Flashcards in Biochemistry Lecture 2 - DNA Replication, Repair, and Mutagenesis Deck (54):
What is acyclovir and what does it do?
It is acyclo-guanosine (nucleotide analogue), and it is a potent inhibitor of the DNA polymerase encoded by the herpes simplex virus.
What is Zidovudine (AZT, azidothymidine) and what does it do?
It is a nucleotide analogue and a potent inhibitor of the reverse transcriptase encoded by HIV. It is unfortunately also toxic to Pol gamma involved in mtDNA synthesis. Also, mutant forms of reverse transcriptase have low affinity for the drug.
It stops replication b/c it has no 3'-OH group to nucleophilically attack an adjacent PO4 group.
Where does replication begin and what are important features of this site?
At the origin of replication. H-bonding between DNA bases and backbone and amino acid side chains of protein complexes such as the origin of replication complex (ORC) is critical. This takes place in the major groove.
What constitutes an origin of replication in humans?
We don't know!
What is T-antigen (T-ag)?
It is the origin of replication complex for Simian Virus 40. It encodes its own HELICASE and recruits other proteins that are provided by the host cell.
What does helicase do?
It "unzips" DNA for replication. It uses ATP.
What purpose do the topoisomerases serve in DNA replication?
They unwind positive supercoils as helicase unzips the dsDNA.
What is Bloom's syndrome?
Caused by DNA helicase deficiency, it puts children at risk for malignancies like leukemia.
What is Werner's syndrome?
Caused by DNA helicase deficiency, causes premature aging.
What do single strand binding proteins do? What are some abbreviations for them?
They bind to ssDNA after helicase unzips the DNA for replication so they do not re-anneal. Abbreviations are SSB, RPA!
What does DNA primase do?
It lays down a RNA primer (a ~10 nucleotide cRNA sequence) because DNA polymerase can't initiate DNA synthesis.
Name the 5 human DNA polymerases.
Pol epsilon, Pol alpha, Pol delta (3 main)
Pol gamma is responsible for mtDNA synthesis.
Pol beta plays a role in DNA repair.
What does Pol alpha do?
It initiates all Okazaki fragments with RNA primers on the lagging strand as it has a RNA primase associated with it.
What replaces Pol alpha's RNA primer on the lagging strand with DNA?
What does Pol Delta do?
It fills in the gaps between Okazaki fragments on the lagging strand and are PCNA (Proliferating Cell Nuclear Antigen) and RFC (Replication Factor C) dependent.
What does Pol epsilon do?
It forms the leading strand of DNA during replication. It is also PCNA and RFC dependent.
What is RFC and what does it do?
Replication Factor C. It loads Pol epsilon and Pol delta onto the DNA strand.
What is PCNA and what does it do?
Proliferating Cell Nuclear Antigen. It helps clamp Pol epsilon and Pol delta onto the DNA strand.
What constitutes a high fidelity polymerase? Which human polymerases are high fidelity?
High fidelity polymerases have proofreading activity by the way of exonuclease. Pol epsilon and Pol Delta are high fidelity polymerases.
What does DNA ligase do?
Covalently links together adjacent base pairs. It works by forming an AMP-enzyme complex, then transfers the AMP to a 5' phosphate at the nick, and the activated phosphate forms a covalent bond with the neighboring 3' OH
What is the direction of exonuclease activity on the leading strand?
3' to 5' direction. It is associated with Pol epsilon. It can back up.
What is the direction of exonuclease activity on the lagging strand?
3' to 5' direction for PROOFREADING.
5' to 3' direction for RNA PRIMER REMOVAL.
How does DNA ligase seal single strand nicks in daughter strands?
DNA ligase becomes activated when ATP donates an AMP unit to form an enzyme-AMP complex. The AMP residue is then transferred to the 5'-phosphate present at the nick and the activated phosphate group forms a covalent link with the neighboring 3'-OH.
What are the cell cycle stages and the Cyclin-dependent kinases associated with the stages?
G1 (growth, G1 Cyclin Cdk)
S (DNA replication)
G2 (growth, Cdk mitotic cyclin)
What are cyclin-dependent kinases (Cdk) and what do they do?
They are a complex of at least two proteins, a kinase, and a cyclin that phosphorylate other molecules and are thought to drive the cell from one stage to another.
What two problems in the S and M stages of the cell cycle, respectively, would normally halt the cell cycle?
S - damaged DNA
M - unassembled spindle
Name the cell cycle checkpoints.
(1) G1-S checkpoint
(2) S checkpoints
(1) G2-M checkpoint
(1) Spindle M phase checkpoint
What is p53 and what/where does it function?
It is known as the tumor suppressor protein and it functions at the G1-S checkpoint to identify damaged DNA. If repair is not possible the cell may undergo apoptosis.
What is Li-Fraumeni Syndrome?
A mutant p53 protein fails to stop cells with damaged DNA from replicating and puts patients at risk for developing tumors at multiple sites.
In what percentage of cancer cases is a mutant p53 tumor suppresor gene present?
More than 50%
Briefly outline the steps of prokaryotic lagging strand DNA synthesis (naming the polymerases).
Primase lays down RNA primer, DNA Pol III builds DNA off the primer, then DNA Pol I and ligase come in to replace the RNA primers with DNA and covalently link the adjacent bases together.
Describe mismatch repair in prokaryotes.
Parental strands have methylated adenine bases, so when a mismatch is detected the cell knows which strand is at fault. Potein Mut S. recognizes the mismatch, Mut H. decides who the parent is and catalyzes strand incision, and Mut L. does something (unknown - thought to provide interface between Mut S and Mut H). The strand error is removed with helicase and exonuclease and is filled with DNA Polymerase (III in the case of prokaryotes), SSBs, and DNA ligase.
What is common in people with mutations in the human homologues of Mut S and L genes?
There is a correlation with hereditary non-polyposis colon cancer (HPCC) and other types of cancer. They have defective mismatch repair pathways (which may also be related to trinucleotide repeats).
The mutations also might play a role in expansion of trinucleotide repeats.
Describe Huntington's disease with regards to trinucleotide repeats.
In a person with Huntington's, the "Huntington's gene" on the short arm of chromosome four contains a higher than average (avg is 11 to 34 repeats) number of CAG repeats (codes for glutamine). Beyond this number, the number of repeats is inversely proportional to the age of onset.
What is Fragile-X syndrome?
A disease that results in moderate to severe mental retardation stemming from failure of normal neural development. Expansion to > 200 trinucleotide repeats (within FMR gene on Chromosome 11).
What is myotonic dystrophy?
Multisystem disorder (muscle weakness, endocrine dysfunction, etc.). Dramatic expansions of repeats (~1000).
What is a thymine dimer?
When an environmental mutagen causes covalent bond formation between adjacent thymine bases. This prevents double helix formation and must be corrected before replication.
What are the steps of prokaryotic excision repair?
Describe the excision repair process in prokaryotes.
The pyrimidine dimer is recognized by the products of the uvrABC gene. The uvrABC complex cuts of the dimer piece, then the gap is filled in by Pol I (no primer needed because nucleotides exist on both sides already). Then ligase bonds in the bases.
What causes Xeroderma pigmentosum?
Defective excision repair.
Describe the DNA Uracil removal process.
uracil-DNA glycosidase goes around and looks for U, and cuts out the backbone adjacent to the uracil nucleotide. Sites where bases have been removed are called "AP" sites. Repair is performed as normal.
Cytosine spontaneously deaminates to form what?
Adenine can be chemically deaminated. What is that product and how does it base pair?
Hypoxanthine is the product and it base pairs like Guanine.
What effect does alkylation (methylation) of the N7 of Guanine have on DNA?
The result is effectively the loss of the guanine residue from the DNA.
Polycyclic hydrocarbons may intercalate between adjacent base pairs in DNA, causing bulky adducts (distortions in the 2x helical structure) which can lead to deletion or insertion mutations. Proflavin and ethidium bromide are examples of chemical mutagens.
What is the prokaryotic analogue to DNA Pol epsilon and delta?
DNA Polymerase III (Polymerase I and ligase seal Okazaki fragments in prokaryotes)
Where do protein/base interactions occur in dsDNA?
The major groove.
What protein regions fit into the major groove of DNA?
Alpha helical regions.
What is ORC?
The origin of recognition complex is a protein that binds to the origin of replication and recruits other proteins to begin the replication process.
What are polymerases gamma and beta involved in?
Gamma - mtDNA synthesis
Beta - DNA repair
What is the relationship between the mismatch repair system and trinucleotide repeats?
An effective mismatch repair system is thought to stabilize trinucleotide repeats.
What can proflavin and ethidium bromide do to DNA?
They can intercalate!
What is the MCM complex?
It is thought to bind to the origin of replication and uses it's helicase to unwind the dsDNA.