DNA Replication/Repair Flashcards
What is DnaA?
A protein that initiates replication in both prokaryotes and eukaryotes.
Prokaryotes
DnaA binds to a specific chromosomal origin, called oriC, to start replication. DnaA accumulates during growth and uses ATP to destabilize AT-rich repeats in the origin. This binding causes the DNA to loop and separate, preparing it for melting by helicase DnaB. The concentration of DnaA determines the start of the replication initiation phase.
Eukaryotes
DnaA monomers form a complex that marks replication origins and acts as a scaffold to recruit more initiator proteins. This complex is similar to the hexameric ORC in lower eukaryotes, and both bind to ATP/ADP. The ATP-bound form is active during initiation.
What are the three major DNA polymerases in E. coli and their primary functions?
Brief descriptions
- DNA Pol III is the major replicative enzyme
- DNA Pol II functions in DNA repair
- DNA Pol I fills gap after removal of RNA primer
What is the function of DNA polymerase I?
DNA pol I removes the RNA primer one ribonucleotide at a time from the 5’ end of the primer (5’->3’ exonuclease activity). DNA pol I also fills in the resulting gaps by synthesizing DNA from the 3’ end of the neighboring Okazaki fragment.
What is DNA gyrase?
A type II topoisomerase that facilitates negative supercoiling. DNA gyrase inserts negative supercoils by nicking both strands of DNA, passing the DNA strands through the nick and then resealing both strands.
How is DNA gyrase clinically relevant?
Examples given on handout
- Quinolones and fluoroquinolones (e.g. Nalidixic acid, ciprofloxacin) are used as antibiotics because they block the action of DNA gyrase and prevent prokaryotic replication. Eukaryotic type II topoisomerases are different from the prokaryote DNA gyrase and are not affected by quinolones.
- Anthracyclins drugs (e.g. etoposide, doxorubicin) that inhibit eukaryotic topoisomerases II are used as anti-cancer agents. They slow the replication of cancer cells and cause them to induce apoptosis (programmed cell death).
What are the eukaryotic DNA polymerases and their functions?
o DNA polymerase α has a subunit with primase activity and is involved
in generating primers for DNA replication.
o DNA polymerase β is used in DNA repair.
o DNA polymerase δ elongates Okazaki fragments on the lagging strand.
o DNA polymerase ε elongates the leading strand.
The RNA primers are removed by flap endonuclease 1 and RNase H and filled by DNA polymerase δ.
o DNA polymerase γ replicates mitochondrial DNA.
What is the repeating DNA sequence found in human telomeres?
TTAGGG
Why are telomeres incorporated at the ends of chromosomes?
As replication reaches the end of a chromosome, a problem arises in the lagging strand. Either primase cannot laydown a primer at the very end of the chromosome, or after DNA replication is complete, the RNA at the end of the chromosome is degraded. As a result, the newly synthesized strand is shorter at the 5’-end, and there is a 3’-overhang in the DNA strand being replicated. If this were allowed to happen, the chromosome shortens with each successive replication and genes at the end of the chromosome would eventually be damaged.
What is the function of telomerase?
- Telomerase lengthens the 3’ overhang with a repeating sequence of bases so that primase can bind and synthesize the complementary strand.
- Telomerase acts as an RNA-dependent DNA polymerase (a reverse transcriptase). It has an RNA subunit which it uses as a template to synthesize the repeating sequence of DNA.
- Telomerase mainly functions in highly replicative cell types like germ cells and stem cells. Most somatic cells do not express telomerase, thus they have a limited capacity to replicate. Tumors arising from somatic cells often activate telomerase in order to continue replicating indefinitely.
What are examples of clinically relevant nucleoside analogs?
o Dideoxynucleosides (e.g. dideoxyinosine and dideoxyadenosine).
Lack 3’-OH group of deoxyribose. Used to treat human immunodeficiency virus (HIV) infection. Incorporation by the HIV reverse transcriptase leads to disruption of viral DNA synthesis.
o Azidothymidine, AZT, a thymidine analogue with a 3’-N3 in place of
the 3’-OH, also used in HIV treatment.
o Cytosine arabinoside (cytarabine, araC) resembles cytidine but has
arabinose instead of ribose as the sugar. Used in some cancer
chemotherapies.
o Adenine arabinoside (vidarabine, araA) resembles adenine but has
arabinose instead of ribose as the sugar. Used as an anti-viral agent
to treat Herpes simplex.
A mutant E. coli strain is found to have a defective DNA polymerase III but functional DNA polymerases I and II. Which of the following is most likely to occur in this mutant strain?
DNA polymerase III is the primary replicative polymerase in E. coli. If it’s defective, the cell would be unable to efficiently extend DNA primers, leading to an accumulation of unprocessed Okazaki fragments on the lagging strand.
What are the categories of point mutations and some clinical examples?
i. Transitions (purine->purine) or (pyrimidine->pyrimidine) .
ii. Transversions (purine->pyrimidine) or (pyrimidine->purine)
iii. Implicated in many genetic diseases, two well-known examples:
1. A to T transversion in the β-globin gene changes glutamic acid to valine in Sickle cell anemia.
2. G to T transversion creates a premature stop codon in the CFTR gene in cystic fibrosis (CF).
What are the cuase of frame shift mutations? Also, what is a clinical example of this mutation?
Frameshift mutations are usually the result of an insertion or deletion.
Examples in genetic disorders:
1. Insertions in the reading frame of
the dystrophin gene cause
Duchenne muscular dystrophy.
2. The most common CF mutation (delF508) is a deletion of three bases in the CFTR gene.
What are missense mutations? Give a clinical example?
A missense mutation results in the substitution of one amino acid for another. For example, in phenylketonuria (PKU), various missense mutations in the PAH gene lead to a defective phenylalanine hydroxylase enzyme.
What are nonsense mutations? Give a clinical example?
A nonsense mutation results when a single base pair change, deletion or insertion, generates a new stop codon and causes premature termination of translation. For example nonsense mutations can cause β-thalassemia when they occur in the β-globin gene, resulting in truncated and non-functional hemoglobin proteins.
What are sense mutations? Give a clinical example?
A sense mutation results in the conversion of a stop codon to an amino acid codon, thus producing a longer than normal protein. While less common, sense mutations can extend proteins abnormally. In Huntington’s disease, CAG repeat expansions result in an extended polyglutamine tract in the Huntington protein.
What are silent mutations? Give a clinical example?
A silent mutation is a change in a codon sequence that does not result in a changed amino acid sequence. I.e. Due to the degeneracy of the genetic code, the new codon specifies the same amino acid. Although silent mutations don’t change the amino acid sequence, they can still have medical implications. For instance, a silent mutation in the CFTR gene can affect mRNA splicing, contributing to cystic fibrosis.
What is damage specific repair?
Aka “direct reversal”
Used to repair alkylated (commonly methylated) DNA
* Typically via suicide enzymes - Resource intensive - reserved for only highly mutagenic or cytotoxic lesions
Example:
O 6-alkylguanine transferase (AGT) directly reverses alkylation at O 6 of guanine. AGT transfers the O6-alkyl group to a Cys residue in the AGT active site (Fig 7). The AGT protein is inactivated by the alkylation and degraded.
How are double-stranded breaks repaired?
Nonhomologous end-joining (NHEJ) utilizes a complex of proteins to
bring the ends of two DNA fragments together and a DNA ligase joins
the ends. Only used in emergency situations and is error prone and
often mutagenic:
Homologous recombination repair (HR) takes sections of broken
strands and exchanges them with an undamaged duplex using the
same proteins which normally function in genetic recombination
between homologous chromosomes during meiosis. HR is less error
prone than NHEJ because any DNA lost is replaced using the
homologous strand as a template.
What are the promoter regions for prokaryotic transcription?
What RNA polymerase is responsible for transcription in prokaryotes?
In prokaryotes, a single type of
RNA polymerase generates all
3 types of RNA
i. The RNA polymerase core enzyme contains 4 subunits (α2ββ’). It cannot
recognize a promoter by itself.
ii. Another subunit, sigma (σ) factor, binds the core enzyme and directs binding to a Pribnow box. Core + σ = holoenzyme
iii. There are several different σ-factors that recognize the promoters of different groups of genes. The major σ-factor is σ70 (designation relates to its mw of 70 kDa)
