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1

Nucleic Acid Structure
and Organization
Learning Objectives
❏❏ Explain information related to nucleotide structure and nomenclature
❏❏ Use knowledge of organization of DNA versus RNA
❏❏ Understand general features of a chromosome

CENTRAL DOGMA OF MOLECULAR BIOLOGY
An organism must be able to store and preserve its genetic information, pass
that information along to future generations, and express that information as it
carries out all the processes of life
...
 
Replication

Translation

Transcription
RNA

DNA

Protein

Reverse
transcription
FigureI-1-1
...
Central
Central
Dogma
of Molecular
Biology
Figure
Dogma
of Molecular
Biology

Genetic information is stored in the base sequence of DNA molecules
...
 
HY
Classically, a gene is a unit of the DNA that encodes a particular protein MY
or RNA
molecule
...


Gene Expression and DNA Replication

High-Yield

Gene expression and DNA replication are compared below
...
If the RNA molecule is a messenger RNA, then the process known as
translation converts the information in the RNA base sequence
to the amino
FUNDAMENTALS
acid sequence of a protein
...
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Immunology

Part I

●

Biochemistry

Biochemistry

When cells divide, each daughter cell must receive an accurate copy of the
genetic information
...


Medical Genetics

Table I-1-1
...
The M phase (mitosis) is the time in which the cell
divides to form 2 daughter cells
...
Gene expression occurs throughout all stages of interphase
...
Cells that have stopped cycling, such as muscle and nerve cells, are
said to be in a special state called G0
...
This is a frequently
tested area on the exam
...
At the end of S phase, each chromosome has
doubled its DNA content and is composed of 2 identical sister chromatids linked at the centromere
...
Replicated DNA is checked for any errors before cell
division
...

The
Eukaryotic
Cycle
Figure
I-1-2
...
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Chapter 1

●

Nucleic Acid Structure and Organization

Control of the cell cycle is accomplished at checkpoints between the various
phases by strategic proteins such as cyclins and cyclin-dependent kinases
...

Reverse transcription, which produces DNA copies of an RNA, is more commonly associated with life cycles of retroviruses, which replicate and express
their genome through a DNA intermediate (an integrated provirus)
...


NUCLEOTIDE STRUCTURE AND NOMENCLATURE
Nucleic acids (DNA and RNA) are assembled from nucleotides, which consist
of 3 components: a nitrogenous base, a 5-carbon sugar (pentose), and ­phosphate
...
If the pentose is ribose, the nucleic acid is RNA
MY(ribonucleic acid); if the pentose is deoxyribose, the nucleic acid is DNA (deoxyriboLY
nucleic acid)
...


LOW YIELD

Purines
NH2

N

N

N
H

N

Adenine

Pyrimidines
FUNDAMENTALS
O
N

HN
H 2N

N

N
H

Guanine

NH2
N
O

NH
N
H

Cytosine

O

O REINFORCEMENT
O
HN

N
H
Uracil

O

MY
LY
HIGH YIELD

High-Yield

Bases

HY

MEDIUM YIELD
LOW YIELD
FUNDAMENTALS

CH3

REINFORCEMENT

N
H
Thymine

Figure I-1-3
...
Bases Commonly Found in Nucleic Acids
• Purines contain 2 rings in their structure
...
Other purine metabolites, not usually found in
nucleic acids, include xanthine, hypoxanthine, and uric acid
...
Cytosine (C) is present in both DNA and

RNA
...


5

USMLE Step 1 Biochem
...
The numbers identifying the carbons of the sugar are labeled with
“primes” in nucleosides and nucleotides to distinguish them from the carbons
of the purine or pyrimidine base
...
Examples of Nucleosides
Figure I-1-4
...
Nucleoside di- and triphosphates are high-energy compounds because of the hydrolytic energy associated with the acid anhydride bonds
...
High-Energy Bonds in
Figure
I-1-6
...

I-1-5
...
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Chapter 1

●

Nucleic Acid Structure and Organization

The nomenclature for the commonly found bases, nucleosides, and nucleotides
is shown below
...
, is sometimes understood and not expressly stated because thymine is
­almost always found attached to deoxyribose
...
Nomenclature of Important Bases, Nucleosides, and Nucleotides
Base

Nucleoside

Nucleotides

Adenine

Adenosine
(Deoxyadenosine)

AMP (dAMP)

ADP (dADP)

ATP (dATP)

Guanine

Guanosine
(Deoxyguanosine)

GMP (dGMP)

GDP (dGDP)

GTP (dGTP)

Cytosine

Cytidine
(Deoxycytidine)

CMP (dCMP)

CDP (dCDP)

CTP (dCTP)

Uracil

Uridine
(Deoxyuridine)

UMP (dUMP)

UDP (dUDP)

UTP (dUTP)

Thymine

(Deoxythymidine)

(dTMP)

(dTDP)

(dTTP)

Names of nucleosides and nucleotides attached to deoxyribose are shown in parentheses
...
Each strand has a distinct 5′ end and 3′ end, and thus
has polarity
...


• Nucleotides linked by 3′, 5′
phosphodiester bonds

The base sequence of a nucleic acid strand is written by convention, in the 5′→3′
direction (left to right)
...
Exceptions occur in certain viruses, some of
which have ssDNA genomes and some of which have dsRNA genomes
...
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Immunology

Part I
Biochemistry

●

Biochemistry
5´- Phosphate

3´- Hydroxyl

O
O
Medical Genetics

P

O

O
5´CH2

5´
Behavioral Science/Social Sciences

H

H 3C

O
T

O

N

N

N

N

H

A

N

H

N

3´

N

O

3´

OH

O
5´CH2

O
O

O
H

O

P

N

O
5´CH2

C

O

N

H
H

O

O

N

O

H

N

3´

G

N

N

3´

N

N

O

P

N

O

O

5´CH2

H

N

A

N

H
H

P

3´
O

O
P

O
3´

H

O

N

5´CH2

5´CH2

H

N

O

O

G
N

N
N

N

H

N

H

O

O
O

C

O

P

N

O
3´

H

O

3´

O

O

3´
O

O

N

O

O

O

T

N

N

5´CH2

CH3

O

N

O

P
O

H

O
O

3´

OH

5´CH2
O

O

P

O

5´

O
3´- Hydroxyl

5´- Phosphate

Figure
I-1-7
...
Hydrogen-Bonded Base Pairs in DNA

8

USMLE Step 1 Biochem
...

• The 2 strands are complementary
...
Thus, the base
sequence on one strand defines the base sequence on the other strand
...
Thus, total
purines equals total pyrimidines
...


In dsDNA (or dsRNA)
(ds = double-stranded)
% A = % T (% U)
%G=%C
% purines = % pyrimidines
A sample of DNA has 10% G;
what is the % T?

With minor modification (substitution of U for T) these rules also apply to
­dsRNA
...
The hydrophilic sugar-phosphate backbone
of each strand is on the outside of the double helix
...
There are about 10 base pairs per
complete turn of the helix
...
The biologic function of
­Z-DNA is unknown, but may be related to gene regulation
...
They exert
their effects by intercalating between
the bases of DNA, thereby interfering
with the activity of topoisomerase II
and preventing proper replication of
the DNA
...


FigureFigure
I-1-8
...

B-DNADouble
DoubleHelix
Helix

9

USMLE Step 1 Biochem
...

Denaturation
Figure
I-1-9
...
No covalent bonds are broken
in this process
...

Denatured single-stranded DNA can be renatured (annealed) if the denaturing
condition is slowly removed
...

Such renaturation or annealing of complementary DNA strands is an important
step in probing a Southern blot and in performing the polymerase chain r­ eaction
(reviewed in Chapter 7)
...
The mixed sample is denatured
and then renatured
...


Recall Question
Methotrexate affects which portion of the cell cycle?
A
...
  G2 phase
C
...
  S phase
Answer: D

ORGANIZATION OF DNA
Large DNA molecules must be packaged in such a way that they can fit inside
the cell and still be functional
...
These molecules may exist as relaxed circles or as supercoiled
structures in which the helix is twisted around itself in 3-dimensional space
...
This form is required for most
biologic reactions
...


10

USMLE Step 1 Biochem
...
They make transient breaks in DNA strands
alternately breaking and resealing the sugar-phosphate backbone
...


Nucleosomes and Chromatin
Without H I

+H I

H2A

H2B
H3

H4
H1
Expanded view

LY
HIGH YIELD

MEDIUM YIELD

MEDIUM YIELD

Sensitive
YIELD
to LOW
nuclease

REINFORCEMENT

H2B

MY

High-Yield

10 nm
FUNDAMENTALS

30 nm

HY

LOW YIELD
FUNDAMENTALS
REINFORCEMENT

H3
Expanded view of
a nucleosome

H4

H2A

Figure
I-1-10
...

Nucleosome
and Nucleofilament
Structure
in
Eukaryotic
DNA DNA
Structure in Eukaryotic

Nuclear DNA in eukaryotes is found in chromatin associated with histones and
nonhistone proteins
...

• Histones are rich in lysine and arginine, which confer a positive charge

on the proteins
...


• DNA is wound around the outside of this octamer to form a nucleo-

some (a series of nucleosomes is sometimes called “beads on a string”
but is more properly referred to as a 10nm chromatin fiber)
...


• Further condensation occurs to eventually form the chromosome
...


Cells in interphase contain 2 types of chromatin: euchromatin (more opened
and available for gene expression) and heterochromatin (much more highly
condensed and associated with areas of the chromosomes that are not expressed)
...
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Immunology

Part I

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Biochemistry

Biochemistry

Less active

More active

Medical Genetics

DNA double helix

Behavioral Science/Social Sciences

10 nm chromatin
(nucleosomes)

30 nm chromatin
(nucleofilament)

30 nm fiber forms loops attached Higher order
to scaffolding proteins
packaging

Euchromatin

Heterochromatin

Figure
FigureI-1-11
...
DNA
DNAPackaging
PackagingininEukaryotic
EukaryoticCell
Cell

Euchromatin generally corresponds to the nucleosomes (10-nm fibers) loosely
associated with each other (looped 30-nm fibers)
...
The figure below shows an electron
micrograph of an interphase nucleus containing euchromatin, heterochromatin,
and a nucleolus
...


Euchromatin
Heterochromatin

Nucleolus

Figure
I-1-12
...

InterphaseNucleus
Nucleus

During mitosis, all the DNA is highly condensed to allow separation of the
sister chromatids
...
Chromosome abnormalities may be assessed on mitotic
chromosomes by karyotype analysis (metaphase chromosomes) and by
banding techniques (prophase or prometaphase), which identify aneuploidy,
translocations, deletions, inversions, and duplications
...
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Chapter 1

●

Nucleic Acid Structure and Organization

Review Questions
Select the ONE best answer
...
A double-stranded RNA genome isolated from a virus in the stool of a child
with gastroenteritis was found to contain 15% uracil
...
15
B
...
35
D
...
85
2
...
Purine nucleotide
B
...
Pyrimidine nucleoside
D
...
Deoxyadenosine
3
...
Which of the following chromosome structures would most likely be degraded first in an apoptotic cell?
A
...
10-nm fiber
C
...
Centromere
E
...
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Immunology

Part I
Biochemistry

Medical Genetics

●

Biochemistry

4
...
The results of her
analysis show 10% adenine, 40% cytosine, 30% thymine and 20% guanine
...
Bacterial chromosome
B
...
Mitochondrial chromosome
D
...
Viral genome

14

USMLE Step 1 Biochem
...
During DNA replication, the
2  complementary strands of parental DNA are pulled apart
...
During cell division, each daughter cell
receives one of the 2 identical DNA molecules
...
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Immunology

Part I

●

Biochemistry

Biochemistry

Replication of Prokaryotic and Eukaryotic Chromosomes
The process of DNA replication in prokaryotes and eukaryotes is compared
­below
...
DNA Replication by a
Semi-Conservative,
Bidirectional
Mechanism
Figure
I-2-1
...

• Nucleases are enzymes that hydrolyze PDE bonds
...

–– Endonucleases cut within the
nucleic acid and release nucleic
acid fragments
...
Separation of the 2 parental strands of
DNA creates 2 replication forks that move away from each other in opposite
directions around the circle
...
The
2 replication forks eventually meet, resulting in the production of 2 identical
circular molecules of DNA
...
Bidirectional replication occurs by means of a
pair of replication forks produced at each origin
...
DNA replication occurs in the nucleus during the S phase of the
­eukaryotic cell cycle
...


18

USMLE Step 1 Biochem
...

Panel A
Cell
division
M
G2

G1
S

Centromere

2 ds DNA
(sister chromatids)

ds DNA

Panel B
3

p 2
1
1
q

Drawing of a
replicated
chromosome

2
3
4
Drawing of a
stained replicated
chromosome
(metaphase)

Photograph of a stained replicated
chromosome
...

PanelA:A:Eukaryotic
Eukaryotic Chromosome
Chromosome Replication
Figure
I-2-2
...
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Immunology

Part I

●

Biochemistry

Biochemistry

COMPARISON OF DNA AND RNA SYNTHESIS
The overall process of DNA replication requires the synthesis of both DNA and
RNA
...
 

Medical Genetics

Behavioral Science/Social Sciences

DNA
Polymerase

RNA
Polymerase

DNA Template

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'

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'

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'

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

Mispaired deoxynucleotide
removed (3'→5' exonuclease)

dTMP

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 3'

Mispaired nucleotide
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'
5' A-U-C-G-G-U 3'
Low-fidelity
RNA synthesis

High-fidelity
DNA synthesis
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-C-T-T-G-A-G-A-C 3'

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

Figure
I-2-3
...

Synthesize
DNA
andand
RNARNA

Table I-2-1
...
These enzymes are most commonly associated with viruses
...
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Chapter 2

●

DNA Replication and Repair

Similarities between DNA and RNA synthesis include:
• The newly synthesized strand is made in the 5′→3′ direction
...

• The newly synthesized strand is complementary and antiparallel to the

template strand
...
Pyrophosphate (PPi, the last two
phosphates) is released during this reaction
...

• DNA contains thymine, whereas RNA contains uracil
...


That is, DNA polymerases cannot initiate strand synthesis, whereas
RNA polymerases can
...
 DNA polymerases have 3′ → 5′ exonuclease
activity for proofreading
...
The sequence of
events is as follows:
1
...

2
...
This
allows the two parental strands of DNA to begin unwinding and forms 2
replication forks
...
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
...
Primase synthesizes a short (about 10 nucleotides) RNA primer in the
5′→3′ direction, beginning at the origin on each parental strand
...
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
...
DNA polymerase III begins synthesizing DNA in the 5′→3′ direction, beginning at the 3′ end of each RNA primer
...

This strand can be made continuously in one long piece and is known as
the “leading strand
...

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
...


21

USMLE Step 1 Biochem
...


Medical Genetics

Behavioral Science/Social Sciences

6
...
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
...
Both eukaryotic and prokaryotic DNA polymerases have the ability to
“proofread” their work by means of a 3′→5′ exonuclease activity
...

8
...

9
...
As helicase unwinds the DNA at the replication forks, the
DNA ahead of it becomes overwound and positive supercoils form
...

­Quinolones are a family of drugs that block the action of topoisomerases
...
Inhibitors of
­eukaryotic topoisomerase II (etoposide, teniposide) are becoming useful as
anticancer agents
...

However, the details have not yet been completely worked out
...


Eukaryotic DNA Polymerases
• DNA α and δ work together to synthesize both the leading and lagging

strands
...


H

HY

• DNA polymerases β and ε are thought to participate primarily in DNA

MY

repair
...


HIGH YIEL

High-Yield

Note

Telomerase

Telomerase

Telomeres are repetitive sequences at the ends of linear DNA
molecules
MEDIUM
YIELDin eukaryotic chromosomes
...
This contributes to the aging of cells, because
eventually the telomeres become so short that the chromosomes
cannot funcFUNDAMENTALS
tion properly and the cells die
...
It
­contains a short RNA template complementary to the DNA telomere sequence,
as well as telomerase reverse transcriptase activity (hTRT)
...
Normally telomerase activity is present only in embryonic cells, germ
­(reproductive) cells, and stem cells, but not in somatic cells
...
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Chapter 2

Cancer cells often have relatively high levels of telomerase, preventing the telomeres from becoming shortened and contributing to the immortality of malignant cells
...
Steps and Proteins Involved in DNA Replication
Step in Replication

Prokaryotic Cells

Eukaryotic Cells
(Nuclei)

Origin of replication (ori)

One ori site per
chromosome

Multiple ori sites per
chromosome

Unwinding of DNA double
helix

Helicase

Helicase

Stabilization of unwound
template strands

Single-stranded
DNA-binding
protein (SSB)

Single-stranded
DNA-binding protein
(SSB)

Synthesis of RNA primers

Primase

Primase

Synthesis of DNA
Leading strand
Lagging strand
(Okazaki fragments)

DNA polymerase III
DNA polymerase III

DNA polymerases α + δ
DNA polymerases α + δ

Removal of RNA primers

DNA polymerase I
(5′→3′ exonuclease)

RNase H
(5′→3′ exonuclease)

Replacement of RNA
with DNA

DNA polymerase I

DNA polymerase δ

Joining of Okazaki fragments

DNA ligase

DNA ligase

Removal of positive
supercoils ahead of
advancing replication forks

DNA topoisomerase
II (DNA gyrase)

DNA topoisomerase II

Synthesis of telomeres

Not required

Telomerase

DNA Replication and Repair

Bridge to Pharmacology
Quinolones and fluoroquinolones
inhibit DNA gyrase (prokaryotic
topoisomerase II), preventing DNA
replication and transcription
...


HY
MY
LY

Reverse Transcriptase

●

High-Yield

Bridge to Pharmacology
One chemotherapeutic treatment of
HIV is the use of AZT (3′-azido-2′,3′dideoxythymidine) or structurally
related compounds
...

The replacementHY
of an azide instead of
a normal hydroxyl group at the 3′
MY
position of the deoxyribose prevents
further replication byLYeffectively causing
chain termination
...


Reverse transcriptase is an RNA-dependent DNA polymerase
that requires
MEDIUM
YIELD an
RNA template to direct the synthesis of new DNA
...
DNA synthesis
reverse
transcriptase in retroviruses can be inhibited by AZT, ddC, and ddI
...
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Immunology

Part I

●

Biochemistry

Biochemistry

5'

3'

3'

5'
Helicase

Medical Genetics

Leading Strand Synthesis (Continuous)
1
...
DNA polymerases α and δ extend the primer, moving
into the replication fork (Leading strand synthesis)
...
Helicase (
Behavioral Science/Social Sciences

) 5' to 3'
...


Lagging Strand Synthesis (Discontinuous)
1
...


2
...

3
...

4
...


5'

3'

3'

5'

5'
3'
5'
3'

+

3'
5'
3'
5'

RNase H (5' exoribonuclease activity) digests
the RNA primer from fragment 1
...

In prokaryotic cells DNA polymerase 1 has both
the 5' exonuclease activity to remove primers, and
the DNA polymerase activity to extend the next
fragment (2) to fill in the gap
...

This whole process repeats to remove all RNA
primers from both the leading and lagging strands
...
DNA Replication
Figure I-2-4
...
  DNA gyrase
B
...
  DNA ligase
D
...
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Chapter 2

●

DNA Replication and Repair

DNA REPAIR

Bridge to Pathology

The structure of DNA can be damaged in a number of ways through exposure
to chemicals or radiation
...
Multiple repair systems have evolved, allowing cells to maintain the
sequence stability of their genomes
...
Thus any defect in DNA repair carries an increased risk of
cancer
...

Mismatch repair occurs in the G2 phase to correct replication errors
...
DNA Repair
Damage

Cause

Thymine
dimers (G1)

UV radiation

Mismatched
base (G2)

DNA replication
errors

Cytosine
deamination
G1

Spontaneous/
heat

Repair of Thymine Dimers

Recognition/
Excision Enzyme

Repair
Enzymes

Excision endonuclease
(deficient in Xeroderma pigmentosum)

DNA polymerase

A mutation on one of
two genes, hMSH2 or
hMLH1, initiates
defective repair of DNA
mismatches, resulting
in a condition known
as hereditary nonpolyposis colorectal
cancer—HNPCC
...

Inactivation or deletion associated
with Li Fraumeni syndrome and
many solid tumors
...
ATM is
inactivated in ataxia telangiectasia,
characterized by hypersensitivity
to x-rays and predisposition to lymphomas
...


HY
DNA polymerase

• The retinoblastoma Rb gene was
the first tumor suppressor gene
cloned, and is a negative regulator
of the cell cycle through its ability
to bind the transcription factor E2F
and repress transcription
of genes
HY
required for S phase
...
The formation of
LOW
YIELD
thymine dimers interferes with DNA replication and normal gene
expression
...

FUNDAMENTALS
REINFORCEMENT

LY
HIGH YIELD
MEDIUM YIELD
LOW YIELD
FUNDAMENTALS
REINFORCEMENT

25

USMLE Step 1 Biochem
...
Thymine Dimer Formation and Excision Repair
Figure I-2-5
...

• DNA polymerase fills in the gap by synthesizing DNA in the 5′→3′

direction, using the undamaged strand as a template
...


26

USMLE Step 1 Biochem
...
The uracil is recognized and removed (base excision) by a uracil glycosylase enzyme
...


Mismatch
repair
• MSH2
• MLH1

M
G2

G1
S

DNA polymerase
proofreads during
DNA synthesis

G0
Thymine dimer
(bulky lesion) repair
• XP
• Nucleotide excision
repair (cytosine
deamination)
Genes controlling
entry into S-phase
• Rb
• p53

HY

HY

Figure I-2-6
...
Important Genes Associated with Maintaining Fidelity
Maintaining Fidelity of Replicating DNA
of Replicating DNA

MY

LY

Diseases Associated with DNA Repair

High-Yield

Inherited mutations that result in defective DNA repair mechanisms
are associMEDIUM YIELD
ated with a predisposition to the development of cancer
...

FUNDAMENTALS
The most common deficiency occurs in the excinuclease enzyme
...


LY
HIGH YIELD
MEDIUM YIELD
LOW YIELD
FUNDAMENTALS
REINFORCEMENT

27

USMLE Step 1 Biochem
...
Carcinomas and melanomas appear early in life,
and most patients die of cancer
...

A 6-year-old child was brought to the clinic because his parents were
concerned with excessive lesions and blistering in the facial and neck area
...
The physician noted excessive freckling throughout the child’s
body, as well as slight stature and poor muscle tone
...
Patients with the
disease should avoid exposure to any source of UV light
...


Hereditary nonpolyposis colorectal cancer (HNPCC) results from a mutation
in one of the genes (usually hMLH1 or hMSH2) encoding enzymes that carry
out DNA mismatch repair
...
In families with HNPCC, individuals may
­inherit one nonfunctional, deleted copy of the hMLH1 gene or one nonfunctional, deleted copy of the hMSH2 gene
...
This causes
chromosomes to retain errors (mutations) in many other loci, some of which
may contribute to cancer progression
...


For example, TGTGTGTG may occur at a
particular locus
...
g
...
This
variation is microsatellite instability
...
In a patient with HNPCC, cells from the resected tumor show
microsatellite instability, whereas normal cells from the individual (which still
retain mismatch repair) do not show microsatellite instability
...


Note

28

USMLE Step 1 Biochem
...

1
...
Which proofreading activity is critical
in determining the accuracy of nuclear DNA replication and thus the
base substitution mutation rate in human chromosomes?
A
...

C
...

E
...
The proliferation of cytotoxic T-cells is markedly impaired upon infection with a newly discovered human immunodeficiency virus, designated
HIV-V
...
Which protein is a potential substrate for the viral enzyme?
A
...

C
...

E
...
The deficiency of an excision endonuclease may produce an exquisite
sensitivity to ultraviolet radiation in xeroderma pigmentosum
...

B
...

D
...


Removal of introns
Removal of pyrimidine dimers
Protection against DNA viruses
Repair of mismatched bases during DNA replication
Repair of mismatched bases during transcription

4
...
Which of the
following enzymes participates in bacterial DNA replication and is
directly inhibited by this antibiotic?
A
...

C
...

E
...
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Immunology

Part I
Biochemistry

Medical Genetics

Behavioral Science/Social Sciences

●

Biochemistry

5
...

In certain cases, resistance is related to an increase in the enzyme cytidine
deaminase in the tumor cells
...

B
...

D
...


cytosine
cytidylic acid
thymidine arabinoside
uracil arabinoside
cytidine

6
...
The defect
has been traced to inadequate production of an enzyme needed for chromosome duplication in the nuclei of rapidly dividing cells
...
Which step
in DNA replication is most likely deficient in DKC patients?
A
...

C
...

E
...
Single-strand breaks in DNA comprise the single most frequent type of
DNA damage
...

This type of break is repaired by a series of enzymes that reconstruct the
sugar and ultimately reform the phosphodiester bonds between
­nucleotides
...

B
...

D
...


DNA glycosylases
DNA helicases
DNA ligases
DNA phosphodiesterases
DNA polymerases

30

USMLE Step 1 Biochem
...
For any particular
gene, only one strand of the DNA molecule (the template strand) is copied by
RNA polymerase as it synthesizes RNA in the 5′ to 3′ direction
...
RNA polymerase recognizes start signals (promoters) and stop signals (terminators) for
each of the thousands of transcription units in the genome of an organism
...


3´
5´

Figure I-3-1
...
Transcription of Several Genes on a Chromosome

33

USMLE Step 1 Biochem
...
The major types of RNA are:

Medical Genetics

Behavioral Science/Social Sciences

• Ribosomal RNA (rRNA), which is the most abundant type of RNA in

the cell
...
Ribosomal RNA associates with ribosomal proteins to form the complete,
functional ribosome
...
Its function is to carry amino acids to the ribosome, where they
will be linked together during protein synthesis
...
Messenger RNA is
the only type of RNA that is translated
...


• Heterogeneous nuclear RNA (hnRNA or pre-mRNA), which is found

only in the nucleus of eukaryotic cells
...


• Small nuclear RNA (snRNA), which is also only found in the nucleus

of eukaryotes
...


• Ribozymes, which are RNA molecules with enzymatic activity
...


RNA POLYMERASES
There is a single prokaryotic RNA polymerase that synthesizes all types of RNA
in the cell
...
A protein factor called sigma (σ) is required for the
initiation of transcription at a promoter
...
Termination of transcription sometimes requires
a protein called rho (ρ) factor
...
Actinomycin D binds to the DNA, preventing transcription
...

• RNA polymerase I is located in the nucleolus and synthesizes 28S, 18S,

and 5
...


• RNA polymerase II is located in the nucleoplasm and synthesizes

hnRNA/mRNA and some snRNA
...


Transcription factors (such as TFIID for RNA polymerase II) help to initiate
transcription
...
All transcription can be inhibited by actinomycin D
...


34

USMLE Step 1 Biochem
...
Comparison of RNA Polymerases
Prokaryotic

Eukaryotic

Single RNA polymerase
(α2 ββ’)

RNAP 1: rRNA (nucleolus) Except 5S rRNA
RNAP 2: hnRNA/mRNA and some snRNA
RNAP 3: tRNA, 5S rRNA

Requires sigma (σ) to
initiate at a promoter

No sigma, but transcription factors (TFIID) bind
before RNA polymerase

Sometimes requires
rho (ρ) to terminate

No rho required

Inhibited by rifampin

RNAP 2 inhibited by α-amanitin (mushrooms)

Actinomycin D

Actinomycin D

TRANSCRIPTION: IMPORTANT CONCEPTS AND
TERMINOLOGY
RNA is synthesized by a DNA-dependent RNA polymerase (uses DNA as a template for the synthesis of RNA)
...

• RNA polymerase locates genes in DNA by searching for promoter regions
...
Binding establishes
where transcription begins, which strand of DNA is used as the template,
and in which direction transcription proceeds
...


• RNA polymerase moves along the template strand in the 3′ to 5′

direction as it synthesizes the RNA product in the 5′ to 3′ direction
using NTPs (ATP, GTP, CTP, UTP) as substrates
...
The RNA product is complementary and
antiparallel to the template strand
...
It is

identical in sequence to the RNA molecule, except that RNA contains
uracil instead of the thymine found in DNA
...


• In the vicinity of a gene, a numbering system is used to identify the

location of important bases
...


–– To the left (5′, or upstream) of this starting point for transcription,
bases are –1, –2, –3, etc
...

• Transcription ends when RNA polymerase reaches a termination signal
...
indb 35

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Immunology

Part I

●

Biochemistry

Biochemistry

Upstream Downstream
Transcription unit
Start
site

Medical Genetics

Behavioral Science/Social Sciences

Terminator

-10 +1+10 Coding (antitemplate) strand

5'
3'

Promoter
DNA
RNA

Template strand

5'

3'
5'

RNA polymerase
transcribes DNA
template strand
RNA transcript is
synthesized 5'
3'

3'

H

HY

Transcription
of DNA
FigureFigure
I-3-2
...

Transcription
of DNA

MY
LY

Flow of Genetic Information from
DNA to Protein

HIGH YIEL

High-Yield

For the case of a gene coding for a protein, the relationship among
theYIELD
sequencMEDIUM
es found in double-stranded DNA, single-stranded mRNA, and protein is
LOW YIELDIt is
­illustrated below
...

complementary and antiparallel to the template strand of DNA
...

REINFORCEMENT

DNA

Coding strand
5' A T G G G G C T C A G C G A C 3'

Transcription 3' T A C C C C G A G T C G C T G 5'
Template strand
mRNA

Translation

Gly

Leu

Ser

FUNDAMENT

REINFORCEM

DNA template strand
is complementary and
antiparallel to the mRNA

Codons
Met

LOW Y

DNA coding strand is
identical to the mRNA
(except T for U)

5' A U G G G G C U C A G C G A C 3'

Protein NH2

MEDIUM YIE

Asp COOH

Direction of
transcription
Direction of
translation

Figure I-3-3
...
Flow of Genetic Information from DNA to Protein

36

USMLE Step 1 Biochem
...
During RNA synthesis, the DNA template sequence TAGC would be transcribed to produce which of the following sequences?
A
...
GCTA
C
...
AUCG
E
...
RNA is antiparallel and complementary to the template strand
...

Approach:
• Cross out any option with a T (RNA has U)
...

• What is the complement of this base? (A)

Examine the options given
...
Repeat the procedure for the 3′ end of the DNA
...

2
...
What would be the base sequence
of the mRNA produced?
3’ CGTCAGC 5’
Transcription → Which product?
5′…GCAGTCG…3′
A
...
5′…CGUGAGC…3′
C
...
5′…CGUCAGC…3′
E
...
Because all nucleic acids are synthesized in the 5′ to 3′ direction, mRNA and the coding strand of DNA must each be oriented 5′ to 3′, i
...
,
in the direction of transcription
...
The top strand is the template strand
...

• Identify the coding strand of DNA from the direction of transcription
...


• Alternatively, if you prefer to find the complement of the template

strand, you will get the same answer
...
indb 37

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Immunology

Part I
Biochemistry

Medical Genetics

Behavioral Science/Social Sciences

●

Biochemistry

PRODUCTION OF PROKARYOTIC MESSENGER RNA
The structure and expression of a typical prokaryotic gene coding for a protein
are illustrated in Figure I-3-4
...
With the help of sigma factor, RNA polymerase recognizes and binds to
the promoter region
...

The promoter identifies the start site for transcription and orients the
enzyme on the template strand
...

2
...
Sigma factor is released as soon
as transcription is initiated
...
The core polymerase continues moving along the template strand in the 3′
to 5′ direction, synthesizing the mRNA in the 5′ to 3′ direction
...
RNA polymerase eventually reaches a transcription termination signal, at
which point it will stop transcription and release the completed mRNA
molecule
...
These two structural features of the newly synthesized
RNA promote dissociation of the RNA from the DNA template
...


• Rho-dependent termination requires participation of rho factor
...
Rho then displaces
RNA polymerase from the 3′ end of the RNA
...
Transcription and translation can occur simultaneously in bacteria
...
Ribosomes bind to a sequence called the Shine-Dalgarno
sequence in the 5′ untranslated region (UTR) of the message
...

6
...


38

USMLE Step 1 Biochem
...

Expression
ofof
a aProkaryotic
Gene
Figure
I-3-4
...
That is, it is transcribed from a single gene and codes for only a single
protein
...
Some bacterial operons
(for example, the lactose operon) produce polycistronic messages
...

The mRNA in this case contains information from several genes and codes for
several different proteins
...

I-3-5
...
indb 39

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Immunology

Part I

●

Biochemistry

Biochemistry

PRODUCTION OF EUKARYOTIC MESSENGER RNA
In eukaryotes, most genes are composed of coding segments (exons) interrupted by
noncoding segments (introns)
...
Introns are removed during processing of the RNA molecule in the nucleus
...
The mature mRNA is translated in the
cytoplasm
...
Transcription of this gene occurs as follows:

Medical Genetics

1
...
The basal promoter region
of eukaryotic genes usually has two consensus sequences called the TATA
box (also called Hogness box) and the CAAT box
...
RNA polymerase II separates the strands of the DNA over a short region to
initiate transcription and read the DNA sequence
...
Both exons and introns are transcribed
...
RNA polymerase II ends transcription when it reaches a termination signal
...

DNA

5´
3´

ATG
Exon 1

Promoter
–70 –25
CAAT TATA
box box
+1

Poly A addition
signal

TAG
Intron

Exon 2
3´
5´

5´ Untranslated
region (UTR)

3´ Untranslated
region (UTR)
Transcription
terminates

Transcription
AUG
Exon 1
Pre-mRNA

5´
5´ UTR

UAG
Intron

Poly A addition
signal

Exon 2
3´
3´ UTR

FigureFigure
I-3-6
...

Eukaryotic
Transcription
Unit
A Eukaryotic
Transcription
Unit

H

HY
MY
LY

Processing of Eukaryotic
Pre-Messenger RNA

HIGH YIEL

High-Yield

The primary transcript must undergo extensive posttranscriptional
MEDIUM processing
YIELD
inside the nucleus to form the mature mRNA molecule
...
A 7-methylguanosine cap is added to the 5′ end while the RNA molecule
FUNDAMENTALS
is still being synthesized
...

REINFORCEMENT

MEDIUM YIE

LOW Y

FUNDAMENT

REINFORCEM

40

USMLE Step 1 Biochem
...
A poly-A tail is attached to the 3′ end
...
The poly-A tail protects the message against rapid degradation
and aids in its transport to the cytoplasm
...

AUG
Exon 1
5´

Pre-mRNA

Poly A addition
signal

UAG
Intron

Exon 2
3´

5´ UTR

3´ UTR
Capping and Poly-A
addition (nucleus)

AUG

hnRNA

5´

Exon 2

Exon 1

Cap

Poly A addition
signal

UAG

Intron

Me
Gppp

AAAAAAAA 3´
Poly-A tail

5´ UTR

5´ Splice 3´ Splice 3´ UTR
donor
acceptor
site
site
Splicing by spliceosome (snRNA) (nucleus)

Excised intron
(lariat) degraded
in nucleus
AUG
Cap
mRNA

5´

3´

Poly A addition
signal

Exon 1

UAG Exon 2

Me
Gppp

5´ UTR

AAAAAAAA 3´
Poly-A tail
Transport to cytoplasm
and translation

3´ UTR

H2N–Protein–COOH
Figure I-3-7
...
Processing
Eukaryotic
Pre-mRNA

41

USMLE Step 1 Biochem
...
For example,
Medical Genetics
mutations that interfere with proper
splicing of β-globin mRNA are
responsible for some cases of
β-thalassemia
...
Introns are removed from hnRNA by splicing, accomplished by spliceosomes (also known as an snRNP, or snurp), which are complexes of snRNA
and protein
...
The intron is excised in the form of a
lariat structure and degraded
...

4
...

5
...


ALTERNATIVE SPLICING OF EUKARYOTIC PRIMARY
PRE-mRNA TRANSCRIPTS
For some genes, the primary transcript is spliced differently to produce two or
more variants of a protein from the same gene
...
Variants of the muscle proteins tropomyosin and troponin T are
produced in this way
...

The primary transcripts from a large percentage of genes undergo alternative
splicing
...
By alternative splicing, an organism can make many more different proteins than it has genes to encode
...
Alternative splicing can be detected by Northern
blot, a technique discussed in Chapter 7
...


AUG
5´ Exon 1

Intron 1

Exon 2

Intron 2

Splicing
variation #1
5´ Exon 1

Exon 2

mRNA #1

Exon 4 3´

Exon 3

Intron 3

UAA
Exon 4 3´

Splicing
variation #2
5´ Exon 1 Exon 3 Exon 4 3´
mRNA #2

Figure I-3-8
...
indb 42

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Chapter 3

●

Transcription and RNA Processing

RIBOSOMAL RNA (rRNA) IS USED TO
CONSTRUCT RIBOSOMES
Eukaryotic ribosomal RNA is transcribed in the nucleolus by RNA polymerase I
as a single piece of 45S RNA, which is subsequently cleaved to yield 28S rRNA,
18S rRNA, and 5
...
RNA polymerase III transcribes the 5S rRNA unit
from a separate gene
...
Eukaryotic ribosomal subunits
are 60S and 40S
...


Prokaryotic Ribosome
50S
subunit

70S
5S RNA
23S RNA

30S
subunit

Eukaryotic Ribosome

16S RNA

80S
5S RNA
60S
subunit 5
...

The
andEukaryotic
EukaryoticRibosomes
Ribosomes
Figure
I-3-9
...
coli), inactivate
the 28S rRNA in the 60S subunit of the
eukaryotic ribosome
...
This prevents
aminoacyl-tRNA binding to the
ribosome, halting protein synthesis
...
The complete prokaryotic ribosome is a 70S particle
...
They are a
function of both size and shape, and therefore the numbers are not additive
...
Each tRNA carries only one type of
activated amino acid for making proteins during translation
...
The
tRNAs enter the cytoplasm where they combine with their appropriate amino
acids
...


43

USMLE Step 1 Biochem
...


A
A

U

Anticodon sequence (CAU)
pairs with codon in mRNA
...
Transfer RNA (tRNA)
Figure I-3-10
...
Posttranscription editing events may include insertion,
deletion, and base alterations of nucleotides (such as adenine deamination)
within the edited RNA molecule
...
 
An important example is cytosine-to-uracil deamination in the apoprotein B
gene
...
In the intestines, the mRNA is edited from a CAA sequence to
be UAA, a stop codon, thus producing the shorter apoprotein B48 form
...
indb 44

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Chapter 3

●

Transcription and RNA Processing

Recall Question
Which of the following is a co-transcriptional event in RNA
synthesis?
A
...
  Splicing
C
...
Important Points About Transcription and RNA Processing

Gene regions

RNA polymerase

Prokaryotic

Eukaryotic

May be polycistronic

Always monocistronic

Genes are continuous coding regions

Genes have exons and introns

Very little spacer (noncoding) DNA
between genes

Large spacer (noncoding) DNA between
genes

Core enzyme: α2ββ′

RNA polymerase I: rRNA
RNA polymerase II: mRNA; snRNA
RNA polymerase III: tRNA, 5S RNA

Initiation of transcription

Promoter (–10) TATAAT and (–35)
sequence

Promoter (–25) TATA and (–70) CAAT
Transcription factors (TFIID) bind promoter

Sigma initiation subunit required to
recognize promoter
mRNA synthesis

Template read 3′ to 5′; mRNA synthesized 5′ to 3′; gene sequence specified from coding
strand 5′ to 3′; transcription begins at +1 base

Termination of transcription

Stem and loop + UUUUU

Not well characterized

Stem and loop + rho factor
Relationship of RNA
t­ ranscript to DNA

RNA is antiparallel and complementary to DNA template strand; RNA is identical (except
U substitutes for T) to DNA coding strand

Posttranscriptional ­processing
of hnRNA (pre-mRNA)

None

In nucleus:
5′ cap (7-MeG)
3′ tail (poly-A sequence)
Removal of introns from pre-RNA
• Alternative splicing yields variants of
protein product

Ribosomes

tRNA

70S (30S and 50S)

80S (40S and 60S)

rRNA and protein

rRNA and protein

Cloverleaf secondary structure
• Acceptor arm (CCA) carries amino acid
• Anticodon arm; anticodon complementary and antiparallel to codon in mRNA

45

USMLE Step 1 Biochem

---