biochem usmle 1-3

Silent mutation

A single nucleotide change that does not alter the amino acid due to the degeneracy of the genetic code.

Missense mutation

A single nucleotide change that alters the amino acid.

Nonsense mutation

A single nucleotide change that results in a stop codon, causing premature termination of translation.

Frameshift Insertion

The insertion of a single nucleotide causes a frameshift, altering the entire reading frame downstream.

Frameshift Deletion

The deletion of a single nucleotide causes a frameshift, altering the entire reading frame downstream.

Transition

Purine to purine (eg, A to G) or pyrimidine to pyrimidine (eg, C to T).

Transversion

Purine to pyrimidine (eg A to T) or pyrimidine to purine (eg C to G).

Frameshift mutation

Deletion or insertion of any number of nucleotides not divisible by 3 turn to misreading of all nucleotides downstream.

Splice site mutation

Retained intron in mRNA turns to protein with impaired or altered function.

Duchenne muscular dystrophy

An example of a frameshift mutation.

Tay-sachs disease

An example of a frameshift mutation.

Cystic fibrosis

An example of a frameshift mutation.

GAG

Original coding DNA sequence.

GAA

Coding DNA sequence in a silent mutation.

GTG

Coding DNA sequence in a missense mutation.

TAG

Coding DNA sequence in a nonsense mutation.

GATG

Coding DNA sequence with an extra 'T' inserted in a frameshift insertion.

GAC

Coding DNA sequence missing 'G' in a frameshift deletion.

Glutamic Acid

Amino acid coded by the original sequence GAG.

Valine

Amino acid that replaces Glutamic Acid in a missense mutation.

Stop

Resulting amino acid in a nonsense mutation.

Aspartic Acid

Starting amino acid in altered sequence due to frameshift.

Frameshift mutations

Insertions or deletions that alter the reading frame, leading to significant downstream changes.

Lac operon

Classic example of a genetic response to an environmental change, activated to switch to lactose metabolism when glucose is absent.

Mechanism of shift

Low glucose = increase adenylate cyclase activity and turns to increased generation of cAMP from ATP which activates catabolite activator protein (CAP) and increased transcription.

High lactose effect

Unbinds repressor protein from repressor/operator site and increases transcription.

Inducible operon

The lac operon is an inducible operon that regulates lactose metabolism.

LacI

Repressor gene.

CAP site

Catabolite activator protein binding site.

Promoter

RNA polymerase binding site.

Operator

Repressor protein binding site.

Structural genes

LacZ, LacY, LacA (encode enzymes for lactose metabolism).

High Glucose, Lactose Unavailable

Glucose inhibits adenylate cyclase, preventing cAMP production, leading to Lac genes not being expressed.

High Glucose, Lactose Available

Lactose is present and converted to allolactose (inducer), allowing basal expression of the lac genes.

Low Glucose, Lactose Unavailable

Low glucose activates adenylate cyclase, producing cAMP, but the repressor remains active, blocking transcription.

Low Glucose, Lactose Available

Low glucose activates adenylate cyclase, increasing cAMP levels, allowing strong expression of lac genes.

CAP Binding

Condition where catabolite activator protein binds to the CAP site, enhancing RNA polymerase binding.

Repressor Binding

Condition where the repressor protein binds to the operator, blocking transcription.

Strong expression of lac genes

Occurs when low glucose and lactose are present.

Lac genes not expressed

Occurs under high glucose, lactose absent conditions.

Very low (basal) expression

Occurs under high glucose, lactose present conditions.

Key Points

1. CAP activation (low glucose) and repressor inactivation (presence of lactose) are both required for strong lac operon expression.

Glucose effect on CAP

Glucose inhibits CAP binding via low cAMP levels.

Allolactose function

Inactivates the repressor.

Nonhomologous end joining

Brings together 2 ends of DNA fragments to repair double-stranded breaks. Homology not required, part of the DNA may be lost or translocated. May be dysfunctional in ataxia telangiectasia.

Homologous recombination

Requires 2 homologous DNA duplexes, a strand from damaged dsDNA is repaired using a complementary strand from intact homologous dsDNA as a template. Defective in breast/ovarian cancers with BRCA1 or BRCA2 mutations and in Fanconi anemia. Restores duplexes accurately without loss of nucleotides.

Nucleotide excision repair

Specific endonucleases remove the oligonucleotides containing damaged bases; DNA polymerase and ligase fill and reseal the gap, respectively. Repairs bulky helix-distorting lesions (eg, pyrimidine dimers). Occurs in G1 phase of cell cycle. Defective in xeroderma pigmentosum (inability to repair DNA pyrimidine dimers caused by UV exposure) presents with dry skin, photosensitivity, skin cancer.

Base excision repair

Base-specific Glycosylase removes altered base and creates AP site (apurinic/apyrimidinic). One or more nucleotides are removed by AP-Endonuclease, which cleaves 5' end. AP-Lyase cleaves 3' end. DNA Polymerase-B fills the gap and DNA Ligase seals it. Occurs throughout the cell cycle. Important in repair of spontaneous/toxic deamination.

Mismatch repair

Mismatched nucleotides in newly synthesized strand are removed and gap is filled and resealed. Occurs predominantly in S phase of cell cycle. Defective in Lynch syndrome (hereditary nonpolyposis colorectal cancer [HNPCC]).

Nucleotide Excision Repair (NER)

Purpose: Repairs bulky DNA lesions, such as pyrimidine dimers caused by UV radiation. 1. Damage occurs: UV light causes the formation of pyrimidine dimers (e.g., thymine dimers). 2. Recognition: Endonucleases detect the distortion in the DNA helix. 3. Excision: Endonucleases remove the damaged segment of nucleotides. 4. Repair synthesis: DNA polymerase synthesizes a new DNA segment using the undamaged strand as a template. 5. Ligation: DNA ligase seals the newly synthesized segment into the DNA backbone. Clinical correlation: Defective NER causes xeroderma pigmentosum (UV light hypersensitivity and skin cancer).

Base Excision Repair (BER)

Purpose: Repairs non-bulky, small base damage, such as deaminated or oxidized bases. 1. Damage occurs: A single base is altered, such as deamination of cytosine (C) to uracil (U). 2. Recognition: A DNA glycosylase recognizes and removes the damaged base, creating an AP (apurinic/apyrimidinic) site. 3. Backbone removal: Endonuclease cuts the 5' end of the damaged site, and lyase cleaves the 3' end. 4. Repair synthesis: DNA polymerase fills in the gap with the correct nucleotide. 5. Ligation: DNA ligase seals the strand. Clinical correlation: BER is crucial for repairing spontaneous base damage caused by cellular metabolism.

Mismatch Repair (MMR)

Purpose: Corrects mismatched nucleotides introduced during DNA replication. 1. Damage occurs: A mismatched base pair (e.g., G paired with A) is incorporated during DNA replication. 2. Recognition: Mismatch repair proteins identify the error on the newly synthesized strand. 3. Excision: The mismatched segment is removed by endonucleases. 4. Repair synthesis: DNA polymerase replaces the missing segment with the correct nucleotides. 5. Ligation: DNA ligase seals the repaired segment into the backbone. Clinical correlation: Defective MMR causes Lynch syndrome (hereditary nonpolyposis colorectal cancer).

NER Clinical correlation

Defective NER causes xeroderma pigmentosum (UV light hypersensitivity and skin cancer).

BER Clinical correlation

BER is crucial for repairing spontaneous base damage caused by cellular metabolism.

MMR Clinical correlation

Defective MMR causes Lynch syndrome (hereditary nonpolyposis colorectal cancer).

Key Summary of NER

NER: Repairs bulky lesions (e.g., UV-induced thymine dimers).

Key Summary of BER

BER: Repairs small base damage (e.g., deamination or oxidation).

Key Summary of MMR

MMR: Corrects replication errors (e.g., mismatched base pairs).

DNA replication

Occurs in 5' to 3' direction (5ynthe3sis) in continuous and discontinuous (okazaki fragment) fashion. Semiconservative. More complex in eukaryotes than in prokaryotes, but shares analogous enzymes.

Origin of replication

Particular consensus sequence in genome where DNA replication begins, may be single (prokaryotes) or multiple (eukaryotes). AT-rich sequences (eg, TATA box regions) are found in promoters (often upstream) and origins of replication (ori).

Replication fork

Y-shaped region along DNA template where leading and lagging strands are synthesized.

Helicase

Unwinds DNA template at replication fork. Helicase halves DNA. Deficient in BLooM (BLM gene mutation).

Single-stranded binding proteins

Prevent strands from reannealing or degradation by nucleases.

DNA topoisomerases

Creates a single (topoisomerase 1) or double (topoisomerase 2) stranded break in the helix to add or remove supercoils (as needed due to underwinding or overwinding of DNA).

Topoisomerase inhibitors in eukaryotes

Irinotecan/topotecan inhibit topoisomerase (TOP) 1, etoposide/teniposide inhibit TOP 2.

Topoisomerase inhibitors in prokaryotes

Fluoroquinolones inhibit TOP 2 (DNA gyrase) and TOP 4.

Primase

Makes RNA primer for DNA polymerase 3 to initiate replication.

DNA polymerase 3

Prokaryotes only, elongates leading strand by adding deoxynucleotides to the 3' end. Elongates lagging strand until it reaches primer of preceding fragment. DNA polymerase 3 has 5'- 3' synthesis and proofreads with 3' - 5' exonuclease.

Chain termination drugs

Drugs blocking DNA replication often have a modified 3' OH, thereby preventing addition of the next nucleotide.

DNA polymerase 1

Prokaryotes only, degrades RNA primer and replaces it with DNA. Same functions as DNA polymerase 3, also excises RNA primer with 5' - 3' exonuclease.

DNA ligase

Catalyzes the formation of a phosphodiester bond within a strand of double-stranded DNA. Joins Okazaki fragments.

Telomerase

Eukaryotes only, a reverse transcriptase (RNA dependent DNA polymerase) that adds DNA (TTAGGG) to 3' end of chromosomes to avoid loss of genetic material with every duplication. Upregulated in progenitor cells and also often in cancer; downregulated in aging and progeria.

Steps of DNA Replication

1. Origin of Replication (A) - DNA replication begins at a specific origin of replication. 2. Topoisomerase (E) - Reduces supercoiling and tension ahead of the replication fork. 3. Helicase (C) - Unwinds the double-stranded DNA. 4. Single-Stranded Binding Proteins (D) - Stabilize unwound DNA strands. 5. Primase (F) - Synthesizes short RNA primers. 6. DNA Polymerase III (G) - Synthesizes leading strand continuously and lagging strand in fragments. 7. DNA Polymerase I (H) - Removes RNA primers and replaces with DNA. 8. DNA Ligase (I) - Seals gaps between Okazaki fragments.

Leading strand

Synthesized continuously in the 5' → 3' direction.

Lagging strand

Synthesized discontinuously in fragments (Okazaki fragments).

Enzymes involved in DNA replication

Topoisomerase, helicase, primase, DNA polymerases (III and I), and DNA ligase.

Chromatin

DNA condensed for fitting in the nucleus.

H1 Histone

Binds nucleosome and linker DNA for stabilization.

DNA Charge

Phosphate groups impart a negative charge.

Histone Charge

Lysine and Arginine provide a positive charge.

Mitosis

Process where DNA condenses into chromosomes.

S Phase

Period of DNA and histone synthesis.

Mitochondrial DNA

Circular DNA not utilizing histones.

Heterochromatin

Highly condensed, darker appearance on EM.

Euchromatin

Less condensed, transcriptionally active DNA.

DNA Methylation

Reversible expression change without sequence alteration.

CpG Islands

Methylation here typically silences gene transcription.

Fragile X Syndrome

Dysregulated methylation implicated in this condition.

Histone Methylation

Can suppress or activate transcription based on location.

Histone Acetylation

Increases transcription by relaxing DNA coiling.

Thyroid Hormone Acetylation

Altered synthesis due to receptor acetylation.

Histone Deacetylation

Tightens DNA coiling, decreasing transcription.

Huntington's Disease

Altered expression linked to histone deacetylation.

Nucleotide

Base, deoxyribose, and phosphate form the building block.

Nucleoside

Base plus deoxyribose, lacking phosphate.

Phosphodiester Bond

Links nucleotides via 3'-5' connection.

Purines

Nitrogenous bases with two rings.

Pyrimidines

Nitrogenous bases with one ring.

C-G Bond Strength

Stronger due to three hydrogen bonds.

A-T Bond Strength

Weaker with two hydrogen bonds.