DNA #17-20 part 2 Flashcards
What are the functions of DNA Topoisomerases?
REVERSIBLY add themselves COVALENTLY to the DNA backbond, breaking the backbond phosphodietster bond so that it can releive the strain on the ds DNA ahead of the replication fork
What is Topoisomerase I and function?
Uses Tyrosine hydroxyl group to insert into the backbone, creating Tyr-O-Phosphate linkages; now strained DNA can rotate freely around remaining backbond
ONce strain releieved, reaction is reversed, re-creating phosphodietser bond in backbone
Step is reversible and no ATP is consumed
What is the function of topoisomerase II?
3 steps:
Untangles the DNA strands (since there are multiple 46 cm strands of dobule helix in a 10-20 um dimater
- creates ds break in one piece of DNA
- causes the intact double helix to pass through the break
- reseasl the break
Driven by ATP Hydrolysis
What are drug inhibitors of Topoisomerase I (2)
Irinotecan
Topotecan
What are drug inhibitors of Topoisiomearse II
Etoposide
Tenipososide
What antibiotics are inhibitors of PROKARYOTIC Topoisomearse II
Novobiocin
Nalidixic acid
Ciprofloxacin
selecftive inhibitors
AKA prok Topoisomerase II
DNA gyrase
What are the special repetitive sequences at teh ends of chromosomes?
Telomeres
What happens when replication reaches the end of a telomere?
What does this enzyme do?
repetiive sequences attract an enzyme called TELOMERASE, which brings along its own RNA template as a primer
Telomerase then adds multiple copies of the short repetitive sequence to the chromosome ends–> This serves ast eh template for DNA polymerase
How do telomeres solve another problelm?
ends of chorocomes could appear to cell as a DS DNA break that needs to be repaired
repetitive telomere sequences are recognized by the cell as true chromosoe ends
What is sickle cell anemia?
What is the result?
result of a single nucleotide mutation in the Beta Globin gene
Result” very water-solutble glutamate on the surface of the protein is changed to valline
Glutamate (-)–> Valine (h-phobic)
Causes multible Hgb moleucles to adhere to each otehr in insoluble fibrous precipitates that can deforme the RBCs adn make them fragile
What is the error rate for DNA polymerase? for mistmatch repair mechanisms?
DNA polymerase 1/10,000,000
Mismatch repair mechanisms fix 99% of errors
overall is 1/1,000,000,000
avg is 6 bp/replication
Mismatch (replication) eros, what are two remedies?
- DNA poly has a proofreading activity,
2. There is a strand-directed mismatch repair system, making use of MutS and MutL proteins
What causes Post-replication mismatch?
What happens t them moleculalry?
Chemical, UV, or radiation-induced damage
Common problems are depurination, deamination, and thymine dimerization
or ds breaks
In newly synthesized DNA, mismatches that escape proofreading are detected by which protein (prok? euk?)
MutS (Pro) or MSH2 (euk)
What is a mismatch?
cuases some mismatch in the double helix conformation
How does mismatch repair machinery knwo which strand is the correct one?
newly synthesized strand has a numbe ro fnicks (backbone breas), makring it as the one in need of repair. NIcks only stay for short time after replication fork passes, so repair system must happen very quickly
When MutS detects a problem what protein associates it with to scan the DNA for the nick ? in pro and euk
in prokarytoes, MutL ass. wth MutS and scans along DNA until nick. A NUCLEASE is then recurited and it cleaves nucleotides off the newly synthesized strang back to MutS
DNA Polymerase rebuils new strand, and LIGASE completes repair
MLH1 or MLH3 in Euk
How can DNA be damaged?
Thermal Collisions with other molecules, encounter with chemically reactive metabolic intermediates or xenobiotics, or exposure with chemically reactive metabolic intermediates or xenobiotics, or exposure to high energy radiation (UV or Xrays)
What is depurination and its consequences
(loss of one of the purine bases)
This leaves the backbone intact, but there is a free hydroxyl group on teh 1 position of deoxyribose
This can lead to loss of nucleotide pair (shortenng of gene by one base, and casuing a frameshift in converting DNA code to protein sequence)
What is deamination and its consequences
loss of amino roup
COnverts cytosine to uracil –> can tlead to CG pair to an AT pair
UV radiation can lead to?
thymines to become covalently attached
results in Thymine Dimer–> stall DNA replication machinery inhigbitn cell replication
Other pyrimidine dimers (T-C and C-C) are possible but less common
yep
What are two main pathways for repairing these kinds of damage?
Base excision repair
nucleotide exicision repair
What is the most common kind of chemical damage?
Depurination
What does Base Excision Repair take care of?
Deamination
Depurination
Some kinds of oxidative damage to DNA
What initiates Base Excision Repair?
Initiated by family of DNA glycosylase enzymes that scan along the DNA “flipping out” one base at a time and examining it
when damaged base is recognized, its GLYCOSYL linkage is borken
Now missing tooth is recognized by ENDONUCCLEASE –>deoxyribose phosphate removed –> DNA polymerase addes new nucleotide –> DNA LIGASE seals nick
What is nucleotide Excision Repair?
When larger structural damage takes place –> nucletodie excision repair comes to rescue
Pyrimidne Dimer CT
or Polycyclic aromatic H-carbons which are known carcinogens
Process of Nucleotide Excision Repair
multienzyme complex scans DNA for large distortions in double helix
When it encoutners such a situation, cleaves phosphodiester backbone on both sides of the lesion
DNA helicase removes the damaged strand
Repairs are carreid out with DNA polymearsa nd DNA ligase
what enzyme removes a damaged strand in Nucleotide Excision Repair?
DNA Helicase
Summary of Repairing DNA Damage (3 enzymes
- Recognize and excise –NUCLEASE
- Resynthesize- DNA Polymerase
- Reconnect backbone —LIGASE
What disease ffrom defects in repair of thymine dimers ?
Xeroderma Pigmentosum
affected inviducals are highly susceptible to basal cell carcinoma and other skin malignancies
What is the most common rmechanism for repairing double stranded beaks in somatic cells?
nonhomologous end-joining
ds breaks are very dangerous
How does non-homologous end joining work?
Enzyme complex brings the ends together and joins them with a ligase, although some bp may be lost in the process
This is a quick and dirty fix! ,
Net resulg: ds break repaired, with deletion of nucleotides at the repair site
What if ds damage is in a critical spot?
it is likely that it will be signaled for apoptosis
What is homologous recombinatino?
More conservative way to repair the break, ensuring that no base pair is lost
makes use of the “backup” copy that is the toehr copy of the chrosomeome
Proces of homologous recombination
broken DNA must locate a highly homolgous ds DNA (not necessarily identical)
- nuclease needs to process broken ends sot hat tehre is a stretch of ss DNA on each
- Loose ends must FIND highly homolous ds DNA on teh other copy. ds DNA dissociaets enough for single strand to move in and pair up –> forming Holliday junction
- Invading strand is extended by repair DNA Polymerase
- Ligase links the backbone break, completing repiar without any loss of nuceotides
Homologus recominbation occurs most offten very soon after a short stretch of NDA has been replciated ; before packaged into chromatin
What the branch point that is established in homologus recombination called?
Holliday Junction
What extends the invading strand?
repair DNA plymerase
What is homologous recobination and its process?
- Nucleasuse trims loose ends ot produce streteches of ss DNA
Branch points form with intact partner –> HOLLIDAY JUNCITONS
- set of recombination proteins stabilize junctions (helicase powered by ATP hydroylsis) that can cause branch point to migrate along the chain, effectively increaing the size of DNA segment being swapped
- Holliday junciton is RESOLVED, by inducing ss cuts then connecting loose ends witha ligase
What is gene conversion?
during misosi, half of the genes parceled out should be maternal and half paternal, but on rare occasions, it is possible for this law to be “bypassed”
Thisis gne coversion, that it apears that one copy of a gene is coverted to the other copy
How can gene conversions happen?
- During homologous recombination processs
tumor suppressor protein, that was itially heterozygours, with one good copy and one mutated copy. If copiees utated copy ont both strands, then adverse
- durin MISMATCH repair for slight mismatches foloowing homolgous recobination (X chromosome )
either strand can be emplate, the other strand is converted to the other allele
What are Trasnposable Elements
“jumping genes”
short, specialized segments of DNA that contain informatino necessary for themt o insert themselves virtually anywhere in the genome
In eukaryotes, typically can’t leave the cell, so effects are limited to cellt hey reside in and its progeny
IN bacteria, these mobile elemetns serve to transmit antibiotic resistance from one cell to another
What is the function of transposase gene?
has the enzymatic activ ity necessary to carry out insertino of the whole segment into a new location
What is the fucntion of the DNA recognition sequence
located on each end of the tranposon
they are recognized by the transposase as teh boundearies of DNA to be moved
IN bacteria, these mobile elemetns serve to transmit antibiotic resistance from one cell to another
What mechanisms are used to transfer DNA transposons?
- cut and paste?
2. Replicative
What are retrotransporsomes?
Unique to Euk!
Much larger group of transposabe elements
Info flows from RNA to DNA in one stge of with a reverse transcriptase
What two groups are Retrotransposons?
- Long Terminal repeats (LTR) retrotranpsosons
LTR has shown no activity/little activity
- Non-LTR Retrotransposons
most activity seen is due to non-LTR retrotransposn - there are 3 major groups and many smaller groups
What are three main groups of non-LTR Retrotransposons
- Long Interspersed Element 1 (LINE-1 or L1)
- Alu elements (named for a restriction enzyme)
- SVA elements
What are L1 elements/
part of non-LTR Retrotrasnsposons
6,000 bp long
L1 elements contain two open reading frames (seqeunces that can be transcribed/translated to proteins)
ORF1 codes for an RNA binding protein
ORF2 codes for a protein with ENDONUCLEASE and REVERSE Transcriptase activiteis
start with RNA polymerase promotoer, so that RNA polymerase can make copies of entire L1 element
Activities enable L1 elements to copy themselves and re-insert copies to new locations in the genome
How many L1 copies are the human genome?
~500,000 copies
most hae mutated over time and lost teh ability to move
What are ALU sequences?
shorter (280 bp) and do not contain coding sequences
What are SVA elements?
about 2,800 bp in lenght and also do not code for pretines
REly on L1 elemnts or other sources of revertse transcriptase in order to be copied and transposed;
There are over 1 millino copies of ALu and 3,000 copies of SVA in the human genome
How can the different transposable elements lead to disease?
Transposon or retrotransposon can insert early in embryogenisis or can damage gene and lead to genetic disease;
inserts in DNA repair gene or tumor suppressor gene –> cancer cell line
- L1 promotor can up-regulate expression of a gene
- Alu promoter may cuase production of micro-RNA which can lea to suppression of affected gene
- some TE sequences can inroduce p5k3 binding sites, altering gene expression
- insertion of repeated sequences in opposite orientations can cause the resulting mRNA copy to form ds RNA, with consequences for RNA editing enyzmes
**TEs have potentia to distrupt genes, gene expression, and protein expression if they are inserted in the wrong places
2.
How does DNA protect itself from transposable elements?
DNA meythylation!
Suppresses gene exptression
Some mRNA editing machinery (APOBECs) may also inhibit L1 and Alu retrotranposition
What are vriuses?
mobile genetic elements tha tcan escape from cells and infect other cells. Theya re more than pieces of genetic info (RNA or DNA) with a protetive coat
How do viruses carry genetic info
s DNA, ss DAN ,ss RNA
What is a retrovirus
Use RNA from genome ,
use Reverse Transcriptase to make DNA copy then INTEGRASE to integrae the new DNA into host cell’s DNA
virus is not part of host cell in a LATENT form (called provirus)
When is viral DNA expressed?
when host cell’s RNA polymerase makes RNA copies of the viral DNA, many factors can affect length of latency period
RNA copies have new copies of viral genome and mRAN coding for viral proteins
What are virus specific proteins?
reverse transcripatse,
integrase,
and proteases sometimes
can be used as targets during drug development
Retverse transcriptase has NO PROOFREADING capability , many copies mutated
What kindof drugs against RT
coctail that attacks three different enzymes
Usually protease inhibitors against resistant strains of HIV
What is Xeroderma Pigmentosum
Genetically heterogenous autosomal recessive disorder characterized by incerased sensitivity to sunlight
development of carcinomas at an early age
some patienst develop neurologic symptims or a more severe clinical phenotype known as de Sanctis-Cacchione syndrom
what is TRICHOTHIODYSTROPHY (TTD)
sulfur-deficient brittle hair
patients with trichothidystrophy have gbrittle hair and nails (b/c of reduced content of cystein-rich matrix proteins)
ichtyotic skin, and phsyical/mental retardation
half patients display photosensitivey, correlated with a nucleotide excision repair (NER) defect
Cockayne Syndrome
abnormal and slow growth and development tha tbeocme evidnet within first few years after birth
Cachetctic dwarfism describes the outward appearance of afflicated individuals
Cutaneous photosensitivey, thin, dry hair, progeroid appearance, porgressive pigmentary retinopathy, sensoineural hearing loss, dental caries, and characteristic stance in the ambulatory patient
What mutation cause Xeroderma Pigmentosum, Trichothiodystropy, Cockayne Syndrome
Caused by mutations in any of several enzymes that are involved in Nucleotide Excision Repair
What syndromes are caused by mutatinos in DNA Helicases called the RECQ helicase
- Werner Syndrome
- Bloom Syndrome
- Rothmund-Thomas Syndrome
What is Werner Syndrome
AKA adult progeria
foloowing puberty, patients age rapidly
Patients reveal a propensity to develop chromosomal abberrations, including translocations, inversions, and deltions
What is Bloom Syndrome
Autosomal recessive disorder characterized by proportionate pre- and postnatal growth deficiency
sun deficiency, sun sensitive, telangiectatic, hypo and hyperpigmented skin, predisposition to malignancy, and chromosomal instabiliyt
What is Rothmund-Thomson Syndrome
hereditary dermatosis characterized by atrophy, pigmentation, and telangiectasia and frequencly accopanied by juvenile cataract, saddle nose, congentical bone defects, distrubances of hair gowth, and hypogonadism
chromosome instability and increased cancer susceptibility are also observed
What are BRCA1 and BRCA2?
specific genes assocaited with hereditary breast cancer
both have complex roles in cell growth regulation, but both are associated with repair of double strand breaks
BRCA2 has been assoicated with other cancers a well (ovarian, prostate, others)
What are hereditary nonpolyposis Colorecteral Cancer?
assosciated with mutations in strand-directed mismathc repair system
Most cases of this disease are caused by mutations of MSH2 or MLH1
show proclivity to early onset, predoinant proximal location of coon cancer
