Genome: DNA Structure, Replication, Repair Flashcards
name 2 situations in which DNA recombination occurs
chromosome cross-over during meiosis (accidental, but confers genetic diversity)
immunoglobulin gene rearrangement (for antibody diversity)
name the purines and pyrimidines
purines: adenine, guanine (2 rings like “pure” wedding rings)
pyrimidines: cytosine and thymine in DNA, cytosine and uracil in RNA (1 ring like a “pie” crust)
describe the important anatomical structural difference between RNA and DNA
RNA: hydroxyl (OH) on 2’ C
DNA: hydrogen on 2’ C
hint: DE-oxy-ribose (deoxygenated carbon, now just a hydrogen)
contrast nucleotide and nucleoside
nucleoside = base + 5C sugar
nucleotide = base + 5C sugar + phosphate
aka nucleotide = nucleoside + phosphate
therefore, nucleotide may be called nucleoside mono-, di-, or tri-phosphate
what kind of bond links base to 5C sugar in nucleoside
N-glycosidic bond (nitrogen atom of base is bonded to hydrogen atom of sugar)
acyclovir is a drug used to treat herpes simplex virus infections. what kind of drug is it?
acyclovir: nucleoside analog of deoxyguanosine
converted into a nucleotide and incorporated into DNA during replication but blocks further DNA synthesis
this nucleoside analog of deoxyguanosine is used to treat HSV infection. What is?
acyclovir
this nucleoside analog of deoxythymidine is used to treat HIV/AIDS. What is?
azidothymidine (AZT), or zidovudine
what is azidothymidine (AZT) used to treat and how does it work
AZT (aka zidovudine) is a nucleoside analog of deoxythymidine used to treat HIV/AIDS
what is zidovudine used to treat
zidovudine (aka azidothymidine or AZT) is a nucleoside analog of deoxythymidine used to treat HIV/AIDS
what is another name for azidothymidine and what is AZT an analog of
AZT = zidovudine = nucleoside analog of deoxythymidine
used to treat HIV/AIDS (halts DNA replication after conversion into nucleotide)
describe the polarity of nucleotide polymerization
phosphodiester bonds between 3’ OH on sugar of one nucleotide and 5’ phosphate on another
leaves free 5’ phosphate group and free 3’ hydroxyl group
[3’ hydroxyl is linked to innermost 5’ phosphate]
endonucleases are known as what kind of enzyme
endonucleases are restriction enzymes (site-specific cleavage)
fill in these features of DNA:
- most common form is ___
- ____ - handed
- outer _____ backbone
- ____ base pairs per helical turn
- base pairs _____ to axis of symmetry
DNA:
- most common is B FORM, which is:
- RIGHT - handed
- outer SUGAR-PHOSPHATE backbone
- 10 base pairs per helical turn
- base pairs PERPENDICULAR to axis of symmetry
what base pairs are most common at the ends of a DNA sequence
A=T (2 H bonds) because they are easier to separate than G(3Hbond)C
A=T base pairs are also rich at the origin of replication for the same reason
describe negative supercoils simply (what they are, purpose, etc)
negative supercoils = unwinding of helix (fewer helical turns) without breaking any bonds
facilitate strand separation
energetically favored (less tension than relaxed DNA)
make up the tertiary structure of DNA
explain what DNA topoisomerases do
enzymes that change tertiary structure of DNA (supercoils)
remove positive supercoils (extra tight winding) ahead of strand opening and excess negative supercoils (opposite direction winding) behind strand opening
have nuclease and ligase activity - transiently break one/both DNA strands, pass other strand through break, and rejoin (to remove one loop, and restore helix)
Topo I cuts single strand, Topo II cuts both strands
what 2 enzymatic functions do DNA topoisomerases have
nuclease and ligase activity
(cut strand/s, rejoin them)
this topoisomerase II is found only in prokaryotes and facilitates bacterial DNA replication. what is?
DNA gyrase
what kind of drugs block the activity of DNA gyrase, a topo II?
examples include Novobiocin, Nalidixic acid, Ciprofloxacin
quinolone drugs
inhibit bacterial DNA synthesis by blocking DNA gyrase (which is only found in prokaryotes)
certain chemotherapeutic drugs target eukaryotic topoisomerases. Examples include Camptothecin, which targets Topo I, and Adriamycin and Etoposide, which target Topo II
how do these work?
inhibit ability of topoisomerases to rejoin DNA, converting topoisomerases to DNA breaking agents
cause side effects to any rapidly dividing cells (hair loss, etc)
what are chromatin and nucleosomes comprised of?
chromatin = DNA + histones (small basic proteins, rich in Arg and Lys)
[histones: H1, H2A, H2B, H3, H4]
nucleosomes: repeating units of chromatin with DNA spacer in between (bound to histone H1)
what type of interactions hold together DNA double helix supercoils and histones and how many histones are in each nucleosome core?
positive/negative charge interactions (negative DNA, positive/basic histones)
histone octamer has 2 molecules each of H2A, H2B, H3, H4
solenoid
chromatin wound into helical tubular coil for tighter packaging
after DNA replication is completed, H1 histone binds spacer DNA and nucleosomes pack tightly into ____
solenoid (large DNA loops)
solenoid DNA loops coil around a protein scaffold, with loops radiating from scaffold —> creates classic 4 arm structure with 2 chromatids joined by a centromere
which kind of DNA replication occurs from just a single origin:
prokaryotic
eukaryotic
prokaryotic DNA is replicated from SINGLE origin
(eukaryotic DNA replicated from many origins)
either way, replication proceeds on both sides of origin in opposite directions (replication fork on each side)
function of DNA helicase
binds near replication fork on either side of the origin and catalyzes DNA strand separation
USES ATP to break H bonds
how do inhibitors of HSV helicase-primase work?
stabilize interaction of helicase-primase with its viral DNA substrate, inhibiting progression of HSV DNA replication
effect against HSV strains resistant to nucleoside analog therapy (acyclovir)
how are RNA primers created? what direction do they run and what is their function?
RNA polymerase (aka primase) synthesizes short RNA primers (~10nt) by copying DNA template strand
RNA primers run 5’—>3’
primers provide free 3’-OH as acceptor of the first deoxyribonucleotide (continuously synthesized on lagging strand)
DNA polymerases copy the ___ strand [don’t overthink it] and use ____________ as precursors
DNA polymerases copy TEMPLATE strand, use deoxyribonucleoside triphosphates as precursors
complementary new strand runs 5’-3’, anti parallel to parental strand
DNA pol can ONLY synthesize DNA 5’-3’ direction
leading strands run 5’-3’ [toward/away] replication fork
lagging strands run 5’-3’ [toward/away] replication fork
leading strands synthesized continuously TOWARD replication fork
lagging strand synthesized discontinuously AWAY from replication fork (new Okazaki fragments created near replication fork)
in prokaryotes, chain elongation is is catalyzed by….
and how does it work? (specific organic chemistry mechanism)
DNA polymerase III: catalyzes chain elongation via NUCLEOPHILIC ATTACK of 3’-OH (growing chain) on innermost 5’-phosphate (incoming deoxyribonucleotide triphosphate), resulting in formation of phosphodiester bond
DNA pol III also has 3’-5’ proofreading activity (backwards reading, exonuclease)
what kind of proofreading activity does prokaryotic DNA Pol III have
3’—> 5’ exonuclease activity
removes erroneously introduced nucleotides not complimentary (mismatch repair, MMR)
match the nucleoside analogs with the illness they treat:
analogs: acyclovir, AZT, ddC, gemcitabine, remdesivir
illness: HSV, HIV, cancer, SARS-coV2
acyclovir - treats HSV (deoxyguanosine)
AZT - treats HIV (thymidine)
ddC - treats HIV (deoxycytidine)
gemcitabine - treats cancer (deoxycytidine)
remdesivir - treats SARS-coV2 (adenosine)
all prevent elongation in DNA replication
DNA Pol III elongates DNA strand in prokaryotes until it is blocked by RNA primer… then what happens?
DNA Pol I uses 5’—>3’ exonuclease activity to remove RNA primers from Okazaki fragments, then uses 5’—>3’ polymerase activity to fill in the gap
DNA Pol I then proofreads 3’—>5’ with exonuclease activity (backwards reading)
DNA ligase then joins fragments (ATP DEPENDENT)
a phosphodiester bond is formed between 5’-P on chain made by DNA Pol III and 3’-OH on chain made by DNA Pol I
in [prokaryotic/eukaryotic] DNA replication, there are many origins from which replication forks are created on both sides for bidirectional replication
eukaryotic - MANY origins
prokaryotic - ONE origin
what are the 3 DNA polymerases of eukaryotic replication and their role
Pol alpha: synthesizes primers for both leading and lagging strands (primase and polymerase activity)
Pol delta: synthesizes lagging strand (polymerase and exonuclease activity)
Pol epsilon: synthesizes leading strand (polymerase and exonuclease activity)
which of these function(s) does eukaryotic Pol alpha have:
primase
polymerase
exonuclease
Pol alpha: primase (RNA) and polymerase (DNA) activity
synthesizes short RNA primers, extends primers with DNA
leading and lagging strands
NO exonuclease/proofreading
what does Pol delta need to associate with to synthesize the lagging strand of DNA?
PCNA: proliferating cell nuclear antigen
[PCNA also associates with Pol epsilon]
Pol delta (lagging strand) has 3’-5’ exonuclease activity, and also displaces the 5’ end of primers from Okazaki fragments. What happens to these primer 5’ end?
degraded by FEN1 (flap exonuclease)
what is the function of FEN1
flap exonuclease
degrades primer 5’ ends that are displaced from Okazaki fragments by Pol delta
PCNA associates with what DNA polymerase? (specific)
PCNA (proliferating cell nuclear antigen) associates with Pol delta (lagging strand, eukaryotic)
AND Pol epsilon (leading strand, eukaryotic)
name the two eukaryotic DNA Pols that have 3’-5’ exonuclease proofreading activity
Pol delta (lagging) and Pol epsilon (leading)
NOT Pol alpha
what happens to nucleosomes/histones during DNA replication?
nucleosomes are displaced as replication fork advances
histones remain loosely associated with one parental strand, new histones are synthesized simultaneously with DNA replication
nucleosomes reform behind advancing replication fork
telomeres are rich in which nitrogenous base
G (triple H bond with C for stability)
Without _____, DNA from linear chromosomes would be degraded, recombined, or fuse end-to-end
telomere T-loops
telomerase:
1. is a _______ complex
2. adds G-rich repeats to the single-stranded _____ ends of linear chromosomes
3. has _____ activity
telomerase:
1. ribonucleoprotein (RNA + protein) complex
2. adds G-repeats to ss 3’ ends
3. has reverse transcriptase activity via hTERT (makes DNA from RNA)
An autopsy report reveals that a patient suffered bone marrow failure due to loss of hematopoietic renewal. Defects were noted in tissues with rapidly dividing cells: alopecia, leukoplakia (precancerous oral lesions), fibrosis, cirrhosis, and hypogonadism.
Genetic analysis reveals both stem and germ cells had a mutation in the RNA component of telomerase, reducing its activity. What condition does this describe?
Dyskeratosis congenita
Briefly describe dyskeratosis congenita and its cause
defects in tissues with rapidly dividing cells
patients often die from bone marrow failure (loss of hematopoietic renewal)
inherited disease caused by reduced telomerase activity (stem and germ cells affected)
what DNA repair mechanism would be utilized following a substitution, insertion, or deletion?
mismatch repair (no damage, just wrong pairing)
what type of DNA repair is utilized for bulky lesions, photoproducts, or chemical adducts?
nucleotide excision
global genomic - for transcriptionally inactive DNA
transcription-coupled - for transcriptionally active DNA (damage halts RNA Pol II along template strand of DNA)
how do prokaryotic and eukaryotic cells recognize the strand with the error from the parental strand in mismatch repair (MMR)?
prokaryotic: Mut proteins, recognize the new strand lacks methyl groups
eukaryotic: MSH and MLH proteins, unknown mechanism
mismatch repair pathway uses a endonuclease or exonuclease to fix base pairing?
BOTH:
endonuclease cleaves strand on either side of mismatch
helicase and exonuclease removes DNA between incision and mismatch
DNA Pol III (prokaryotic) or Pol delta/epsilon + PCNA (eukaryotic) fills gap, followed by DNA ligase
microsatellite instability (MSI) is a sensitive diagnostic marker for what
loss of mismatch repair (MMR) activity in tumors - hallmark of MMR-deficient cells
[microsatellite sequences are short repeats found in non-coding regions, MMR defects most frequent in repetitive DNA tracts]
Lynch syndrome is due to a defect in what repair pathway?
Lynch syndrome = HNPCC (hereditary nonpolyposis colorectal cancer)… patients at high risk for colorectal cancer, among others
due to defect in mismatch repair (MMR) - most patients have mutation in MSH2 or MLH1 (recognize error strand from parent strand)
microsatellite instability (MSI) is a hallmark, can be tested via PCR
oxidation, deamination, depurination, or alkylation of nitrogenous bases caused by ROS, carcinogens, or toxins requires what kind of DNA repair?
base excision repair (BER) - for damage to a single base
- glycosylase cleaves N-glycosidic bond
- apurinic/apyrimidic endonuclease cleaves sugar-phosphate backbone
- deoxyribose phosphate lyase removes sugar-phosphate residue
- DNA Pol I (beta) fills gap, followed by DNA ligase
what enzymes (4) are needed for base excision repair (BER)? What do they do (specific)?
- glycosylase: cleaves N-glycosidic bond between base/deoxyribose
- apurinic/apyrimidic (AP) endonuclease: cleaves sugar-phosphate backbone
- deoxyribose phosphate lyase: removes sugar-phosphate residue
- DNA Pol I (beta) / DNA ligase: fills gap
A 35yo M pt presents with colon cancer. Genetic analysis reveals biallelic mutation in a gene encoding a DNA glycosylase MYH. The pt most likely has defective _____ DNA repair
base excision repair (BER)
describe Werner’s Syndrome: its hereditary pattern, phenotype, and cause
Werner’s Syndrome:
-rare autosomal recessive
-causes premature aging (20-30yo), presents with cataracts, atherosclerosis, cancer, etc
-cells have mutations in DNA helicase WRN which is important for base excision repair (BER) and formation of T-loops
[cells sensitive to ROS, chromosomal rearrangements, and telomere fusions and shortening]
A 23yo M pt presents with skin atrophy, cataracts, and atherosclerosis. He is in the 30th % height for his age. Genetic analysis reveals a mutation in the RecQ-family DNA helicase WRN. What is your diagnosis?
Werner’s Syndrome: rare autosomal recessive disorder
mutation in base excision repair (BER) pathway —> cells sensitive to ROS and chromosomal rearrangements
Ultraviolet light induces ____ formation between _____ in the DNA of skin cells. This leads to distortion of the DNA helix and frame shift mutations. Fortunately, these lesions can be correct by _____
Ultraviolet light induces DIMER formation between PYRIMIDINES in the DNA of skin cells. This leads to distortion of the DNA helix and frame shift mutations. Fortunately, these lesions can be correct by NUCLEOTIDE EXCISION REPAIR (NER)
Xeroderma pigmentosum (XP) is a hereditary disorder in which patients show extreme solar sensitivity and high risk of skin cancer as well as internal cancers.
XP results from defects in the Global Genomic or Transcription-coupled NER?
Global Genomic Nucleotide Excision Repair defect —> XP
mutations in damage recognition or helicase activity
[note: XP can also present with neuronal degeneration depending on which protein is affected]
This hereditary developmental and neurological disorder is associated with defects in transcription-coupled NER (TC-NER) which affect recognition of stalled RNA Pol II. Patients present with growth delays, intellectual disabilities, neuron demyelination, and sun sensitivity. However, they do not present with increased risk of skin cancer. What is…
Cockayne syndrome
increased risk of cancer is not seen because transcription does not resume after RNA Pol II is blocked - damaged transcriptionally active cells likely undergo apoptosis
What is Cockayne syndrome and what causes it?
hereditary developmental and neurological disorder associated with defects in TC-NER (transcriptionally coupled nucleotide excision repair)
growth delays, intellectual disabilities, demyelination, sun sensitivity
NOT at higher risk of cancer because transcription does not resume after RNA Pol II is blocked (cancer can’t form)
tumor cells deficient in nucleotide excision repair are very sensitive to this chemotherapeutic drug, which forms bulky intra-strand adducts within DNA
Cisplatin
match:
Global Genomic NER
Transcriptionally Coupled NER
with
neurodegeneration
increased risk of cancer
GG-NER —> increased risk of cancer but NOT CNS disorder (used in DNA that is not transcriptionally active)
TC-NER —> neurodegeneration but NOT increased cancer risk (RNA Pol II is stalled and cannot resume, so cancer can’t form)
HOWEVER: GG-NER and TC-NER feed into common pathway, in which mutations can cause an increased risk of cancer AND neurodegeneration
loss of a single nucleotide and damaged 5’ and/or 3’ termini at the site of the break is repaired by what mechanism
single strand break repair
in SSB Repair:
1. ____ recognizes the SSB
2. ____ is recruited as a molecular scaffold
3. ____ processes/fixes the 3’ / 5’ end damage
4. ____ inserts the missing nucleotide
5. DNA ligation
SSB:
1. PARP-1
2. XRCC1
3. Aprataxin (APTX)
4. DNA Pol beta
this autosomal recessive spinocerebellar ataxia syndrome presents with peripheral neuropathy, hypercholesterolemia, hypoalbuminemia, and cerebellar atrophy. It is caused by a mutation in APTX. What is this condition, and what repair mechanism is deficient?
Ataxia Oculomotor Apraxia - deficient in SSB repair pathway
loss of apraTAXin (APTX) gene causes aTAXia [oculomotor] aPRAXia
what is the hereditary pattern of Ataxia Oculomotor Apraxia, a disease caused by mutation in Aprataxin (APTX) gene, leading to deficient SSB repair pathway?
autosomal recessive
contrast the two types of double strand break (DSB) repair
non-homologous end-joining (NHEJ):
major pathway, can occur any point in cell cycle, does not require sequence homology, error prone and can lead to chromosomal translocations
homologous recombination (HR):
only S and G2 phase of cell cycle (when sister chromatid is present), requires homology of DNA (undamaged chromosome is template), non-mutagenic
what type of DSB repair does this pathway describe?
1. Ku70/Ku80 bind DNA ends
2. frayed ends removed by endonuclease activity of DNA-PKcs:Artemis
3. DNA ligase joins ends
NHEJ (non-homologous end joining)
why can’t homologous recombination occur at any point in the cell cycle?
sister chromatid must be present to use homologous DNA of undamaged chromosome as a template
only S and G2 phase
what kind of DNA repair does this pathway describe?
1. RAD52 binds DNA ends
2. RAD51 recombinase searches for sequence homology
2b. BRCA1 and BRCA2 regulate RAD51
3. single strand invasion, nuclease and helicase activation, ligase joins strands
homologous recombination (HR) double strand repair
BRCA1 and BRCA2 mutations cause susceptibility to breast cancer. However, some chemotherapeutic drugs such as bleomycin (oxidizing agent), camptothecin (topoisomerase I inhibitor), and anthracyclines (Top II inhibitors) may be effective because they induce ____
double strand breaks (DSBs)
BRCA1 and BRCA2 regulate RAD51 in homologous recombination DSB repair
patients with Ataxia Telangiectasia have increased chromosomal abnormalities in T and B cells and a higher propensity to develop lymphoid cancer. They are hypersensitive to ionizing radiation. What mutation is associated with this disorder and what kind of repair mechanism is faulty?
Ataxia Telangiectasia (AT): autosomal recessive, associated with mutation in ATM protein (slows down replication after DSB to allow repair)
faulty double strand break repair
A 36yo F pt presents with immune deficiency and lymphoid tumors. Genetic analysis reveals a mutation in the ATM protein. What does this protein do, and what is your diagnosis?
ATM: activated by DSB, signals to cell-cycle checkpoint to slow cell cycle for repair
ATM also responds to DNA breaks during B and T development/ differentiation (mutation —> immune deficiency)
diagnosis: Ataxia Telangiectasia (AT) - autosomal recessive
T/F: the monomer units in polynucleotide chains are joined together by 3’-5’ phosphodiester bonds
TRUE
all type II topoisomerases have ___ and ___ activity
nuclease and ligase
DNA gyrase is a Top II - can create bubble
which of these processes does NOT require DNA Top II?
replication
recombination
repair
transcription
none - DNA Top II is required for ALL (replication, recombination, repair, transcription)
DNA Top alter supercoiling, negative supercoils facilitate strand separation, which is required in all of these events
A mutation inhibiting the 5’-3’ exonuclease activity of DNA Pol I in a bacteria strain is most likely to cause which of these?
a. increased number of replication errors
b. accumulation of telemeric repeats
c. defect in mismatch repair
d. defect in removal of RNA primers
e. defect in DNA chain elongation
d. defect in removal of RNA primers - 5’-3’ exonuclease activity of DNA Pol I removes RNA primers so it can fill gaps
-DNA pol III and I proofread, so not increased number of replication errors
-bacterial DNA is circular, so not telemeric repeats
-DNA Pol III performs mismatch repair (and lagging strand synthesis)
nucleoside analog work by suppressing ____
DNA chain elongation
nucleoside analog is converted to nucleotide by kinases and incorporated but prevent nucleophilic attack
What repair mechanism is used to repair thymine dimers?
Nucleotide excision repair
Thymine dimers are source of damage in xeroderma pigmentosum
Abasic sites are repaired by what mechanism
Base excisison repair - for damage of one nucleotide
