Lecture 2: DNA Replication/Cell Cycle Flashcards
Origin of Replication
An initiation site for DNA replication.
Not understood what constitutes a human origin of replication.
Helicase
An enzyme that disrupts the hydrogen bonds that hold duplex DNA together.
SSB Protein
Single strand binding protein. Present to prevent reannealing.
RPA
Human single strand binding protein.
ORC
Origin recognition complex.
A group of proteins that are required for identification of the origin of replication.
A syndrome resulting from a DNA helicase deficiency, increasing risks for leukemia.
Bloom’s Syndrome.
Werner’s Syndrome
A premature aging disease associated with a defective helicase.
DNA Primase
Synthesizes about a 10 nt RNA primer.
DNA Polymerase
Adds nucleotides sequentially to the 3’ termini of growing chains.
What direction do polymerases move in?
5’ to 3’.
“Downhill.”
What is the specific mechanism DNA polymerases use add nucleotides?
Catalyze the nucleophilic attack of the 3’-OH of the preceding deoxyribose on the innermost phosphorous atom of a correctly base paired deoxyribonucleotide triphosphate (dNTP). Upon formation of a phosphodiester bond, pyrophosphate (PPi) is released.
dNTP
Deoxyribonucleotide triphosphate
PPi
Pyrophosphate (P207, 4-)
What are the three polymerases that participate in higher eukaryotic DNA replication?
- Pol α-primase complex
- Pol ε
- Pol δ
Leading Strand
Polymerized continuously by pol ε behind DNA helicase
Polymerase α - primase complex
Pol α - Primase complex
Come back to this!***
Pol α is the only known polymerase to be associated with a primase. This specific complex plays a critical role in the initiation of every Okazaki Fragment.
Polymerase ε
Pol ε
Catalyzes polymerization of the LEADING strand.
Is considered a “high fidelity” polymerase due to it’s 3’ to 5’ exonuclease “proofreading” activity.
Polymerase δ
Pol δ
After the RNA primer (laid by Pol α - Primase complex) is removed by an exonuclease, this enzyme catalyzes polymerization of the LAGGING strand by forming Okazaki fragments.
Considered a “high-fidelity” polymerase.
Exonuclease
Cleave individual nucleotides from the ends of polynucleotide chains.
PCNA
Proliferating Cell Nuclear Antigen
Acts as a “clamp” to hold Pol ε and Pol δ onto the DNA.
RFC
Replication Factor C
Helps to load Pol δ and Pol ε onto DNA.
Lagging Strand
Overall growth of the strand is in the 3’ to 5’ direction, but all polymerases move in the 5’ to 3’ direction.
Made up of Okazaki fragments.
Lagging strand fragments that are due to the 5’ to 3’ nature of polymerases working in a 3’ to 5’ direction.
Okazaki Fragment
After activation by formation of an enzyme-AMP (from ATP) complex, this enzyme then catalyzes the covalent linking of each Okazaki fragment by inserting an activated phosphate group.
DNA Ligase
DNA Polymerase γ
Pol γ
Responsible for human mitochondrial DNA synthesis.
DNA Polymerase β
Pol β
Is believed to play a role in certain repair events.
Potent inhibitor of the DNA polymerase encoded by herpes simplex viruses.
Acyclovir
AZT
Azidothymidine
Potent inhibitor of the reverse transcriptase encoded by the HIV virus.
Limited due to toxicity; it is a substrate for DNA polymerase γ and disrupts mitochondrial DNA synthesis.
List the four phases of mitosis.
- G1 (cell growth)
- S (synthesis and DNA replication)
- G2 (cell growth)
- M (mitosis)
Protein Kinases
Enzymes that add phosphate groups to other molecules; phosphorylators.
CDK
Cyclin-dependent protein kinases. Made up of a kinase and a cyclin.
Closely regulate progress through the cell cycle.
Cyclin
Any of a number of proteins associated with the cycle of cell division; thought to initiate certain processes of mitosis.
p53
A protein that functions at the G1/S checkpoint to help prevent cells from entering into S phase if they contain damaged DNA. The DNA will then be repaired.
Alternatively, if the DNA cannot be repaired, apoptosis may occur.
Therefore, p53 is a tumor-suppressing protein. A defective p53 protein may cause cell proliferation (cancer).
Li-Fraumeni Syndrome
Associated with individuals who inherit mutant forms of p53; these people are at risk of developing tumors at multiple sites.
How do enzymes involved in mismatch repair identify the proper “wild-type” versus the mutant?
In E. coli, it has been shown to be “methylation” of adenine residues in the wild type.
Newly replicated DNA strands lack these methyl groups.
What are the 3 mismatch repair proteins in E. coli?
- Mut S
- Mut H
- Mut L
Mut S
Recognizes mismatched bases. (Seeks)
Mismatch repair protein in E. coli.
Mut H
Recognizes the d(GATC) site and subsequently catalyzes the incision. (Hacks)
Mismatch repair protein in E. coli.
Mut L
Unknown function, but is thought to provide a protein-protein interface between Mut S and Mut H.
(Lack of understanding)
Mismatch repair protein in E. coli.
After incision by Mut H in mismatch repair in E. coli, what are the following steps?
- “Unzipping” by DNA helicase and an exonuclease.
- SSB binds.
- Filled in by a DNA polymerase (DNA pol III, E. coli specific.)
- DNA ligase to suture.
- Methylation.
hMSH
human Mut S homologue
hMLH
human Mut L homologue
Trinucleotide Repeat Disorder
Genetic disorders caused by trinucleotide repeat expansion, a kind of mutation where trinucleotide repeats in certain genes exceed the normal, stable threshold, which differs per gene.
Huntington’s Disease
People inherit a Huntington’s gene containing CAG repeats above the normal range. They run a rusk of developing Huntington’s Disease.
Inverse relationship with age of onset and number of repeats. Genetic anticipation.
Excision Repair
The removal of a “faulty” DNA segment and its replacement by DNA synthesis.
Name the four steps of excision repair.
- Incision.
- Excision.
- Resynthesis.
- Ligation.
Which step of excision repair is specific to the type of damage?
Step #1, incision.
uvrABC Complex
The protein/enzyme complex that performs the incision step of excision repair of UV-damage (DNA thymine dimer formation) DNA in E. coli.
What is the purpose of studying repair systems in E. coli?
To demonstrate that many of the same enzymes that function in DNA replication are often involved in DNA repair.
What skin disease results from biochemical defects in the excision repair process of UV-damaged DNA in humans?
Xeroderma Pigmentosum.
People with this condition generally die from metastases of malignant skin tumors before the age of 30.
Which nitrogenous base spontaneously deaminates to form uracil?
Cytosine.
Though this mutation happens at a low rate, it is significant.
Which enzyme removes uracil from DNA in humans?
Uracil-DNA glycosidase.
Name the three common types of mutations:
- Substitutions
- Deletions
- Insertions
Substitution
Mutation that exchanges one base for another.
Insertion
Mutations in which extra base pairs are inserted into a new place in the DNA.
Deletion
Mutation in which a section of DNA is lost, or deleted.
Name three types of chemical mutations.
- Deamination
- Alkylation
- Intercalation (by polycyclic hydrocarbons)
Proflavin and Ethidium Bromide are what types of chemical mutagens?
Intercalators.
Radiation
Exposure of cells to ionizing radiation causing the removal of electrons from molecules. Can alter the structure of DNA.
What repair system repairs alkylations and spontaneous cytosine–>uracil reactions?
Base excision repair.
What is the nucleotide excision repair system used for?
Removal of bulky adducts (thymine dimers), likely from UV damage.
What repair system fixes replication errors, insertions, and deletions?
Mismatch repair system.
Mutations in hMSH or hMLH may cause what pathology?
Hereditary non-polyposis colon cancer (HNPCC).
Name three trinucleotide repeat disorders.
- Huntington’s Disease
- Myotonic Dystrophy
- Fragile X Syndrome
