep it from reforming double stranded DNA.
1. Covalently attaches adjacent Okazaki fragments.- 2. Synthesizes an RNA primer. 3. Unwinds the two strands of DNA to create a single stranded region.--- Helicase 4. Removes positive supercoiling ahead of the replication fork. 5. Removes RNA primers and fills in the region with DNA. 6. Extends DNA from the RNA primer, synthesizes the leading strand and the lagging strand.--- DNA Pol III 7. Binds to single stranded DNA to keep it from reforming double stranded DNA.
List and describe three types of functionally important sequences within bacterial origins of replication
1. DnaA boxes—Binding sites for the DnaA protein. 2. Methylation sites—Sites of adenine methylation that are important for regulating DNA replication. 3. AT-rich region—Site where the DNA initially separates to form an opening that is sometimes called a replication bubble
Summarize process of chromosoal DNA replication in E. Coli
1. DnaA proteins bind to origin of replication, resulting in separation of AT rich region 2. DNA helicase breaks H+ bonds between DNA strands, topoisomerase alleviates supercoiling, and single-strand binding proteins hold parental starts apart. 3. At each two forks, primase synthesizes RNA primer in leading strand and multiple primers in lagging strand. DNA pol III synthesizes the daughter DNA strands. In lagging strand, ozaki fragments are made. DNA pol I removes primers and fills in with DNA, and DNA ligase covalently links ozaki fragments together 4. Processes continue until two replication forks reach each other on other side of circular bacterial chromosome at a ter sequence 5. In some cases, chromosomes are intertwined and separated via topoisomerase II.
T or F: 1. Eukaryotic cells have multiple DNA polymerases. 2. Prokaryotic cells have multiple origins of replication. 3. Eukaryotic cells must replicate the mitochondrial DNA with polymerase gamma. 4. Eukaryotic cells use polymerase I to remove the RNA primers. 5. DNA replication in eukaryotes takes a much longer time than DNA replication in prokaryotes.
1. Eukaryotic cells have multiple DNA polymerases. True 2. Prokaryotic cells have multiple origins of replication. False 3. Eukaryotic cells must replicate the mitochondrial DNA with polymerase gamma. True 4. Eukaryotic cells use polymerase I to remove the RNA primers. False 5. DNA replication in eukaryotes takes a much longer time than DNA replication in prokaryotes. True
At a single origin of replication, how many leading strand primers are required for the complete replication of the DNA strand? Multiple Choice 1 2 3 4
2
Arrange the following proteins in the proper order in which they participate in DNA replication. 1. Primase 2. Helicase 3. Single-stranded binding proteins 4. DNA polymerase
2 3 1 4
DNA polymerases use their ________ activity to remove a mismatched basepair. Multiple Choice 3' -> 5' exonuclease 5' -> 3' exonuclease RNase protease mismatchase
3' -> 5' exonuclease
Which is NOT true? 1. DNA strand can serve as template strand on many occasions 2. Following semiconservative DNA replication, one strand is a newly made daughter strand and the other strand is a parental strand 3. DNA double helix may contain two strands of DNA that were made at the same time 4. DNA double helix obeys ATGC rule 5. DNA double helix could contain one strand that is 10 generations older than its complimentary strand.
3. DNA double helix may contain two strands of DNA that were made at the same time
Which of the following DNA sequences is complementary to 5' TAGAC 3'? Multiple Choice 5' TAGAC 3' 5' ATCTG 3' 5' GTCTA 3' 5' CAGAT 3' None of these responses is correct.
5' GTCTA 3'
DNA polymerase III does not have the following activity: Multiple Choice 5' to 3' DNA polymerase activity 5' to 3' exonuclease activity 3' to 5' exonuclease activity
5' to 3' exonuclease activity
Ability of DNA polymerase to digest a DNA strand from one end is called exonuclease activity. Exonuclease activity is involved in digesting RNA primers and also in proofreading. In which direction is the exonuclease activity occurring during removal of RNA primers?
5' to 3'. DNA pol I does not have reverse direction.
If eukaryotic chromosome has 25 origins of replication, how many forks does it have at the beginning ofDNA replication
50
A short sequence, which may be recognized by primase, is repeated many times throughout E. coli chromosome. It is hypothesized that primase may recognize this as a site to begin synthesis of RNA primer. If the E. Coli chromosome is 4.6m bp, how many copies of the primase recognition would be necessary?
A primer is needed to make each Okazaki fragment. The average length of an Okazaki fragment is 1,000 to 2,000 bp. If we use an average value of 1,500 bp for each Okazaki fragment, then there needs to be approximately 3067 copies
What is a processive enzyme? Explain why processivity is an important feature of DNA polymerase
A processive enzyme is one that remains clamped to one of its substrates. In the case of DNA polymerase, it remains clamped to the template strand as it makes a new daughter strand. This is important to ensure a fast rate of DNA synthesis.
What would results be if Meselson and Stahl experiment was carried out for 4-5 generations? What would results be after 3 generations if mechanism of DNA replication was dispersive? Explain three different bands in CsCl gradient
A. Four generations: 7/8 light, 1/8 half-heavy; five generations: 15/16 light, 1/16 half-heavy. B. All of the DNA double helices would be 1/8 heavy. C. The CsCl gradient separates molecules according to their densities. 14N-containing compounds have a lighter density compared to 15N -containing compounds. The bases of DNA contain nitrogen. If the bases contain only 15N, the DNA will be heavy; it will sediment at a higher density. If the bases contain only 14N, the DNA will be light; it will sediment at a lower density. If the bases in one DNA strand contain 14N and the bases in the opposite strand contain 15N, the DNA will be half-heavy; it will sediment at an intermediate density.
According to Chargaff's rule, which of the following statements about double-stranded DNA is true? rev: 02_26_2015_QC_CS-9127 Multiple Choice amount of A = amount of T; amount of C = amount of G A + G = C + T The total number of purines = the total number of pyrimidines All of these responses are correct.
All of these responses are correct.
Telomerase replicates the ends of linear chromosomes. Which of the following accurately describes telomerase and telomeres? Multiple Choice The telomerase enzyme is made up of both protein and RNA. Telomerase uses reverse transcriptase activity to add the telomere repeats. Telomere sequences vary among organisms. Telomeres prevent the loss of genetic material caused by telomere shortening. All of these responses are true.
All of these responses are true.
Telomeres contain a 3' overhand region. Does telomerase require a 3' overhand to replicate the telomere region?
As shown in Figure 11.24, the first step in replicating a telomere involves binding of telomerase to the telomere. The 3' overhang binds to the complementary RNA in telomerase. For this reason, a 3' overhang is necessary for telomerase to replicate the telomere.
With regard to DNA replication, define bidirectional synthesis
Bidirectional replication refers to DNA replication in both directions starting from one origin.
The compound nitrous acid replaces amino groups with keto groups. When nitrous acid reacts with DNA bases, it can change cytosine to uracil and change adenine to hypoxanthine. A DNA double helix has the sequence: TTGGATGCTGC AACCTACGACC What would the sequence be immediately after adding nitrous acid? Let H represent hypoxanthine and U represent uracil If this was treated with nitrous acid, then removed, the DNA was replicated for two generations. What would be the sequences of the DNA after replication twice?
C4. A. TTGGHTGUTGG HHUUTHUGHUU B. TTGGHTGUTGG HHUUTHUGHUU TTGGHTGUTGG CCAAACACCAA AACCCACAACC HHUUTHUGHUU TTGGHTGUTGG TTGGGTGTTGG CCAAACACCAA CCAAACACCAA AACCCACAACC AACCCACAACC GGTTTGTGGTT HHUUTHUGHUU
What provides the energy for the formation of the bond between an incoming dNTP and the growing DNA chain? Multiple Choice Cleavage of the dNTP into dNMP and PPi Hydrogen bonding between nucleotides Nothing, this reaction is energetically favorable DNA polymerase
Cleavage of the dNTP into dNMP and PPi
Which enzyme replaces the RNA primer with DNA? Multiple Choice Helicase DNA pol I DNA pol III Primase
DNA pol I
In E. coli, which DNA polymerase carries out the majority of the DNA synthesis? Multiple Choice DNA polymerase II DNA polymerase I DNA polymerase III DNA polymerase IV
DNA polymerase III
The enzyme that travels along the leading strand assembling new nucleotides on a growing new strand of DNA is Multiple Choice DNA polymerase I. DNA ligase. RNA primase. DNA polymerase III. helicase.
DNA polymerase III
A DNA strand has the following sequence: 5'- GATCCCGATCCGCATACATTTACCAGATCACCACC-3' In which direction would DNA polymerase slide? If this was a template by DNA polymerase, what would be the sequence of the new strand?
DNA polymerase would slide from right to left. The sequence of the new strand would be 3'-CTAGGGCTAGGCGTATGTAAATGGTCTAGTGGTGG-5'
Which eukaryotic DNA polymerase interacts with the primase? Multiple Choice DNA polymerase ε DNA polymerase α DNA polymerase η DNA polymerase Δ
DNA polymerase α
In eukaryotes, what is meant by DNA replication licensing? How does this process occur?
DNA replication licensing refers to origins of replication that have MCM helicases bound to them and are able to begin the process of DNA synthesis. The licensing process begins when a group of proteins called the origin recognition complex (ORC) bind to an origin. ORC promotes the binding of Cdc6, Cdt1, and a group of six proteins called MCM helicase.
What activity does the replisome has the the primosome lacks? Multiple Choice DNA synthesis ability Helicase activity Primase activity Topoisomerase activity
DNA synthesis ability
Bacterial cells need to tightly regulate DNA replication so that replication does not initiate multiple times before cell division occurs. The cells use several methods to regulate the initiation of DNA replication. In order for replication to initiate which of the following needs to occur? Multiple Choice DnaA is bound to OriC and the GATC sites on one strand are methylated. DnaA is not bound to OriC and the GATC sites on one strand are methylated. DnaA is bound to OriC and the GATC sites on both strands are methylated DnaA is not bound to OriC and the GATC sites on both strands are methylated
DnaA is bound to OriC and the GATC sites on both strands are methylated
What key structural features of DNA underlie its ability to be faithfully replaced?
Double stranded structure following AT/GC rule
T or F: As helicase unwinds the DNA molecule, the separated strands are kept apart by DNA polymerase.
False
T or F: Before DNA replication can occur, the OH bonds between the strands must be broken.
False
T or F: DNA replication can begin at any site along the DNA molecule.
False
T or F: During DNA replication, the lagging strand is synthesized continuously, while the leading strand is synthesized discontinuously.
False
T or F: The newly-synthesized strand of DNA has exactly the same base sequence as that of its template strand.
False
Figure of how helicase works
Fig. 11.6 in book, shows ring of helicase traveling along DNA strand separating two helices.
In bacteria, DNA polymerase I removes RNA primers. What enzyme plays this role in eukaryotic cells? Multiple Choice Flap endonuclease Tus proteins Cdt1 DNA polymerase α
Flap endonuclease
E Coli chromosome contains 4.6m bp. How long would it take to replicate its DNA? Assuming that DNA pol III is primary enzyme and it can actively proofread during synthesis, how many bp mistake will be made in one round of 1000 bacteria?
Given 4,600,000 bp of DNA, and assuming that DNA replication occurs at a rate of 750 nucleotides per second, the time required if there were a single replication fork would be 4,600,000/750 = 6,133 seconds, or 102.2 minutes Because replication is bidirectional the time required is 102.2/2 = 51.1 minutes Actually, this is an average value based on a variety of growth conditions. Under optimal growth conditions, replication can occur substantially faster. With regard to base pair mistakes, if we assume an error rate of 1 mistake per 100,000,000 nucleotides: 4,600,000 1,000 bacteria = 4,600,000,000 nucleotides of replicated DNA 4,600,000,000/100,000,000 = 46 mistakes When you think about it, this is pretty amazing. In this population, only 46 base pair mistakes would be made in 1,000 bacteria, each containing 4.6 million bp of DNA.
The following sentences describe the Meselson and Stahl experiment. Place them in order so they explain the steps of the experiment. Determine where DNA is located in cesium chloride gradient Remove samples of E coli at multiple times points every 30 mins Transfer E coli to N media Isolate DNA from E Coli and load onto cesium chloride density gradient
Grow E coli in presence of heavy nitrogen for several generations Transfer E coli to N media Remove samples of E coli at multiple times points every 30 mins Isolate DNA from E Coli and load onto cesium chloride density gradient Determine where DNA is located in cesium chloride gradient
5 DNA boxes are found within OoR in e coli. Are sequences of 5 DnaA boxes similar to each other? What is most common sequences for DnaA box?
In Figure 11.5, the first, second, and fourth DnaA boxes are running in the same direction, and the third and fifth are running in the opposite direction. Once you realize that, you can see that the sequences are very similar to each other. B. According to the direction of the first DnaA box, the consensus sequence is TGTGGATAA ACACCTATT C. This sequence is 9 base pairs long. Because there are four kinds of nucleotides (i.e., A, T, G, and C), the chance of this sequence occurring by random chance is 4-9, once every 262,144 nucleotides. Because the E. coli chromosome is more than 10 times longer than this, it is fairly likely that this consensus sequence occurs elsewhere. The reason why there are not multiple origins of replication, however, is because the origin has five copies of the consensus sequence very close together. The chance of having five copies of this consensus sequence occurring close together (as a matter of random chance) is very small.
Which of the following best describes the function of telomerase at the telomere? Multiple Choice It makes special primers that do not need to be removed. It synthesizes new DNA without the use of a template. It adds new DNA to both strands of the telomere overhang. It adds new DNA to the shorter strand of the telomere overhang. It adds new DNA to the longer strand of the telomere overhang.
It adds new DNA to the longer strand of the telomere overhang.
Which statement best describes synthesis of the lagging strand? Multiple Choice It proceeds 5'-3' away from the replication fork It proceeds 3'-5' toward the replication fork It proceeds 5'-3' toward the replication fork It proceeds 3'-5' away from the replication fork
It proceeds 5'-3' away from the replication fork
One way bacterial cells regulate DNA replication is throughGATC methlyation sites within the origin of replication. Would this mechanism work if the DNA was conservatively replicated?
No. In a conservative mechanism, one double helix would always be fully methylated so the cell would not have any way to delay the next round of DNA replication via a methylation mechanism.
The short DNA segments formed on the discontinuously replicated strand are called Multiple Choice Kawasaki fragments. Nagasaki fragments. Miyazaki fragments. Okazaki fragments. Suzuki fragments.
Okazaki fragments.
Single strand binding proteins keep the two parental strands of DNA separated from each other until DNA polymerase has an opportunity to replicate the strands. Suggest how they keep the stransa separated and do not impede ability to replicate.
Primase and DNA polymerase are able to knock the single-strand binding proteins off the template DNA.
Prokaryotic chromosomes do not have telomeres because Multiple Choice Prokaryotic chromosomes do not have repetitive sequences. Prokaryotic chromosomes are circular. Prokaryotic chromosomes only have one origin of replication. Prokaryotic cells have another mechanism to replicate the ends of their chromosomes.
Prokaryotic chromosomes are circular.
Before the lagging strand can begin assembling new DNA nucleotides, which of the following must occur? Multiple Choice DNA polymerase III adds deoxyribonucleotides. DNA polymerase I removes some material and replaces it with DNA. OH bonds must be broken between the two strands of DNA. DNA ligase forms a phosphodiester bond between the 3' OH of the growing strand and the 5' phosphate in front of it. RNA primase constructs a short RNA primer.
RNA primase constructs a short RNA primer.
DNA replication is said to be Multiple Choice dispersive. semiconservative. conservative. liberal. inconclusive.
Semiconservative
If DNA adenine methyltransferase (Dam) had been discovered via isolation of temperature sensitive mutants defective in DNA replication would it have been a rapid-stop mutant or a slow-stop mutant? Multiple Choice Slow-stop mutant Rapid-stop mutant Neither; this mutation would not affect DNA replication.
Slow-stop mutant
Explain proofreading of DNA polymerase
The active site of DNA polymerase has the ability to recognize a mismatched nucleotide in the newly made strand and remove it by exonuclease cleavage. Proofreading occurs in a 3 to 5 direction. After the mistake is removed, DNA polymerase resumes DNA synthesis in the 5 to 3 direction.
A digram of a linear chromosome is shown. The end of each strand is labeled A-D. Which ends could not be replicated by DNA polymerase and why? 5'-A------------------------B-3' 3'-C------------------------D-5'
The ends labeled B and C could not be replicated by DNA polymerase. DNA polymerase makes a strand in the 5 to 3 direction using a template strand that is running in the 3 to 5 direction. Also, DNA polymerase requires a primer. At the ends labeled B and C, there is no place (upstream) for a primer to be made.
You have engineered additional ter sequences into the bacterial chromosome. They are spaced throughout the chromosome. What effect do you predict this will have on DNA replication in this cell? Multiple Choice The entire chromosome will not be replicated. The chromosome will be replicated more than once. Extra ter sequences should have no effect on the cell.
The entire chromosome will not be replicated.
What enzymatic features of DNA polymerase prevent it from replicating one of the DNA strands at the ends of linear chromosomes? How is telomerase different in its ability to synthesize a DNA strand? What template does telomerase use? Does it still result in a telomere sequence that is tandemly repetitive?
The inability to synthesize DNA in the 3 to 5 direction and the need for a primer prevent replication at the end of the DNA strands. Telomerase differs from DNA polymerase in that it uses a short RNA sequence, which is part of its structure, as a template for DNA synthesis. Because it uses this sequence many times in row, it produces a tandemly repeated sequence in the telomere at the ends of linear chromosomes.
Discuss similarities and differences in DNA lagging and leading strands. What is advantage of primosome and replisome as opposed to having all replication enzymes functioning independently of each other?
The leading strand is primed once, at the origin, and then DNA polymerase III synthesizes DNA continuously in the direction of the replication fork. In the lagging strand, many short pieces of DNA (Okazaki fragments) are made. This requires many RNA primers. The primers are removed by DNA polymerase I, which then fills in the gaps with DNA. DNA ligase then covalently connects the Okazaki fragments together. Having the enzymes within a complex such as a primosome or replisome provides coordination among the different steps in the replication process and thereby allows it to proceed faster and more efficiently.
Which of the following is not true about ARS (autonomously replicating sequence) elements? Multiple Choice They are found in yeast. They are about 50 base pairs long. They are required for the initiation of chromosome synthesis. They have the same sequence as oriC. They are primarily made up of A and T base pairs.
They have the same sequence as oriC.
What is the function of the enzyme primase during DNA replication? Multiple Choice To unwind the double helix to prime it for replication To prime DNA for replication by preventing the formation of knots To add a DNA primer to enable replication of DNA To add a RNA primer to enable replication of DNA To correct errors made during DNA replication
To add a RNA primer to enable replication of DNA
T or F: Following base removal, DNA polymerase can add nucleotides in the 5' to 3' direction.
True
T or F: Improper base-pairing during DNA replication causes a pause in chain elongation.
True
T or F: In the absence of telomerase activity, chromosomes are shortened slightly after every round of replication.
True
T or F: Telomeres consist of direct repeat sequences
True
T or F: The DNA replication machinery is assembled at the replication fork.
True
Telomerase is unique because it contains Multiple Choice a DNA molecule. an RNA molecule. different RNA molecules. different DNA molecules. both DNA and RNA molecules.
an RNA molecule.
Enzymes that are processive Multiple Choice does not dissociate from the growing strand once it has joined two nucleotides. have a high degree of accuracy. are very error-prone.
does not dissociate from the growing strand once it has joined two nucleotides.
he enzyme that unwinds a segment of the DNA molecule is Multiple Choice DNA polymerase. DNA ligase. RNA primase. DNA polymerase III. helicase.
helicase
The proofreading function of DNA polymerase reduces the error rate from about one in a million basepairs to about one in a ________ basepairs. Multiple Choice hundred thousand ten thousand thousand ten million hundred million
hundred million
Proofreading by DNA polymerase involves the removal of Multiple Choice only the mismatched base on the old strand of DNA. only the mismatched base on the newly-synthesized strand of DNA. the mismatched basepair on both strands of DNA. several bases on the newly-synthesized strand of DNA. several bases on the old strand of DNA.
several bases on the newly-synthesized strand of DNA.
DNA polymerases cannot replicate Multiple Choice the 5' ends of linear DNA strands. the 3' ends of linear DNA strands. sequences synthesized by telomerase. sequences recognized by endonuclease.
the 3' ends of linear DNA strands.
As shown in 11.24, telomerase attaches additional DNA, 6 nucleotides at a time, to the end of eukaryotic chromosomes. However, it makes only one dna strand. Describe how the opposite strand is replicted.
the opposite strand is made in the conventional way by DNA polymerase using the strand made via telomerase as a template.