Biology 330: Replication of Genomic DNA (Exam 2 Study, part 1)

Which two elements were identified in an acidic compound that was isolated from the nucleus by Johann Miescher?

Miescher found nitrogen and phosphorus in a compound that he called nuclein, which would later be renamed deoxyribonucleic acid (DNA).

Why were proteins initially thought to encode information that pertains to inheritable traits?

Because of their complex polypeptide chains made of 20 different amino acids.

Which two macromolecules make the Bacteriophage structure?

Protein and DNA

In a cell, which macromolecules are labeled using 35S-methionine and 32P, respectively?

35S-methionine is used to label proteins, and 32P is used to label DNA.

Describe Hershey and Chase experiments. What conclusions were made from this study?

Hershey and Chase radioactively labeled the proteins and DNA in bacteriophages. The radioactive bacteriophages infected bacteria, injecting the DNA labeled with 32P rather than proteins labeled with 35S-methionine. They concluded DNA carries inheritable information.

What is the DNA nucleotide made of?

A DNA nucleotide is made of a deoxyribose sugar, a phosphate group, and a nitrogenous base

Which bases are purines and pyrimidines, respectively? Explain why they are classified this way.

The purine bases are adenine and guanine; purines have two rings. The pyrimidine bases are cytosine, thymine, and uracil; pyrimidines have one ring.

Which base is found only in RNA and not in a DNA molecule?

Uracil

In his study of DNA structure, what major findings were made by Erwin Chargaff?

Chargaff found that the amount of adenine relative to guanine differs in species, but the amount of adenine is always equal to the amount of thymine, and the amount of guanine is always equal to the amount of cytosine. Chargaff’s findings indicate that purines bind specifically to pyrimidines.

Linus Pauling built a DNA model and it failed. What was wrong with his proposed model?

In Pauling’s model of DNA, the negatively charged phosphate groups faced inward, which would cause them to repel each other and make DNA unstable.

In their study of DNA structure, which three points did Watson and Crick focus on?

Watson and Crick wanted to find the shape of DNA, its size, and its bonding requirements (how strands are held together).

What type of DNA did Maurice Wilkins laboratory work with?

Dehydrated DNA.

What types of DNA did Rosalind Franklin’s laboratory work with?

Both dry and wet DNA.

Based on Franklin’s data, what were the calculated principal dimensions of the DNA molecule?

DNA is a helix, and each full twist is 3.4 nm. The diameter is 2 nm. The distance between each base pair is 0.34 nm.

Based on Franklin’s data, how many bases are per turn of the helix?

10 bases per turn of the helix

Based on Franklin’s data, how many strands were thought to be twisted into a helix?

Franklin knew that there was more than one strand but had not yet found the precise number.

Based on Franklin’s data, what was the orientation of phosphate groups in the DNA molecule?

Franklin deduced that the phosphate groups faced the exterior of the DNA molecule.

Who recognized the significance of Rosalind Franklin’s data when she presented her findings?

Maurice Wilkins recognized the significance of Rosalind Franklin’s data and presented it to James Watson and Francis Crick without her knowledge.

Based on the Watson-Crick model, how many strands is a DNA molecule made of and in which direction do DNA strands run?

Two strands that run antiparallel

Based on the Watson-Crick model, which groups or parts of a nucleotide are located on the outside in the DNA molecule?

The sugar and phosphate groups

Based on the Watson-Crick model, where are nitrogenous bases located in the DNA molecule, and what holds the bases together?

The nitrogenous bases are located on the inside, held together by hydrogen bonds (A-T double H-bonds, G-C triple H-bonds).

Which bacterial DNA polymerase was described by Arthur Kornberg?

DNA polymerase I in E. coli

Explain what the origins of replication (ori) is.

Ori (origins of replication) are sites where DNA replication begins. Ori are the binding sites of DNA polymerases.

What is the function of DNA polymerase III in bacteria?

In bacteria, DNA polymerase III synthesizes both the leading and lagging strands of DNA

What is the function of DNA polymerase I in bacteria?

DNA polymerase I removes the RNA primers used in DNA synthesis, and it repairs damaged DNA.

In eukaryote cells, which DNA polymerases are found in the nucleus?

DNA Polymerases alpha (α), delta (δ), and epsilon (ε)

In eukaryote cells, which DNA polymerase is found in the mitochondria?

DNA Polymerase gamma (γ)

Describe the two fundamental properties that are shared by all DNA polymerases.

1. DNA polymerases require RNA primers to add nucleotides to the parent DNA template.

2. DNA polymerases always synthesize from 5’ to 3’

What is the enzyme called, which unwinds the parent DNA strand? How?

Helicase – breaks hydrogen bonds between bases and unwinds DNA.

What is the overall direction called in which the parent DNA strand unwinds?

Replication fork movement

In which direction is a parent DNA strand copied?

DNA is copied from 3’ to 5’

What is the function of small DNA binding proteins in DNA replication?

Bind to single strands of DNA to stabilize them while DNA polymerase copies the template

What are the functions of Topoisomerase I and II?

Topoisomerases can break and rejoin DNA strands to fix twisting. Topoisomerase I breaks just one strand of DNA. Topoisomerase II breaks both strands simultaneously. Transient breaks allow DNA strands to rotate freely.

Explain what semiconservative replication is

One parent strand of DNA is used to form two strands of DNA, each containing half of the old template and half of the newly synthesized DNA.

Describe Reiji Okazaki’s findings in regard to the copying of parental DNA template strands.

Okazaki found that DNA synthesis is only continuous on one strand. On the other strand, DNA assembly is discontinuous, proceeding in short nucleotide segments, which were later called Okazaki fragments.

Describe the continuous assembly of DNA

DNA is synthesized from 5’ to 3’ in the same direction as the replication fork movement with no gaps.

Describe the discontinuous assembly of DNA

DNA is synthesized from 5’ to 3’ in short segments called Okazaki fragments. This occurs in the opposite direction of replication fork movement. The Okazaki fragments are joined together by ligase.

What are leading and lagging DNA strands?

The leading strand is the DNA strand synthesized continuously in the same direction as the replication fork movement. The lagging strand is the DNA strand synthesized discontinuously in the opposite direction of the replication fork movement.

What is the difference between Okazaki fragments and the DNA lagging strand?

Okazaki fragments are short stretches of DNA that make up the lagging strand. The entire lagging strand is formed as ligase joins the Okazaki fragments.

In DNA replication, what is the role of a primase?

Primase synthesizes RNA primers so that DNA polymerase can function. On the lagging strand, primase forms a complex with DNA pol. alpha to extend the primers into Okazaki fragments

What are newly synthesized Okazaki fragments made of?

Newly synthesized Okazaki fragments contain RNA-DNA segments

What is the function of DNA polymerase ε ?

Synthesizes the leading strand of DNA in the eukaryotic cell nucleus

What are the functions of Primase and DNA polymerase α ?

Primase and DNA polymerase α form a complex to synthesize Okazaki fragments (RNA-DNA segments)

What are the functions of RNase H and 5′ to 3′ exonucleases?

RNase H degrades the RNA strand of RNA-DNA segment.

5′ to 3′ exonucleases remove RNA in the RNA-DNA segment from the 5′ end.

What is the function of DNA polymerase δ ?

Fills gaps that result from the removal of RNA

What is the function of DNA ligase?

Joins segments of DNA together to form an intact strand; joins Okazaki fragments to form the lagging strand.

DNA polymerases have proofreading mechanism that fix errors in DNA replication and strand breaks. What type of exonuclease activity do replicative DNA polymerases in eukaryotes (DNA pol. ε and δ) possess?

DNA polymerases ε and δ can hydrolyze DNA in the 3’ to 5’ direction. Incorrect bases can be removed by the 3’ to 5’ exonuclease activity. Because it runs in the opposite direction of DNA synthesis, this mechanism is proofreading newly synthesized DNA.

In the synthesis of a new DNA strand, what is the error frequency in which a wrong nucleotide is added and base paired to the DNA template?

Less than one incorrect base per billion nucleotide bases

What are the three forms of DNA?

B-DNA, A-DNA, and Z-DNA

Which form of DNA was described in the Watson-Crick structure?

B-DNA

Which forms of DNA are right-handed helix rotation?

A-DNA and B-DNA

Which form of DNA is left-handed helix rotation?

Z-DNA

Which form of DNA is favored in solutions that are devoid of water?

A-DNA

How many base pairs per helical turn are found in A, B, and Z forms of DNA?

A-DNA: 11, B-DNA: 10.5, Z-DNA: 12

What are the two ways in which mutations can be introduced into DNA?

Exposure to radiation or chemicals

What major type of DNA damage is induced by UV light, and how is this damage directly reversed by photoreactivation?

UV light causes pyrimidine dimers in DNA, where adjacent thymine bases form double bonds, making a cyclobutane ring that disrupts transcription. Photoreactivation is the process in which the enzyme photolyase repairs the thymine dimer using energy from visible light to break the cyclobutane ring. Photoreactivation is used by plants, bacteria, and yeast, but is absent in human cells.

What are the three types of excision repair that correct damaged DNA in both prokaryotic and eukaryotic cells?

Base-excision repair, nucleotide-excision repair, and mismatch repair

Explain base-excision repair of DNA.

DNA glycosylase recognizes and removes single damaged/abnormal bases (like uracil in DNA). Glycosylase activity creates an apurinic (AP) site on the DNA. The AP site is then repaired by AP endonuclease by cutting adjacent to the AP site. The remaining deoxyribose sugar is removed enzymatically as well. The gap is then filled with the correct base by DNA polymerase and sealed by DNA ligase.

Explain how UV-induced thymine dimers can be repaired using nucleotide-excision repair.

In nucleotide-excision repair, the damaged DNA is recognized and unwound by helicase. The damaged strand of DNA is then cleaved on both sides by nucleases, resulting in excision of oligonucleotides. The gap is filled by DNA polymerase and ligase.

Uracil is found only in RNA. Explain two ways in which uracil can be introduced into DNA.

In DNA, uracil can sometimes be placed instead of thymine, or it can form from the deamination of cytosine, wherein the removal of the amino group from cytosine results in uracil

Which enzyme catalyzes the excision of uracil in DNA?

Glycosylase

What is the site in DNA called where uracil is excised by DNA glycosylase and only the deoxyribose sugar remains?

An apurinic (AP) site

What is the function of an AP endonuclease?

repairs the AP site by cleaving the DNA backbone adjacent to it.

Name two enzymes that fill the single-base gap following AP endonuclease activity and the removal of deoxyribose sugar.

DNA polymerase and DNA ligase

What are the terminal sequences of linear DNA molecules called and what are they made of?

Telomeres. They are made of repeat sequences of guanine residues on one strand (TTAGGG in humans) and a complimentary strand, which are repeated hundreds or thousands of times, terminating in a 3’ overhang of single-stranded DNA.

What is the function of a telomerase?

Maintains telomeres by extending the repeat sequence in the absence of a DNA template.

Telomerase is a reverse transcriptase. Explain what a reverse transcriptase is.

An enzyme that synthesizes DNA from an RNA template, which is the opposite of transcription.

Why does telomerase carry its own RNA template?

Telomerase carries its own RNA template, complementary to the repeat sequence, to extend the 3’ end of telomeres by one repeat unit without a DNA template.

Which two enzymes catalyze the synthesis of the DNA strand that is complementary to telomere repeat sequence?

Primase and DNA Polymerase α

After removal of the RNA primer from the newly synthesized DNA strand, what happens to the overhanging 3′ end of chromosomal DNA?

The 3’ end can form loops, which bind to the protein shelterin. Shelterin protects the DNA ends from degradation.

Most somatic cells do not have high levels of telomerase to maintain telomere length for an indefinite number of cell divisions. What effect does the shortened telomeres have on cells?

Shortened telomeres are associated with human disease and aging, because telomere length determines a cell’s lifespan and ability to divide.