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What is the central dogma of molecular biology?
DNA transcribes into RNA which then translates into protein
What are the exceptions to the central dogma of molecular biology?
1.) Reverse Transcription: Single-strand RNA viruses (retroviruses) can convert mRNA back DNA
2.) RNA Replication (ssRNA virus): SARS-Cov-2 (Covid-19)
What is the structure of DNA?
1.) Nitrogen Base
2.) Carbon Sugar
3.) Phosphate group
How are nucleic acid chains read?
5’ → 3’
The 3’ end always has a what?
hydroxyl group
How is the double helix stabilized?
hydrogen bonds and hydrophobic stacking
How many hydrogen bonds between adenine and thymine?
2
How many hydrogen bonds between guanine and cytosine?
3
What are Chargaff’s Rules?
The amount of adenine equals the amount of thymine, and the amount of guanine equals the amount of cytosine, and the total amount of purines equals the total amount of pyrimidine
What makes up the DNA double helix?
Phosphodiester Bond: The bond between the 3’ hydroxyl of a sugar group in a nucleotide and a phosphate group attached to the 5’ carbon of another sugar group (links nucleotides together to make up the backbone)
Glycosidic Bond: The nitrogen-carbon linkage between the 9’ nitrogen of purine bases or 1’ nitrogen of pyrimidine bases and the 1’ carbon of the sugar group (links the base pairs onto the deoxyribose-phosphate backbone)
What are intercalating agents?
Hydrophobic molecules containing flat aromatic and fused heterocyclic rings can be inserted between the stacked base pairs of DNA.
They are potential cancer-inducing agents
What are examples of intercalating agents?
Ethidium bromide
Acridine Orange
Actinomycin D
Cyber Green
What is the function of Helicase?
Opens up the DNA at the replication fork
What is the function of Topoisomerase?
Works at the region ahead of the replication fork to prevent supercoiling. (nicks the helix to release the tension)
What is the function of Primase?
Synthesizes RNA primers complementary to the DNA strand.
What is the function of DNA polymerase III?
Extends the primers, adding on to the 3' end, to make the bulk of the new DNA.
What is the function of DNA polymerase I?
RNA primers are removed and replaced with DNA (Exonuclease)
What is the function of DNA ligase?
The gaps between DNA fragments are sealed.
What are the differences between Type I and Type II DNA topoisomerases?
Type I cleaves one strand, while Type II cleaves both strands.
Type I does not require ATP while Type II does
How does the antimicrobial Ciprofloxacin and Quinolones work?
By inhibiting DNA gyrase (topoisomerase II), ciprofloxacin prevents bacteria from replicating their DNA, leading to cell death.
What are the main differences between eukaryotic and prokaryotic DNA replication?
1.) Multiple origins vs single origin
2.) Occurs in S phase of cell cycle
3.) Multiple Polymerases
4.) Multiple Enzymes
What does Rnase H do in eukaryotic replication?
Removes RNA primers
What are anti-cancer drugs that target topoisomerases in eukaryotic DNA replication?
Etoposide: human topoisomerase II
Camptothectin: topoisomerase I
Indenoisoquinoline: topoisomerase I
What is a nucleosome?
histones + DNA
How are the 8 histones of the core particle arranged?
They arranged as an octamer composed of (H3)2(H4)2 tetramer and a pair of (H2A-H2B) dimers
What is telomerase?
The telomerase enzyme is an RNA-dependent DNA polymerase (reverse transcriptase) that uses RNA template for the addition of DNA sequences on to the telomere, thereby compensating for the loss of DNA that occurs with each round of replication
TTAGGG
New synthesis (extension of 3’ overhang)
How can you inhibit DNA synthesis?
Nucleoside analog: modification on the hydroxyl group at the 3’-carbon of the deoxyribose
What is Cisplatin?
A chemotherapeutic agent that reacts with DNA such that nitrogens of adjacent purines (guanines) replace the chloride atoms. This modification disrupts DNA structure by cross-linking two purines and leads to cell death
What is a transition substitution point mutation?
A→G Purine →Purine
C→T Pyrimidines→Pyrimidines (Methylation then deamination)
What is a transversion substitution point mutation?
Purine←→Pyrimidines
What is the most common point mutation in cancer cells?
C:G → T:A (transition)
Reactive oxygen species can do what?
Induce DNA damage
What is alkylation?
A chemical reaction that entails transfer of an alkyl group
Methylation is the most common type of alkylation
What are the 4 main ways that bases are altered?
Deamination, oxidation, methylation, and depurination
What is a thymine dimer?
Ultraviolet radiation leads to crosslinking of adjacent pyrimidines (thymine) along one strand of DNA
What can cause a double-strand break?
Ionizing radiations, including X-rays and radioactive decays, and free radical products of oxidative metabolism
What us the function of retinoblastoma protein (RB)?
In G0 and early G1, Rb physically associates with E2F factors and blocks their transactivation domain
In late G1, Rb-p releases E2F, allowing the expression of genes that encode products necessary for S-phase progression
What is direct reversal repair?
Do not require a template
Are specific to the type of damage incurred and do not involbe breakage of the phosphodiester backbone
Methylation of guanine bases, is directly reversed by the protein methyl guanine methyl transferase (MGMT)
What are the three types of DNA repair?
1.) Mismatch repair
2.) Base excision repair
3.) Nucleotide excision repair
How does DNA repair UV-induced Thymine Dimer
1.) Identify mismatched or mutated DNA strand
2.) An enzyme cuts out and removes the damaged DNA (excision endonuclease)
3.) DNA polymerase fills the gap
4.) DNA ligase forms the phosphodiester linkage
What pathway is Hereditary Nonpolyposis Colorectal Cancer (HNPCC) associated with?
DNA mismatch repair pathway
What pathway is Xeroderma Pigmentosum (XP) associated with?
Nucleotide-excision repair
What pathway is Ataxia Telangiectasia (AT) associated with?
Response to dsDNA breaks
What pathway are familial breast and ovarian cancer associated with?
Homology-directed repair of dsDNA breaks
What is the ames test-assay for carcinogenicity?
Salmonella his — strain must be grown in the presence of Histidine, and the mutant allele is susceptible to back-mutation to a wild-type allele
many non-carcinogens are converted to carcinogens in the liver
Mix test compound and homogenized rat liver together, add salmonella bacteria unable to grow without added histidine in culture medium, count colonies that were able to grow beause they underwent mutation
What are the two important tumor suppressing genes?
p53 and Rb
Does RNA synthesis require primers?
RNA chains can be initiated de novo so no primers are required
What does a holoenzyme do in RNA synthesis?
Recognizes promoter regions on the DNA
Why is the sigma subunit of RNA polymerases important?
The sigma subunit of the holoenzyme helps the polymerase locate the promoter sites
What are the three steps of RNA synthesis?
1.) Initiation: binding of the RNA polymerase holoenzyme to the promoter region
2.) Elongation
3.) Termination: The RNA polymerase stops moving on the DNA template. The RNA transcript falls off from the transcription complex
What is the pribnow box in E.coli?
In E. coli, 2 DNA sequences that act as a promoter for many genes are the -10 sequence (Pribnow box) and the -35 sequence
What is Rho-independent (intrinsic termination)
RNA transcript forms a stable hairpin turn (stem-loop); RNA transcripts contains a string of U’s
What is Rho-dependent (protein dependent)
rho-factor binds to. aC-rich region; contains helicase activity to unwind the 3’-end of the transcript from template; displace the DNA template strand
What is the TATA box?
Recognized by proteins other than RNA polymerase itself
closely resembles the bacterial -10 sequence but is further from the start site . Pribnow box (in prokaryotes)
What are enhancers?
Cis-acting elements that have no promoter activity of their own yet can excert their stimulatory actions over distances of several thousand base pairs
What do ribozymes do?
-Catalytic RNAs that function as enzymes and do not require proteins for catalysis
-occur mostly within self-splicing introns and RNA encoded parasites
limited to cleavage and ligation of RNA
What is Rifampin?
inhibits initiation and elongation
What is actinomycin D?
Actinomycin intercalates between bases of the DNA double helix, preventing the DNA from being used as a template
What is alpha-amanitin?
High specificity for eukaryotic RNA Polymerase II, made by mushrooms
