3/24/2020

The Cell Cycle

USMLE Molecular Biochemistry Handout M: Cell Division (Mitosis)
Sister chromatids split, no gene expression

G0: Resting phase

(senescence)
G2: Growth (Gap) Exs: Neurons
2 identical copies of
2 sets of homologous
chromosomes G1: Growth (Gap)
2 homologous
Joshua D. Brooks NOTE: Gene expression during chromosomes
interphase (all but M) Ex: Intestinal epithelial
Associate Director of Pre-Clinical Academics, Kaplan Med
Signal = growth factor
Instructor of Pharmacology, Biochemistry,
S: DNA Replication (Synthesis)
Behavioral Sciences, and Integrated Cases Identical chromatids made of homologues
PHARM: -tinib
joshua.brooks@kaplan.com tyrosine kinase inhibitors
NOTE: Only time DNA synthesis occurs
(stop cell in G1)
P. 4-5
1 2

Cell Cycle Pharmacology Cell Cycle Pharmacology

Cell Division (Mitosis) Cell Division (Mitosis)

Resting phase Resting phase

Growth (Gap) (senescence) Growth (Gap) (senescence)
Growth Growth
(Gap) (Gap)

DNA Replication (Synthesis) DNA Replication (Synthesis)

PHARMACOLOGY: Anti-cancer drugs PHARMACOLOGY: Anti-cancer drugs
Non-Cell Cycle Specific S Phase M Phase Non-Cell Cycle Specific S Phase M Phase
(NCCS) (NCCS)
Cyclophosphamide Methotrexate Paclitaxil Alter DNA structure Target nucleotide Target spindle fibers
Cisplatin 5-flurouracil Vinblastine synthesis or DNA and microtubules
Hydroxyurea Vincristine synthesis
P. 4(m)
3 4
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Nucleotide Structure and Nomenclature The Nitrogenous Base Structures

Only in RNA Only in DNA
• Nucleotides are the building blocks of both DNA and RNA
• Nucleotides consist of three components:
• Five-carbon sugar (ribose ring)
• Nitrogenous base
• Phosphate
MNEMONIC: PURe As Gold MNEMONIC: CUT the Pye

MNEMONIC: PURe As Gold MNEMONIC: CUT the Pye

Think wedding Think pie
Gold rings – need 2! Pie has 1 ring of crust!
MNEMONIC: Which purine? MNEMONIC: Which pyrimidine?
Amine (NH) only? Adenine! Methyl (CH3)? Thymine!
One oxygen? Cytosine!

P. 5
5 6

The Five Carbons of the Ribose Ring Comparing the Polymerases

DNA Sugar RNA Sugar Nucleic acid DNA RNA
synthesized (5 →3 )
Required template DNA* DNA*
(copied 3 →5 )
Required substrates dATP, dGTP, dCTP, ATP, GTP, CTP, UTP
dTTP
Required primer RNA (or DNA – if PCR) None

Proofreading activity Yes No

(3 →5 exonuclease)
Carbons of the Sugar: S phase: Add –PO43- on 5’ to –OH on 3’
1’: Nitrogenous base (not shown) NOMENCLATURE: Nuclease types
PHARM: NRTIs + “-ovirs”
3’: -OH Exonuclease: Cleaves a nucleotide from the end of DNA/RNA
Missing 3’-OH  added to DNA but
5’: Phosphate (if present)
no new phosphodiester (PDE) bonds Endonuclease: Cleaves inside DNA/RNA strand creating fragments
(USMLE CLUE: “chain terminator”) P. 20-21
7 8
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Comparing the Polymerases Comparing the Polymerases

Nucleic acid DNA RNA Nucleic acid DNA RNA
synthesized (5 →3 ) synthesized (5 →3 )
Required template DNA* DNA* Required template DNA* DNA*
(copied 3 →5 ) (copied 3 →5 )
Required substrates dATP, dGTP, dCTP, ATP, GTP, CTP, UTP Required substrates dATP, dGTP, dCTP, ATP, GTP, CTP, UTP
dTTP dTTP
Required primer RNA (or DNA – if PCR) None Required primer RNA (or DNA – if PCR) None

Proofreading activity Yes No Proofreading activity Yes No

(3 →5 exonuclease) (3 →5 exonuclease)
NOMENCLATURE: Polymerase types NOTE: Certain polymerases require RNA template .
DNA polymerase reads DNA, it is a DNA-dependent DNA polymerase RNA-dependent DNA-polymerase (telomerase – eukaryotes;
RNA polymerase reads DNA; it is a DNA-dependent RNA polymerase reverse transcriptase – HIV, retroviruses)
P. 20-21 P. 20-21
9 10

Replication: Origin of Replication Replication: DNA Strand Synthesis

• Sequence of origin of Strand Synthesis
Origin of Replication
replication is found • Primase synthesizes RNA Primer (5’—3’)
3’
Helicase • Helicase breaks the H- Polymerase 5’ • In eukaryotes, DNA polymerase δ & α
bonds Going into fork extend primer
Primer
3’
• Single stranded binding MNEMONIC: Make DNA with δNα
proteins (SSBs) keep the
strands apart Helicase • MICRO: Proks use DNA poly III for this.
MNEMONIC: III letters in DNA
Leading vs. Lagging strand?
Leading: helicase leads polymerase (top) – one primer, one polymerase
Lagging: helicase and polymerase opposite (bottom) ---
multiple primers, multiple polymerases.
P. 21-22, 24(diagram) P. 21-22 (reading), 24
11 12
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Replication: Removing Primers The Replication Fork

Removing Primers / Joining Fragments
(Eukaryotic)
• RNAse H (5’ exonuclease) digests primer Direction of
5’
Replication Fork Leading Strand
• DNA polymerases replaces primer with DNA Read into the fork
Topoisomerase II 5’

• DNA ligase joins fragments (important for Helicase 5’

Primer removed lagging)
and replaced

• MICRO: DNA Poly I BOTH removes primers

(5’ exonuclease) and replaces RNA with DNA Lagging Strand
Read away from fork
MNEMONIC: Poly I goes after Ist nt Topoisomerases
Lagging strand
gaps filled
Unwind DNA (negative supercoiling) to relax & prevent breaks
P. 21-22 (reading), 24
13 14

The Replication Fork The Checkpoint (G1/S)

G1S; No Damage G1S; Some Damage
Direction of Damage sensors
5’
Replication Fork
Cyclins Cyclins p53
Topoisomerase II 5’

Helicase 5’ Increased cyclin

Cyclin-dependent Cyclin-dependent
kinases (CDKS) kinases (CDKS) kinase inhibitor
(p21)
P
Rb E2F Rb E2F
PHARMACOLOGY: Fluoroquinolones (“-floxacin”) “off” “on  to S!” “on” “off (stay in G1)”
Antibiotics leads to dsDNA fragmentation – inhibit DNA gyrase/Topo IV
NOTE: Too much damage?
PHARMACOLOGY: Topoisomerase II inhibitors “-poside” Can’t stay in G1 forever  p53 starts
Anti-neoplastics leads to dsDNA fragmentation S-phase specific apoptosis.

P. 23(m)
15 16
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Tumor Suppressors and Known Defects Base Excision: Cytosine Deamination

Gene Function Disease Damage Cause Recognition & Repair
p53 Prevents damaged cells Li-Fraumeni – family history Excision Enzymes Enzymes
from entering S phase, turn SBLA (Sarcoma, breast,
on cyclin kinase inhibitor leukemia, adrenal gland, Cytosine Spontaneous, Uracil glycosylase, DNA
p21; chromosome 17 early age, multiple tumors) deamination heat, nitrates apyrimidinic (AP) polymerase,
Rb Negative regulator of Retinoblastoma, (G1) endonuclease + lyase DNA ligase
transcription factor E2F, osteosarcoma 5’ 3’ 5’ 3’ 5’ 3’
Heat, Uracil
keeps cell in G1, GGG G G nitrates GGG G G glycosylase GGG G G
CCC CC CCUC C CC CC
chromosome 13

endonuclease
Apyrimidinc
3’ 5’ 3’ 5’ 3’ 5’
PATHOLOGY: Human Papilloma Virus (HPV) induces:
• Oncoprotein E7 – binds to/inhibits Rb 5’ 3’ 5’ 3’ 5’ 3’
• Oncoprotein E6 – binds to/inhibits p53 GGG G G GGG G G GGG G G
CCCCC DNA ligase CCCC C DNA Poly β, ε
CC CC
GENETICS: Tumor suppressor cancers show autosomal dominant inheritance.
3’ 5’ 3’ 5’ 3’ 5’
P. 25
17 18

Nucleotide Excision: Mismatch Repair Lynch Syndrome & Microsatellites

Damage Cause Recognition & Repair Enzymes On Chromosome 7, there is a small tandem repeat (GATA); you get a
Excision Enzymes number of repeats from mom and dad. # of repeats shown.
Mismatch Replication hMSH2 or hMLH1 DNA polymerase HEALTHY LYNCH
TUMOR CELL
Repair (G2) errors initiates repair of β, ε 4 6 4 6
mismatch DNA ligase
PATHOLOGY: Hereditary Nonpolyposis colorectal cancer (Lynch)
4 6 4 6 4 6 6 7
• Familial clustering of cancers (colon, uterine, endometrial, etc.)
• Deficiency in a mismatch repair enzyme
• Mutation on chromosome 2 or 3
4 6 4 6 4 6 4 6 4 6 5 7 7 9 6 7
• Mean age of colon cancer is 40-50 years old
• Problems copying microsatellites
Every healthy cells all pass on One tumor cell loses ability to
same number of repeats. correct; passes on different # of
All cells should have the same #. repeats every generation.
P. 27-28
19 20
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Tumor Suppressors and Known Defects Tumor Suppressors and Known Defects
Gene Function Disease Gene Function Disease
ATM Sensor kinase, detects Ataxia telangiectasia (Louis- APC Binds to and targets Familial adenomatous
damage and relays that info Bar Syndrome) – transcription factor β- polyposis colon cancer
to p53, chromosome 11 hypersensitive to x-rays, catenin for degradation --
predisposed to lymphomas chromosome 5

BRCA-1 Repair single stranded and BRCA-1: Breast, ovary,

PATHOLOGY: Familial adenomatous polyposis (FAP)
BRCA-2 double stranded breaks in prostate cancer
• Mean age of colon cancer is <40 years old
DNA BRCA-2: Breast cancer
• Many (100+) polyps in the colon
BRCA 1—chromosome 17
• APC Gene found in chromosome 5
BRCA 2—chromosome 13
MNEMONIC: Inherited colon cancer
Colon has 5 letters, and 2 + 3 = 5

21 22

Structure of a Chromosome Structure of a Chromosome

3’ 5’ 3’ 5’
Non-template #1 Non-template #2 Template #3 Coding #1 #1
Non-template Coding #2 #2
Non-template Template #3

Template #1 Template #2 Non-template #3 Template #1 Template #2 Coding #3 #3

Non-template
5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’

Genes are double-stranded, but only one is converted to RNA. EXAMPLE

Synonyms: Coding: 5’–AATTGGCC–3’ Synonyms:
METHOD: Find the template strand in dsDNA
Non-template strand Template: 3’–TTAACCGG–5’ Template strand
1st: Find direction polymerase moves (promoter to terminator) Coding strand mRNA: 5’–AAUUGGCC–3’ Non-coding strand
2nd: Find strand with correct polarity in that direction (read 3’  5’) Sense strand Antisense strand
P. 33 P. 33
23 24
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Coding and Template DNA Strands Eukaryotes & RNA Editing

• USMLE TESTED EXAMPLE: ApoB Gene in liver and intestine

NOTE:
…One gene =
one mRNA =
two proteins =
TAKEAWAY: Gene Location Clues RNA editing
+1 is the first nt copied – promoter is before +1; it is not transcribed.
Mutate (+) nucleotides, mutate gene – change protein structure.
Mutate (-) nucleotides, mutate promoter – change protein amount.
P. 35-36 P. 44
25 26

Structure of tRNA The Genetic Code

Universal: Except in mitochondria TIP #1: # of nucleotides
BOARD CLUE: If a question
mentions odd nucleotides, Degenerate: UUU and UUC = Phenylalanine in exons = 3 x # of amino
it’s asking about tRNA! Unambiguous: UUU = Always Phenylalanine acids in protein
CCA added to 3’ end,
then amino acid added to CCA
FOUR CODONS TO REMEMBER:
Start Codon: Stop Codon 1:
RNA = AUG RNA: UAA (You are annoying)
Coding DNA: ATG Coding DNA = TAA
T: ribothymidine Stop Codon 2: Stop Codon 3:
ψ: pseudouridine
RNA: UGA (You go away) RNA: UAG (You are gone)
D: dihydrouridine
Coding DNA: TGA Coding DNA: TAG
Anticodon (H-bonds with codon)
TIP #2 If a Q offers a sequence and no codon chart – it’s about stop codons!
Cm: acetylated cytosine
P. 43-44 P. 50
27 28
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Mutations Mutations

Type of Mutation Effect on Protein Type of Mutation Effect on Protein

Silent: new codon specifies same None Missense: new codon specifies Possible decrease in function
amino acid different amino acid (may increase function,
Sickle-Cell (Glu6Val, GAG  GTG) possible evolutionary change)
See size difference in DNA/RNA? NO
See size difference in DNA/RNA? NO
See size difference in protein? NO
See size difference in protein? NO
See charge difference in protein? MAYBE

P. 51-52 P. 51-52
29 30

Mutations Mutations

Type of Mutation Effect on Protein Type of Mutation Effect on Protein

Missense: new codon specifies Possible decrease in function Nonsense: addition of new stop Shorter than normal, usually
different amino acid (may increase function, codon nonfunctional
Sickle-Cell (Glu6Val, GAG  GTG) possible evolutionary change)
Two types of missense: See size difference in DNA/RNA? NO
Conservative: Wrong but similar type of AA (EX: glu  asp) See size difference in a protein? YES – smaller
Non-conservative : Wrong but different type of AA (EX: glu  val) TAKEAWAY: Silent, missense, & nonsense mutations are examples of
point mutations – nucleotides are changed (DNA size same)
P. 51-52
31 32
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Mutations Mutations
Type of Mutation Effect on Protein
In-frame: addition/deletion of Depends on where the
multiple of 3 bases addition/deletion is made
See size difference in DNA/RNA? YES
Type of Mutation Effect on Protein See size difference in protein? YES
Frameshift: addition/deletion of Loss of function, often shorter In-frame deletion: Cystic fibrosis (CFTR gene)
bases (not a multiple of 3 bases) than normal or missing entirely In-frame addition: Huntington’s disease (huntingtin gene)

TAKEAWAY: Add/delete nucleotides (change DNA size) causes –

See size difference in DNA/RNA? YES • Frameshift (1 or 2 nts): All codons after the mutation are different.
See size difference in protein? Maybe – where’s the new stop codon? • In-frame (3 nts): An extra (or missing) codon; other codons same.

P. 51-52 P. 51-52
33 34

Special Mutations Translation: Initiation

Type of Mutation Effect on Protein
Large segmental deletion Loss of function, shorter than
(unequal crossover in meiosis) normal or entirely missing
Cri-du-chat (terminal, Chr. 5p)

5’ splice site (donor) or 3’ splice Variable effects ranging from

site (acceptor) addition or deletion of a few Required: energy, initiation factors, mRNA, start codon, and Met.
β –thalassemia (long mRNA) amino acids to deletion of an PHARMACOLOGY: Antibiotics that inhibit initiation
entire exon 1) Aminoglycosides: inhibit 30S subunit from assembling, insert
misreads (”frameshift-like”) into the AA code
See size difference in DNA? Possibly – point vs. addition/deletion 2) Linezolid: inhibit 50S subunit from assembling
See size difference in RNA? YES
See size difference in protein? YES

P. 51-52 P. 56-57
35 36
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Translation: Elongation Translation: Elongation

PHARMACOLOGY: Block aminoacyl-tRNA from binding to A site PHARMACOLOGY: Block peptidyl transferase
1) Tetracyclines: 30S 1) Chloramphenicol: 50S
2) Streptogramins: 50S

37 38

Translation: Elongation Modification at the ER and Golgi

PATHOLOGY: Lysosome enzyme
deficiencies

Tay-Sachs, lack of enzyme

Hexoaminidase A  build up
ganglioside GM2 (inclusion
body)

Gaucher, lack of enzyme

glucocerebrosidase  build up
of glucocerebroside (inclusion
PHARMACOLOGY: Block translocation from A site  P site body)
1) Macrolides: 50S
2) Clindamycin: 50S

P. 61, 63(m)
39 40
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Modification at the ER and Golgi Collagen Synthesis

PATHOLOGY: I-Cell Disease Collagen structure: Gly – X – Y
• Phosphotransferase in Golgi
doesn’t work 1) Translation begins in
• No mannose-6-P cytoplasm; N-terminal
• Lysosomal enzymes get hydrophobic signal
secreted (none in lysosome) sequence translated,
• All enzymes missing  growing prepro-α chain
nothing broken down  targeted to RER
inclusion body is VARIETY of
material

I-cell: Golgi (not lysosome) issue

P. 61, 63 P. 64-65
41 42

Collagen Synthesis Collagen Synthesis

2) Signal sequence cleaved 4) Hydroxylysines are
by signal peptidase in ER, glycoslyated
creates pro-α chain
5) Three pro-α chains
3) Lysines & prolines are hydrogen bond together
hydroxylated; chains H- (triple helix); disulfide
bond links added

6) Procollagen secreted from

Enzymes: Lysyl and proyly fibroblast via Golgi
hydroxylase
Required: Vitamin C
PATHOLOGY: Vitamin C
deficiency? Scurvy
P. 64-65 P. 64-65
43 44
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Collagen Synthesis Chromatin Structure and Gene Expression

In eukaryotes, genes are
7) Disulfide links removed
packaged in nucleosomes.
from the ends to form
collagen
Transcription factors bind to
DNA, recruit proteins to
8) Collagen helices are
remodel (e.g. histone acetylase)
assembled into fibrils
through crosslinking
Histone acetylase targets lysine
lysines (Cu++ required)
in histone, releasing DNA
(euchromatin)
Enzymes: Lysyl oxidase
GOAL: make genes more
Required: Copper
available for transcription
PATHOLOGY: Copper deficiency?
Menkes Disease
P. 64-65 P. 75-77
45 46

Bending During Transcriptional Activity Comparing Blotting Techniques

Specific TFs (hormone receptors/downstream signaling)
MNEMONIC:
SNoW DRoP

S: South D: DNA
Enhancer (Response)
N: North R: RNA
Elements
W: West P: Protein

Transcription
General TFs Factor II D

Basal Promoter
Elements

Nuclear factor 1 ANOTHER BLOT? Southwestern Blot

Specificity Protein 1 TATA-Binding Protein Used for finding DNA-binding proteins (uses) 32P-DNA probe
P. 75-77 P. 104
47 48