Biochem - Molecular (DNA Replication, Mutations, & Repair) Flashcards
Pg. 69-71 in First Aid 2014 Sections include: -DNA replication -Mutations in DNA -DNA repair
Compare/Contrast DNA replication in eukaryotes and prokaryotes.
Eukaryotic DNA replication is more complex than the prokaryotic process but uses many enzymes analogous to those listed below. In both prokaryotes and eukaryotes, DNA replication is semiconservative and involves both continuous and discontinuous (Okazaki fragment) synthesis.
What is the origin of replication, and what purpose does it serve? Is it single or are their multiple origins?
Particular consensus sequence of base pairs in genome where DNA replication begins. May be single (prokaryotes) or multiple (eukaryotes).
What is the Replication fork, and what purpose does it serve?
Y-shaped region along DNA template where leading and lagging strands are synthesized.
What is the function of Helicase?
Unwinds DNA template at replication fork
What is the function of Single-stranded binding proteins?
Prevent strands from reannealing
What is the function of DNA topoisomerases?
Create a single- or double-stranded break in the helix to add or remove supercoils
What is the mechanism of Fluoroquinolones?
Fluoroquinolones - inhibit DNA gyrase (prokaryotic topoisomerase II)
What is the function of Primase?
Makes an RNA primer on which DNA polymerase III can initiate replication
In what organisms is DNA polymerase I versus III found?
DNA polymerase I (Prokaryotic only) & DNA polymerase III (Prokaryotic only)
What is the function of DNA polymerase III?
Elongates leading strand by adding deoxynucleotides to the 3’ end. Elongates lagging strand until it reaches primar of preceding fragment. 3’ => 5’ exonuclease activity “proofreads” each added nucleotide. DNA polymerase III has 5’ => 3’ synthesis and proofreads with 3’ => 5’ exonuclease.
What is the function of DNA polymerase I? How does it compare/contrast to DNA polymerase III?
Degrades RNA primer; replaces it with DNA; Has same functions as DNA polymerase III but also excises RNA primer with 5’ => 3’ exonuclease.
What is the function of DNA ligase?
Catalyzes the formation of a phosphodiester bond within a strand of double-stranded DNA (i.e., joins Okazaki fragment). Seals.
What is Telomerase, and what function does it have?
An RNA-dependent DNA polymerase that adds DNA to 3’ ends of chromosomes to avoid loss of genetic material with every duplication.
Draw a zoomed in version of DNA replication, including and labeling the following: (1) Fork movement (2) Leading strand (3) Lagging strand (4) Area of interest (5) Origin of replication.
See p. 69 in First Aid 2014 for visual at bottom left of page
Draw DNA replication, including and labeling the following: (A) Origin of replication (B) Replication fork (C) Helicase (D) Single-stranded binding protein (E) Topoisomerase (F) Primase (G) DNA polymerase III (H) DNA polymerase I (I) DNA ligase.
See p. 69 in First Aid 2014 for visual at bottom right
List the 4 major types of DNA mutations in order of increasing severity.
Severity of damage: silent «_space;missense < nonsense < frameshift
What is a transition versus transversion? To what kinds of mutations do they apply?
For silent, missense, and nonsense mutations: TRANSITION - purine to purine (e.g., A to G) or pyrimidine to pyrimidine (e.g., C to T), TRANSVERSION - purine to pyrimidine (e.g., A to T) or pyrimidine to purine (e.g., C to G)
What defines a silent DNA mutation? Where does it often occur, and why?
Nucleotide substitution but codes for same (synonymous) amino acid; often base change in 3rd position of codon (tRNA wobble)
What defines a missense DNA mutation? In what context is it considered conservative?
Nucleotide substance resulting in changed amino acid (called conservative if new amino acid is similar in chemical structure)
What is a clinical example of a missense DNA mutation?
Sickle cell disease
What defines a nonsense DNA mutation?
Nucleotide substitution resulting in early stop codon; Think: “STOP the NONSENSE!”
What defines a frameshift DNA mutation? What is usually the result of this? What is a clinical example of this?
Deletion or insertion of a number of nucleotides not divisible by 3, resulting in misreading of all nucleotides downstream, usually resulting in a truncated, nonfunctional protein; Duchenne muscular dystrophy
Describe the process of Nucleotide excision repair. For what general purpose is it used?
Specific endonucleases release the oligonucleotide-containing damaged bases; DNA polymerase and ligase fill and reseal the gap, respectively; Repairs bulky helix-distorting lesions.
In what condition is Nucleotide excision repair defective? What characterizes this condition?
Defective in Xeroderma pigmentosum, which prevents repair of pyrimidine dimers because of ultraviolet light exposure.
Describe the process of Base excision repair, including key factors and their functions.
Base-specific glycosylase recognizes altered base and creates AP site (apurinic/apyrimidinic). One or more nucleotides are removed by AP-endonuclease, which cleaves the 5’ end. Lyase cleaves the 3’ end. DNA polymerase-Beta fills the gap and DNA ligase seals it.
In what context is Base excision repair important?
Important in repair of spontaneous/toxic deamination
Briefly describe the process of Mismatch repair.
Newly synthesized strand is recognized, mismatched nucleotides are removed, and the gap is filled and resealed.
In what condition is Mismatch repair defective?
Defective in hereditary nonpolyposis colorectal cancer (HNPCC)
What are 3 types of single strand DNA repair?
(1) Nucleotide excision repair (2) Base excision repair (3) Mismatch repair
What is a type of double strand DNA repair?
Nonhomologous end joining
What is the purpose of Nonhomologous end joining? Is homology required?
Brings together 2 ends of DNA fragments to repair double-stranded breaks. No requirement for homology.
In what condition is Nonhomologus end joining mutated?
Mutated in ataxia telangiectasia
