Amniocentesis Flashcards
What is semiconservative DNA replication?
Semiconservative DNA replication means that when DNA is replicated its two strands are separated and each serve as a template for the daughter strand.
How are deoxynucleotide monophosphates added to the newly synthesized chain?What bond is formed? What is the direction of DNA synthesis?
dNMPs are added to the newly synthesized chain by the DNA polymerases that breaks off the last two phosphates in the in the formation of the phosphodiester bond which forms between the 3’ end of the chain dNMP and 5’ end of the new dNMP.
A pyrophosphate group is released (PPi) which is hydrolyzed to two inorganic phosphates (2Pi) by pyrophosphatase and leads to the removal of products.
DNA synthesis moved from 5’ to 3’. The phosphodiester bonds are created from the 3’ end of one dNMP to the to 5’ end of another.
What is a primosome and what are its components?
The primosome is a macromolecule complex that is required for transcription to start, ie for DNApol to start adding dNMPs.
Prokaryotes
DnaA - binds to the origin of replication (oriC) and starts helix unwinding, ATP-dept
DnaB - helicase, continues to open strands in APT-dept maner
DnaC - required for DnaB binding to the DNA strand, ferries DnaB to the strand
SSR - used to ‘paint’ unwound ssDNA so that it is protected, stabilized, and doesn’t fold in on itself
DNAprimase - DnaG - creates RNA primer, acts as an RNA polymerase
Eukaryotes
ORC - origin recognition complex binds to the origin of replication (oriC) and starts helix unwinding, (ATP-dept?)
MCM complex - helicase, continues to open strands in (ATP-dept?)
SSR - used to ‘paint’ unwound ssDNA so that it is protected, stabilized, and doesn’t fold in on itself (not sure if this is in euk as well as prok)
alpha DNA polymerase - creates RNA primer
How are the leading and lagging strands of DNA synthesized? What is an Okazaki fragment?
Leading strands are formed by the continuous transcription of complementary DNA which follows the replication fork, 5’ to 3’, only one RNA primer is required.
Lagging strands are formed by discontinuous synthesis for the strand that is antiparallel and moves in the direction opposite the replication fork, 5’ to 3’, multiple RNA primers are required for each Okazaki fragment. Okazaki fragments are put together by Pol III which removes the RNA primer, adds missing nucleotides and DNA ligase which ligates the pieces together.
What is the replisome and what are its components?
Replicates the DNA once the primer is in place w/ some aspects of the primosome, including helicase and DNAprimase as well as DNA polymerase.
Prok - DNApol I (pol 5’ to 3’, exo 5’ to 3’, exo 3’ to 5’) DNApol III (pol 5’ to 3’, exo 3’ to 5’)
Euk has different DNA pols and RNA primers are removed by RNaseH proteins. The euk DNA pols include alpha (PRIMASE, pol 5’ to 3’) beta (REPAIR pol 5’ to 3’), delta (pol 5’ to 3’, exo 3’ to 5’ of the lagging strand), epsilon (pol 5’ to 3’ and exo 3’ to 5’ of the leading strand), and gamma (pol 5’ to 3’ and exo 3’ to 5’ of the mitochondrial DNA).
Describe bacterial replication, noting the role of each enzyme involved.
- oriC with the primosome.
- DnaA binds to oriC and begins the process of melting in an ATP-dept process.
- DnaB is ferried to the DNA by DnaC, and binds to one of the DNA strands and begins to unravel it in an ATP-dept process.
- SSB stabilizes the single stranded DNA and protects it as well as keeps it from folding in on itself.
- DNAprimase creates the RNA primer that DNApolymerase can extend.
- Replisome is formed with aspects of the primosome (helicase and DNAprimase) as well as DNA-polymerase I and III.
- Replication is stopped when the replication fork runs into a TER SITE and this terminates the unwinding of the DNA.
What is the role of eukaryotic DNA polymerase gamma? How about delta and epsilon? What eukaryotic DNA polymerase has primase activity?
Eukaryotic DNA polymerase gamma polymerizes the Mitochondrial genome and has the ability to polymerize from 5’ to 3’ and proof read and check its work through exonucleases 3’ to 5’.
Delta is a nuclear DNA polymerase that works on the lagging strand and has 5’ to 3’ polymerases and 3’ to 5’ exonucleases.
Epsilon is a nuclear DNA polymerase that works on the leading strand and has 5’ to 3’ polymerases and 3’ to 5’ polymerases. 5’ to 3’ exonuclease activities are preformed by RNaseH in euk. DNA replication.
Alpha DNA polymerase has primase activity in euk. DNA replication and creates the RNA primers.
What are nucleosomes? What are histones?
Nucleosomes are complexes of DNA and histones that make up chromatin and ultimately chromosomes. Histones (/nucleosomes) are highly conserved and are replicated during DNA synthesis.
What are telomeres? What is the role of telomerase? Which strand is elongated by telomerase? Does the RNA component of telomerase function as a template or a primer? Is telomerase typically found in normal somatic cells?
Telomeres are the non-coding end regions that provide structural integrity for euk. chromosomes and keep the ends from being degraded. The are characterized by hundreds of tandem repeats of hexanucleotide sequences. Telomerase is the enzymatic protein of telomeres that elongates the longer strand of the DNA, because it is the only one it can polymerize from 5’ to 3’. Telomeres also have an RNA component which serves as the template for the elongation of the strand. This is not in agreement with the central dogma because you have the production of DNA from RNA. Telomerases are not typically found in normal somatic cells.
What are some examples of DNA damage?
Examples of DNA damage include interaction with reactive oxygen species (ROS) or reactive nitrogen species which can damage the bases, ionizable radiation which can break the DNA strands, alkylation, UV light forming thymine dimers, and base decay forming APs.
What are some examples of DNA mutations?
DNA mutations include:
gene mutations - base substitutions, point mutations, small deletions or insertions, frame shift mutations
chromosome mutations - tri-nucleotide expansions (normal trinucleotide repeats are expanded in mutated gene) insertions or deletions; translocations
genome mutations - loss or gain of whole chromosomes
What are the clinical consequences of the inability to repair DNA damage in mammals? What is HNPCC? XP?
The inability to repair DNA damage will lead to a permanent DNA mutation that can be passed on to future generations. HNPCC and XP are resultant diseases from the inability to DNA damage.
Hereditary nonpolyposis colorectal cancer - MMR-deficiency which results in microssatellite instability of acrosomes.
Xeroderma pigmentosum - mutations in XP protein used in NER in humans - causes extreme light sensitivity and developing skin cancer
Describe the two mechanisms by which ds breaks in DNA are repaired. Which one is error-prone? Why?
Double-stranded DNA breaks from ionizable radiation are repaired either by Homologous repair (HR) or Non-homologous end joining (NHEJ). HR works through recognizing the break and using an exonuclease to resection the DNA. Then RAD51as well as other proteins bind to the DNA and then scan for its homologous partner on the sister chromatid, this is very energy expensive. Once the homologous partner is found, a complex forms where the intact DNA is used as a template for the repair. DNA pol and then lygase repair the DNA. This is error-free.
NHEJ on the other hand occurs when there is a double stranded break and KU70 and KU80 bind the ends and DNA-protein kinases which phosphorylate the complex. This results in end trimming and exonuclease, and ligation by XRCC4 and Ligase IV. DNA can be lost in the process, which is very error-prone.
What are the 3 R’s of DNA repair?
Recognize, remove, replace
Describe nucleotide excision repair (NER). Give an example of DNA damage that is repaired by NER.
NER is much less specific than BER and it scans DNA for structural abnormalities such as bulky adducts and thymine dimers formed by UV radiation. It can also repair structural damage from oxidative damage. Once it recognizes the damage, it recruits helicase to open the strands, excinuclease cleaves the mutation on either side and DNA pol and ligase are recruited to fill in the gaps.
