KAPLAN_USMLE_STEP_1_LECTURE_NOTES_2018_BIOCHEMISTRY_and_GENETICS

Part I ● Biochemistry

4.A medical student working in a molecular biology laboratory is asked by her mentor to determine the base composition of an unlabeled nucleic acid sample left behind by a former research technologist. The results of her analysis show 10% adenine, 40% cytosine, 30% thymine and 20% guanine. What is the most likely source of the nucleic acid in this sample?

A.Bacterial chromosome

B.Bacterial plasmid

C.Mitochondrial chromosome

D.Nuclear chromosome

E.Viral genome

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Chapter 1 ● Nucleic Acid Structure and Organization

Answers

1.Answer: C.

U = A = 15%.

Since A + G = 50%, G = 35%.

Alternatively, U = A = 15%, then U + A = 30% C + G = 70%, and

G = 35%.

2.Answer: D. A nucleoside consists of a base and a sugar. The figure shows the nucleoside adenosine, which is the base adenine attached to ribose.

3.Answer: B. The more “opened” the DNA, the more sensitive it is to enzyme attack. The 10-nm fiber, without the H1, is the most open structure listed. The endonuclease would attack the region of unprotected DNA between the nucleosomes.

4.Answer: E. A base compositional analysis that deviates from Chargaff’s rules (%A = %T, %C = %G) is indicative of single-stranded, not doublestranded, nucleic acid molecule. All options listed except E are examples of circular (choices A, B and C) or linear (choice D) DNA double helices. Only a few viruses (e.g. parvovirus) have single-stranded DNA.

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DNA Replication and Repair 2

Learning Objectives

Explain how DNA and RNA synthesis differ

Know key steps in DNA replication

Know major kinds of DNA repair

DNA REPLICATION

Genetic information is transmitted from parent to progeny by replication of parental DNA, a process in which 2 daughter DNA molecules are produced that are each identical to the parental DNA molecule. During DNA replication, the 2 complementary strands of parental DNA are pulled apart. Each parental strand is then used as a template for the synthesis of a new complementary strand (semiconservative replication). During cell division, each daughter cell receives one of the 2 identical DNA molecules.

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Part I ● Biochemistry

Replication of Prokaryotic and Eukaryotic Chromosomes

The process of DNA replication in prokaryotes and eukaryotes is compared below.

Prokaryotes

Origin of replication

Eukaryotes

Multiple origins of replication

Centromere

Sister chromatids are

separated during mitosis

Note

•Polymerases are enzymes that synthesize nucleic acids by forming phosphodiester (PDE) bonds.

•Nucleases are enzymes that hydrolyze PDE bonds.

–Exonucleases remove nucleotides from the 5′ or the 3′ end of a nucleic acid.

Figure I-2-1. DNA Replication by a Semi-Conservative,

Bidirectional Mechanism

The bacterial chromosome is a closed, double-stranded circular DNA molecule having a single origin of replication. Separation of the 2 parental strands of DNA creates 2 replication forks that move away from each other in opposite directions around the circle. Replication is, thus, a bidirectional process. The 2 replication forks eventually meet, resulting in the production of 2 identical circular molecules of DNA.

Each eukaryotic chromosome contains one linear molecule of dsDNA having multiple origins of replication. Bidirectional replication occurs by means of a pair of replication forks produced at each origin. Completion of the process results in the production of 2 identical linear molecules of dsDNA (sister chro-

–Endonucleases cut within the matids). DNA replication occurs in the nucleus during the S phase of the nucleic acid and release nucleic eukaryotic cell cycle. The 2 identical sister chromatids are separated from each

acid fragments.

other when the cell divides during mitosis.

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Chapter 2 ● DNA Replication and Repair

The structure of a representative eukaryotic chromosome during the cell cycle is shown below.

Panel A

Cell division

M

G2 G1

S

Centromere

2 ds DNA	ds DNA
(sister chromatids)

Panel B

3

p2

1

1

q23

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Drawing of a	Drawing of a	Photograph of a stained replicated
replicated	stained replicated	chromosome. The individual
chromosome	chromosome	chromatids and centromere are
	(metaphase)	difficult to visualize in the photograph

Figure I-2-2. Panel A: Eukaryotic Chromosome Replication During S-Phase

Panel B: Different Representations of a Replicated Eukaryotic Chromosome

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Part I ● Biochemistry

COMPARISON OF DNA AND RNA SYNTHESIS

The overall process of DNA replication requires the synthesis of both DNA and

RNA. These 2 types of nucleic acids are synthesized by DNA polymerases and

RNA polymerases, respectively.

DNA

Polymerase

DNA Template

3' C-A-T-G-A-C-T-A-G-C-C-G-A-A-C-T-C-T-G-G-A 5' 5' A-C-U-G 3'

				Primer required for
				DNA synthesis (5'→3')
	RNA
				using dNTP substrates
	primer

3' C-A-T-G-A-C-T-A-G-C-C-G-A-A-C-T-C-T-G-G-A 5' 5' A-C-U-G-A-T-C-G-G-T 3'

RNA

Polymerase

DNA Template

3' C-A-T-G-A-C-T-A-G-C-C-G-A-A-C-T-C-T-G-G-A 5'

Primer not required for RNA synthesis (5'→3') using NTP substrates

3' C-A-T-G-A-C-T-A-G-C-C-G-A-A-C-T-C-T-G-G-A 5' 5' A-U-C-G-G-U 3'

Mispaired deoxynucleotide

Mispaired nucleotide

dTMP

removed (3'→5' exonuclease)

not removed

3'

C-A-T-G-A-C-T-A-G-C-C

-G

-A-A-C-T-C-T-G-G-A 5'

3'

C-A-T-G-A-C-T-A-G-C-C

-G-A-A-C-T-C-T-G-G-A 5'

5'

3'

5'

A-C-U-G

-A-T-C-G-G

A-U-C-G-G

-U

3'

High-fidelity

Low-fidelity

DNA synthesis

RNA synthesis

3'

C-A-T-G-A-C-T-A-G-C-C

-G-A-A-C-T-C-T-G-G-A 5'

3'

C-A-T-G-A-C-T-A-G-C-C-G-A-A-C-T-C-T-G-G-A 5'

5'

5' A-U-C-G-G-

-U-U-G-A-G-A-C 3'

U

A-C-U-G

-A-T-C-G-G-C-T-T-G-A-G-A-C 3'

Figure I-2-3. Polymerase Enzymes Synthesize DNA and RNA

Table I-2-1. Comparison of DNA and RNA Polymerases

		DNA Polymerase			RNA Polymerase
Nucleic acid synthesized (5′→3′)	DNA			RNA
Required template (copied 3′→5′)	DNA*			DNA*
Required substrates	dATP, dGTP, dCTP, dTTP			ATP, GTP, CTP, UTP
Required primer	RNA (or DNA)			None
Proofreading activity (3′→5′ exonuclease)	Yes			No

* Certain DNA and RNA polymerases require RNA templates. These enzymes are most commonly associated with viruses.

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Chapter 2

Similarities between DNA and RNA synthesis include:

•The newly synthesized strand is made in the 5′→3′ direction.

•The template strand is scanned in the 3′→5′ direction.

•The newly synthesized strand is complementary and antiparallel to the template strand.

•Each new nucleotide is added when the 3′ hydroxyl group of the growing strand reacts with a nucleoside triphosphate, which is basepaired with the template strand. Pyrophosphate (PPi, the last two phosphates) is released during this reaction.

Differences include:

•The substrates for DNA synthesis are the dNTPs, whereas the substrates for RNA synthesis are the NTPs.

•DNA contains thymine, whereas RNA contains uracil.

•DNA polymerases require a primer, whereas RNA polymerases do not. That is, DNA polymerases cannot initiate strand synthesis, whereas RNA polymerases can.

•DNA polymerases can correct mistakes (“proofreading”), whereas RNA polymerases cannot. DNA polymerases have 3′ → 5′ exonuclease activity for proofreading.

STEPS OF DNA REPLICATION

The molecular mechanism of DNA replication is shown below. The sequence of events is as follows:

1.The base sequence at the origin of replication is recognized.

2.Helicase breaks the hydrogen bonds holding the base pairs together. This allows the two parental strands of DNA to begin unwinding and forms 2 replication forks.

3.Single-stranded DNA binding protein (SSB) binds to the single-stranded portion of each DNA strand, preventing them from reassociating and protecting them from degradation by nucleases.

4.Primase synthesizes a short (about 10 nucleotides) RNA primer in the 5′→3′ direction, beginning at the origin on each parental strand. The parental strand is used as a template for this process. RNA primers are required because DNA polymerases are unable to initiate synthesis of DNA, and can only extend a strand from the 3′ end of a preformed “primer.”

5.DNA polymerase III begins synthesizing DNA in the 5′→3′ direction, beginning at the 3′ end of each RNA primer. The newly synthesized strand is complementary and antiparallel to the parental strand used as a template. This strand can be made continuously in one long piece and is known as the “leading strand.”

•The “lagging strand” is synthesized discontinuously as a series of small fragments (about 1,000 nucleotides long) known as Okazaki fragments. Each Okazaki fragment is initiated by the synthesis of an RNA primer by primase, and then completed by the synthesis of DNA using DNA polymerase III. Each fragment is made in the 5′→3′ direction.

● DNA Replication and Repair

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Part I ● Biochemistry

Note

Telomerase

•Completes the replication of the telomere sequences at both ends of a eukaryotic chromosome

•Present in embryonic cells, fetal cells, and certain adult stem cells; not present in adult somatic cells

•Inappropriately present in many cancer cells, contributing to their unlimited replication

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•There is a leading and a lagging strand for each of the two replication forks on the chromosome.

6.RNA primers are removed by RNAase H in eukaryotes and an uncharacterized DNA polymerase fills in the gap with DNA. In prokaryotes DNA polymerase I both removes the primer (5’ exonuclease) and synthesizes new DNA, beginning at the 3′ end of the neighboring Okazaki fragment.

7.Both eukaryotic and prokaryotic DNA polymerases have the ability to “proofread” their work by means of a 3′→5′exonuclease activity. If DNA polymerase makes a mistake during DNA synthesis, the resulting unpaired base at the 3′end of the growing strand is removed before synthesis continues.

8.DNA ligase seals the “nicks” between Okazaki fragments, converting them to a continuous strand of DNA.

9.DNA gyrase (DNA topoisomerase II) provides a “swivel” in front of each replication fork. As helicase unwinds the DNA at the replication forks, the DNA ahead of it becomes overwound and positive supercoils form. DNA gyrase inserts negative supercoils by nicking both strands of DNA, passing the DNA strands through the nick, and then resealing both strands. Quinolones are a family of drugs that block the action of topoisomerases. Nalidixic acid kills bacteria by inhibiting DNA gyrase. Inhibitors of eukaryotic topoisomerase II (etoposide, teniposide) are becoming useful as anticancer agents.

The mechanism of replication in eukaryotes is believed to be very similar to this. However, the details have not yet been completely worked out. The steps and proteins involved in DNA replication in prokaryotes are compared with those used in eukaryotes in Table I-2-2.

Eukaryotic DNA Polymerases

•DNA α and δ work together to synthesize both the leading and lagging strands.

•DNA polymerase γ replicates mitochondrial DNA.

•DNA polymerases β and ε are thought to participate primarily in DNA repair. DNA polymerase ε may substitute for DNA polymerase δ in certain cases.

Telomerase	High-Yield

Telomeres are repetitive sequences at the ends of linear DNA molecules in eukaryotic chromosomes. With each round of replication in most normal cells, the telomeres are shortened because DNA polymerase cannot complete synthesis of the 5′ end of each strand. This contributes to the aging of cells, because eventually the telomeres become so short that the chromosomes cannot function properly and the cells die.

Telomerase is an enzyme in eukaryotes used to maintain the telomeres. It contains a short RNA template complementary to the DNA telomere sequence, as well as telomerase reverse transcriptase activity (hTRT). Telomerase is thus able to replace telomere sequences that would otherwise be lost during replication. Normally telomerase activity is present only in embryonic cells, germ (reproductive) cells, and stem cells, but not in somatic cells.

Chapter 2 ● DNA Replication and Repair

Cancer cells often have relatively high levels of telomerase, preventing the telomeres from becoming shortened and contributing to the immortality of malignant cells.

Table I-2-2. Steps and Proteins Involved in DNA Replication

							Eukaryotic Cells
	Step in Replication			Prokaryotic Cells
							(Nuclei)
	Origin of replication (ori)			One ori site per			Multiple ori sites per
				chromosome			chromosome
	Unwinding of DNA double			Helicase			Helicase
	helix
	Stabilization of unwound			Single-stranded			Single-stranded
	template strands			DNA-binding			DNA-binding protein
				protein (SSB)			(SSB)
	Synthesis of RNA primers			Primase			Primase
	Synthesis of DNA			DNA polymerase III			DNA polymerases α + δ
	Leading strand			DNA polymerase III			DNA polymerases α + δ
	Lagging strand
	(Okazaki fragments)
	Removal of RNA primers			DNA polymerase I			RNase H
				(5′→3′exonuclease)			(5′→3′ exonuclease)
	Replacement of RNA			DNA polymerase I			DNA polymerase δ
	with DNA
	Joining of Okazaki frag-			DNA ligase			DNA ligase
	ments
	Removal of positive			DNA topoisomerase			DNA topoisomerase II
	supercoils ahead of			II (DNA gyrase)
	advancing replication forks
	Synthesis of telomeres			Not required			Telomerase

Reverse Transcriptase	High-Yield

Reverse transcriptase is an RNA-dependent DNA polymeraseMEDIUMthat requiresYIELD an RNA template to direct the synthesis of new DNA. Retroviruses, most notably HIV, use this enzyme to replicate their RNA genomes. DNA synthesis by reverse transcriptase in retroviruses can be inhibited by AZT, ddC, and ddI.

Eukaryotic cells also contain reverse transcriptase activity:

•Associated with telomerase (hTRT)

•Encoded by retrotransposons (residual viral genomes permanently maintained in human DNA) that play a role in amplifying certain repetitive sequences in DNA (see Chapter 7)

Bridge to Pharmacology

Quinolones and fluoroquinolones inhibit DNA gyrase (prokaryotic topoisomerase II), preventing DNA replication and transcription. These drugs, which are most active against aerobic gram-negative bacteria, include:

•Levofloxacin

•Ciprofloxacin

•Moxifloxacin

Resistance to the drugs has developed over time; current uses include treatment of gonorrhea and upper and lower urinary tract infections in both sexes.

Bridge to Pharmacology

One chemotherapeutic treatment of HIV is the use of AZT (3′-azido-2′,3′- dideoxythymidine) or structurally related compounds. Once AZT enters cells, it can be converted to the triphosphate derivative and used as a substrate for the viral reverse transcriptase in synthesizing DNA from its RNA genome.

The replacement of an azide instead of a normal hydroxyl group at the 3′ position of the deoxyribose prevents further replication by effectively causing chain termination. Although it is a DNA polymerase, reverse transcriptase lacks proofreading activity.

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