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BIO 325 Lecture 14: Steps of Replication

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School
UT-Austin
Department
Biology
Course
BIO 325
Professor
G Kim

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Steps of Replication
• Step 1: unwinding the double helix
o DNA Helicase – attaches to the DNA and unwinds the double helix, breaking
hydrogen bonds
▪ The protein that progressively unwinds DNA head of each replication
fork is alled heliase
o Single Stranded Binding Proteins – bind to the unwinded DNA strands and
prevet the strads fro reforig keeps the helix ope
o Topoisomerases – relax the supercoiling caused from DNA unwinding
▪ The protei topoisoerase is a eze that relaes superoils 
nicking strandsr67
• Step 2: involves the use of polymerase
o Pol III Holoenzyme consists of DNA polymerase III and replication proteins
▪ DNA Pol III – recognize primers, replicates the DNA to produce new
strands of complementary DNA
o To start replication, the primase – enzyme synthesis RNA primers
o DNA Pol I – removes RNA primers and replaces them with DNA
o Okazaki fragments – created by the discontinuous replication on the lagging
strand
▪ The discontinuous lagging strand on a replication fork consists of
Okazaki fragets that are later ligated together to for oe strad
of DNA
o DNA Ligase – combine all Okazaki fragments together, to create a single new
replicated strand of DNA
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Related Questions

Order the following steps of the DNA replication process fromfirst to last. (From 1 to 8)

DNA ligase joins Okazaki fragmments together

Elongation of leading strands occurs continuosly and elongationof lagging strands occurs via generation of Okazaki fragments. Bothare synthesized in 5' to 3' direction.

DNA polymerase I removes the RNA primers and replaces them withDNA nucleotides

DNA polymerase III starts adding DNA nucleotides to the 3' endof the existing RNA chain.

Helicase untwists the double helix at the replication fork

Single-stranded binding proteins bind the unpaired DNA strands,keeping them from re-pairing.

Topoisomerase helps releive the strain caused by DNA untwisting.It breaks, swivels and rejoins DNA strands.

Primase starts a complementary RNA chain using parental DNAstrand as a template

1. In addition to identifying the genetic material, the experiments of Avery, MacLeod, and McCarty with different strains of Streptococcus pneumoniae demonstrated that
DNA is a double helix.
DNA may be taken up by bacterial cells and be active.
DNA is present in bacteriophage T2.
eukaryotic cells may be genetically transformed.
DNA binds to the cell wall of the bacterium.
2. In order to show that DNA in cell extracts is responsible for genetic transformation in Streptococcus pneumoniae, important corroborating evidence should indicate that _______ also destroy transforming activity.
temperatures high enough to kill cells
enzymes that hydrolyze proteins
enzymes that hydrolyze DNA
enzymes that hydrolyze polysaccharides
enzymes that hydrolyze RNA
3. Based on what you have learned about the experiments conducted by Griffith and Avery and colleagues with bacteria, which of the following would result in transformation of living R cells?
Pure RNA from S cells
Cell-membrane extracts from S cells
A mixture of pure RNA and protein from S cells
Cell-free extracts from S cells
Pure protein from S cells
4. A-T base pairs in a DNA double helix
form three hydrogen bonds with each other.
are more heat-stable than G-C base pairs.
are of a substantially different length than G-C base pairs.
are not accessible to DNA binding proteins.
are chemically distinct from G-C base pairs.
5. If 23 percent of the bases in a sample of double-stranded DNA are adenine, what percentage of the bases are uracil?
27
50
0
73
23
6. The uniform diameter of the DNA structure provides evidence for
antiparallel DNA strands.
a right-handed helix.
a double-stranded helix.
3′ end phosphorylation.
purine–pyrimidine pairing.
7. If a sequence of one strand of DNA is 5′-TGACTATC-3′, what is the complementary strand?
5′-ACTGATAG-3′
3′-TGACTATC-5′
3′-ACTGATAG-5′
3′-ACTGATAG-3′
5′-TGACTATC-3′
8. What structural aspect of the DNA facilitates dissociation of the two DNA strands for replication?
3′ end phosphorylation
Purine–pyrimidine pairing
Antiparallel DNA strands
Covalent bonds between sugars and phosphate groups
Hydrogen bonding between paired bases
9. If the Meselson–Stahl density gradient experiment had resulted in two bands of DNA molecules after only one round of replication, one containing only 15N and the second only 14N, this result would have indicated that replication was
unidirectional.
dispersive.
conservative.
bidirectional.

semiconservative.
10. The nucleoside analogue acyclovir, which is used to treat herpes simplex virus (HSV) infections, lacks a 3′ hydroxyl group (–OH). Predict what will happen if the host cell DNA polymerase incorporates a molecule of acyclovir into an elongating strand of HSV DNA.
The replication complex will no longer bind to origins of replication.
The DNA polymerase will only incorporate acyclovir molecules.
Acyclovir will bind single-strand binding proteins.
DNA polymerase will not be able to link a successive nucleotide.
Acyclovir will block the activity of the DNA helicase.
11. Which of the following does not demonstrate the stability of the DNA double helix?
Single-strand binding proteins are required to keep the strands apart.
Paired bases form hydrogen bonds.
High temperatures are required to denature it.
DNA synthesis requires energy.
DNA helicases require ATP.
12. What effect would a primase inhibitor have on DNA replication?
DNA polymerase would not be able to add bases to the DNA.
Primase would be blocked from joining the DNA replication complex.
Primase would not be able to provide primers for DNA polymerases.
DNA polymerase would not be able to use DNA as a template.
There would be no effect on DNA replication.
13. To replicate their DNA in a timely manner, most eukaryotic chromosomes
normally have uncoiled DNA.
have multiple origins of replication.
contain linear molecules of single-stranded DNA.
have two replication forks that move in the same direction.
are composed entirely of DNA.
14. Which statement about DNA replication is false?
Okazaki fragments are synthesized as parts of the leading strand.
Error rates for DNA replication are reduced by proofreading of the DNA polymerase.
The sliding clamp increases the rate of DNA synthesis.
Replication forks represent areas of active DNA synthesis on the chromosomes.
Ligases and polymerases function in the vicinity of replication forks.
15. In many eukaryotes, there are repetitive sequences called telomeres at the ends of chromosomes. After successive rounds of DNA replication, the _______ strand becomes shorter. In some cells, an enzyme called _______ repairs the shortened strand.
leading; telomerase
lagging; telomerase
lagging; DNA polymerase I
leading; DNA polymerase I
leading; replicase
16. A researcher studies normal human fibroblast cells. They can be maintained in culture but die off after about 30 cell generations. Unexpectedly, a colony of cells continues to survive and divide past 30 generations. Which scenario is most likely true for these cells?
DNA polymerase is constantly active.
Primase is able to extend the telomeres.
The mismatch repair system is extra efficient.
The telomerase gene is being expressed.
The cells do not need the ends of their chromosomes.
17. If DNA polymerase III introduces an incorrect nucleotide, what is the first corrective action made by the DNA repair system?
The replication complex excises the incorrect nucleotide.
The excision repair proteins remove the incorrect nucleotide.
The mismatch repair system scans for base-pairing mismatches.
There is no correction, because nucleotide errors by DNA polymerases are not repaired.
DNA ligase connects the correct nucleotide.
18. Choose the correct order of the following four events in the excision repair of DNA:
(1) Base-paired DNA is made complementary to the template.
(2) Damaged bases are recognized.
(3) DNA ligase seals the new strand to existing DNA.
(4) Part of a single strand is excised.
1, 2, 3, 4
2, 1, 3, 4
3, 4, 2, 1
2, 4, 1, 3
4, 2, 3, 1
19. Six complete cycles of PCR should result in a _______-fold increase in the amount of DNA.
64
32
12
128
6
20. When double-stranded DNA is heated to temperatures above 90°C, it denatures. Denaturation is a process that
breaks covalent bonds.
hydrolyzes DNA.
separates double-stranded DNA into two single strands.
causes new hydrogen bonds to form.
irreversibly destroys the double helical structure.

1.When a protein binds to DNA at a site defined by a particular nucleotide sequence, with which parts of the DNA does the protein primarily interact?

A. the phosphate and deoxyribose groups in the DNA backbone

B. the phosphate and deoxyribose groups in the DNA backbone, and hydrophobic interactions with the nucleotide bases

C. nucleotide bases within the major grooves of the DNA

D. nucleotide bases within the major and minor grooves of the DNA

E. none of the above

2. Which of the following proteins binds to DNA with sequence specificity?

A. POI Ill holoenzyme

B. DnaA

C. DnaB

D. Dam methylase

E. Both B and D

3. Which of the following is NOT a functional parallel among all known cellular systems of DNA replication?

A. Replication is initiated at specific sites

B. Replication is continuous on the leading strand and discontinuous on the lagging strand

C. DNA replication is semiconservative

D. Replication forks move away from the origin in one direction

E. Nucleases, polymerases and ligases replace RNA primers with DNA and seal the remaining nick

4. A DNA polymerase cannot synthesize a DNA strand de novo (from the beginning) from a mixture of dNTPs. Which of the following explains the role of the primer in the reaction promoted by this enzyme?

A. The primer is an initiation protein with attached nucleotides that binds to the template and to which new nucleotides are added to the 3 ' end.

B. The primer is an initiation protein with attached nucleotides that binds to the template and to which new nucleotides are added to the 5' end.

C. The primer is a preexisting strand of RNA or DNA to which new nucleotides are added to the 3' end.

D. The primer is a preexisting strand of RNA or DNA to which new nucleotides are added to the 5' end.

E. The primer is a preexisting strand of DNA synthesized by DNA pol I, prior to replicative DNA synthesis by DNA pol Ill.

5. Universal features of DNA polymerases include:

A. 5'-3' exonuclease function.

B. Formation of an 'closed' conformation of the polymerase when the template-nucleotide base pair is correct.

C. Use of a single magnesium ion to orient reacting molecules and assist in dissipation of the negative charge that develops during nucleotidyl transfer.

D. Alignment of conserved residues in the active site for attack of the DNA primer by the 3 'OH on the incoming nucleotide.

E. Continuous unbinding and rebinding of the polymerase to the DNA template as each nucleotide is added.