DNA Structure/function/replication/repair Flashcards
Which nucleic acids bases are purines? Pyrimidines?
Purines: A + G
Pyrimidines: C T U
What is a nucleoside?
Nucleic acid base with a 5 carbon sugar attached via a N’glycosidic bond
What is a nucleotide?
Nucleoside + phosphate via a phosphoesterase bond
CLINICAL EXAMPLE:
How does salmonella cause food poisoning?
Inactivation of DNA adenine methylase
- blocks expression of virulence genes
- prevent diz development in mice
- induces an immune response
Clinical correlation:
How are nucleoside analogs used to tx Anti-viral and anti-cancer txs? Give an example:
Incorporates into DNA to prevent chain elongation.
Acyclovir - deoxyguanosine analog (HSV)
Azidothymidine (AZT) - analog of deoxythymidine (zidovudive- HIV)
Zalcitabine (dideoxycytidine) - analog of deoxycytidine (HIV)
How to nucleotides polymerize?
3’-5’ phosphodiester bonds btwn 3’-OH on sugar of one nucleotide and 5’-P on the next
What is chain polarity of the structure?
Free 5’-P at the 5’ end and free 3-OH at 3’end
What is the 1’ sttucture of DNA? 2’? 3’?
Primary - nucleotide chain
Secondary - Double helix
Tertiary - supercoiling
What are nucleases? What kind of nucleases do we have?
Nucleases - hydrolyze phosphodiester bonds
Exonuclease - cut at an end of a polynucleotide chain
Endonuclease - cleave at internal phosphodiester bonds (i.e. Restriction enzymes, site specifc cleavage)
A restriction enzyme is an example of what kind of nuclease?
Endonuclease
What is the most common DNA structure? What is it composed of?
B-form
2 anti-parallel polynucleotide chains
5’-3’ & 3’-5’
Right handed double helix
Outer sugar phosphate back bone
Bases perpendicular to axis
10BP/helical turn
Complete turn Q 34A
Chains held together with hydrogen bonds
Minor (narrow) and Major (wide) grooves
CLINICAL CORRELATION:
How do anti-cx drugs DACTINOMYCIN, actinomycin D exert cytotoxic effects?
Intercalate at MINOR grooves
Thus interfering with DNA/RNA syn
How does DNA size differ between E.coli and Humans?
Ecoli: 1 molecule, circular, DS, 4X10^6bp, 2mm
Human: 46 chromosomes, linear, DS, 6X10^9 BP, 6 geet long
Does a negatively supercoilded DNA double helix have fewer or greater helical turns compared to a relaxed B DNA double helix?
Fewer helical turns
I.e. 100BP->10 helical turns
100 -> 8 turns (neg. supercoiled)
100BP->12 turns (pos. Supercoiled)
Why are negative supercoils so important?
- Facilitate DNA strand separation in double helix (for replication/transcription/repair/recombination)
- Energetically favored
- NRG needed for strand separation are stored in the supercoils.
Do negative supercoils break phosphodiester bonds?
No.
double strand helix is partially unwound (bubbles) and then normal BP is restored.
When eukaryotic histone proteins bind to double helix, histones force DNA to wrap around them . Generates neg supercoil.
What do DNA topoisomerases do?
Change tertiary structure (supercoiling)
Swivel points in DNA helix
How does strand separation influence positive supercoiling?
Ex. If we have 100BP and uncoil 50, the remainder of 50 BP will be forced to have more helical turns —> (+ supercoiling)
How to topoisomerases work?
They have both nuclease and ligase activity.
- Transiently break one or both of the DNA strands
- Pass the strands thru the break.
- Rejoin them
How does TopoI differ from TopoII?
TopoI -> cuts single strand of helix
Topo II ->cuts both strands of helix. Think of a loop and all that happens in first step one side is being pulled underneath other. TOPOII comes in and cuts so that now one part of strand loops over and then other loops behind. Religates and now we have a negative supercoil.
What is bacterial DNA gyrase?
An unusual TopoII
Can remove both (+ & -) supercoils.
Facilitates bacterial DNA replication
REQUIRES ATP!!!
Introduces negative supercoils into relaxed circular DNA
CLINICAL CORRELATION:
How do drugs targeting Topoisomerases differ from ABX to ACX?
ABX -> FQs. Target DNA gyrase (topoII) not found in eukaryotes. Shouldnt have cell cycle specific side effects b/c of this.
Novobiocin - block ATP-binding to gyrase
(Nalidixic acid/Ciprofloxacin - interferes with endonuclease activity of gyrase
ACX ->
Camptothecin -> TopoI
Adriamycin/Etopside ->TopoII
(Converts Topo into DNA breaking agents)
How do prokaryotes and eukaryotes differ in chromatin and chromosome structure?
Pro: DNA associated with non-histone proteins. Condense DNA to form nucleoid (nonmembrane bound region)
Eukary: Associated with histone and non-histone proteins
Condensed in the nucleus. Nucleoprotein complex is called chromatin
What are histones?
Small BASIC Proteins. Typically ARG and LYS rich
What are the 5 classes of histones?
H1, H2A, H2B, H3, H4
What is the structure of the nucleosome?
A. Core: Histone octamer w/ DNA supercoiled around (2 histones of h2a/b,h3, h4)
**140BP dna (1&3/4 superhelical turn) winds around histones
B. DNA spacer = 20-80 bp btwn cores, binds with H1.
Why is H1 important?
As soon as DNA replication is completed, 1 H1 binds spacer DNA and promotes packing of nucleosomes
What is a solenoid?
Helical tubular coil of chromatin
How is compaction of the eukaryotic chromosome completed?
Solenoid loops itself and forms large DNA loops (600A) w/ each loop contained 40,000-80,000 BP
DNA loops around scaffold protein.
DNA loops radiate from scaffold = metaphase chromosome (classic 4 arm structure)
When does replication take place in the cell?
S phase.
Is replication a conservative process?
No, semi-conservative.
How is replication initiated?
Partial opening of double helix beginning at origin of replication
What base pairs do we typically see at origins or replication?
A:T base pairing
What recognizes AT rich areas of initiation? Is this dependent on energy?
DnaA -> induces melting
NEEDS ATP!
How many replication forks are created at origin?
2
What is the role of DnaB in replication? Does it need ATP to function?
DnaB (DNA helicase) unwinds the double helix.
ATP dependent yes
What are SSBBP role in replication?
Single-strand DNA-binding proteins keep DNA single strands apart & protect from nucleases
Where is TopoII in relation to the replication fork during replication?
Works ahead to remove positive supercoils.
CLINICAL Correlation:
How do HSV helicase-primase inhib work?
Inhibit progression of HSV DNA replication.
Effective against resistant HSV strains
Why are RNA primers important to DNA replication?
DNA polymerase cannot recognize ssDNA strands. (Unlike RNA pol)
What enzyme adds primers to DNA?
Primase (RNA pol) about 10nt
What is the polarity of RNA primers?
Run in the 5’->3’ direction. Primers provide a free-3’OH as an acceptor of 1st deoxyribonucleotide
What strand has the most RNA primers>
Lagging strand (Okazaki frags)
What is the polarity of the complementary strand of DNA being synthesized?
5’->3’ remember DNA is antiparallel
What catalyzes chain elongation in prokaryotes?
DNA pol III
Nucleophilic attack of 3’-0H terminus on the innermost 5’posphate
How is DNA replication regulatd?
Proofreading!
There is removal of erroneously introduced nucleotides that are not complementary to the template
What type of exonuclease activity does DNA pol III have?
3’-5’ (looks behind it)
CLINICAL CORRELATION:
What are some examples of drugs that incorporate nucleotide analogs to prevent chain elongation?
Acyclovir -> deoxyguanosine (HSV)
AZT -> thymidine analog (HIV)
DdC -> cytidine. (HIV)
Gemcitabine -> Deoxyguanosine (Cancer
How are RNA primers removed from strands?
5’-3’ exonuclease activity of DNA POL1 removes RNA primers from Okazaki frags.
DNA synthesis cont. with 5’-3’ polymerase activity of DNA Pol I
DNA POL III synthsizes DNA until blocked by primer.
What kind of exonuclease activity does DNA pol I have?
3’-5’
What covalently binds Okazaki fragments? IS this ATP dependent?
DNA Ligase - Yes!
DNA Pol III responsible for 5’-P and DNA Pol I responsible for 3’-OH (from removed RNA primer)
What are some key differences between Eukaryotic and Prokaryotic DNA replication?
- More dna
- Polymerases and proteins involved
- Dna associated with histones
- Presence of telomeres
What is the role of DNA Pol alpha? Any exonuclease activity?
- DNA replication thru primer synthesis (primase).
- Initiates syn. On leading and lagging DNA
No exonuclease activity
Primers get removed anyway
Role of Pol Beta?
DNA repair (Base excision repair -BER)
No exonuclease activity
Role of Pol gamma?
Mitochondrial DNA replication
3’ -> 5’ exonuclease activity
Role of Pol delta?
Lagging strand DNA replication
Associated with PCNA to elongate lagging strand
3’-5’ exonuclease activity (looks behind)
Dissociates 5’ end of primers fro Okazaki frag -> degraded by FEN1
DNA repair (MMR, NER)
Role of Pol sigma?
DNA replication @ leading strand
3’-5’ exonuclease activity
DNA repair (MMR, NER)
What are telomeres? How can we recognize them?
Ends of linear chromosome
Short non-coding G-rich DNA repeat (TTAGGG)
Form t-loops
What are the roles of telomeres?
A.Protecting ends of linear chromosomes
- Recognition as broken DNA and subsequent degradation.
- Recombination
- End-to-end fusion
B. Prevent loss of important coding terminal sequences.
Since primers are needed for DNa replication
What is the “end problem” that telomeres solve?
Primer removal would result in loss of DNA information. This adds info to 3’ end of strand so primer can bind on and initate lagging strand length
How does telomerase work?
Ribonucleoprotein.
Adds G-rich DNA repeats to SS 3’ends of linear chromosomes.
Protein component has REVERSE transcriptase activity.
When is telomerase most active?
Stem/Germ b4 birth. Telomeres shorten with each cell division
What happens when chromosome length declines to a critical point?
End-to-end fusions (no more t-loops)
What is the role of p53 in relation to telomeres?
P53 is a dna damage sensors. Should initiate cell growth arrest
In cancers, telomerase may be reactivated with loss of p53.
CLINICAL correlation:
What is Dyskeratosis congenita?
Inheritied disease with reduced telomerase activity. (Mutations in gene that encodes for RNA component of telomerase)
Affects highly proliferative tissues
Alopecia, gut disprders, lung fibrosis
What is difference betwen Dyskeratosis congenita and Hutchinson-Gilford progeria?
HG progeria = accelerated telomere shortening
(alopecia, aged skin appearance, die from MI
Dyskeratosis = reduced telomerase activity (die from BM failure)
What pathway would replication errors be fixed through?
MMR (single extra nucleotide/mismatch)
How would DNA repair damage to a single base?
BER (deamination/depurination/alkylation/oxidation)
What are the ways DNA could repair bulky DNA adducts
- Global genomic- NER
- Transcription coupled NER
(Induced via radiation/chemicals, distortion of helix)
How are SSB incorporated into DNA?
Oxidative damage
How are DS DNA breaks incorporated into DNA? How would we fix these?
Ionizing radiation (gamma/xrays) Oxidizing agents (bleomycin) Topo inhib (camptothecin ->topoI
Fixed via:
1. Non-homologous end joining
- Homologous recombination
What enzymes are involved in MMR pathway?
Proteins? How do we recognize?
- Endonuclease
- Exonuclease
- Helicase
- Polymerase (POL III / delta/sigma +PCNA)
- Ligase
Proteins:
E.coli - MutS/MutL
Human - MSH2/6 (mismatch)
MSH2/3 (1-4 nt insertions/deletions)
CLINICAL CORRELATION:
What cancers are lynch pts mor elikely to have? What do 90% of these pts have?
Colorectal (+endometrial/skin/ovarian/gastric/renal)
MSH2 or MLH1
What enzymes are involved in the BER pathway?
- Glycosylase
(recognizes damaged base), cleaves n-glycosidic bond) - Endonuclease (apurinic/apyrimidic cleaves sugar backbone)
- Deoxyribose P lyase (removes sugar-phosphate residue)
- Polymerase (DNA POL 1 (Beta) fills in new base
- Ligase
CLINICAL Correlation:
How do defects in BER manifest?
A. Mutation in gene encoding for MYH glycosylase.
Increased risk for colon cancer.
B. Mutations in RecQ DNA helicase WRN
Werner’s syndorme: Rare autosomal recessive disorder
WRN helicase involved with BER
CLINICAL CORRELATION:
UV damage induces thymine dimers & smoking has carcinogens. What pathway corrects this DNA damage? What mutations would these cause if uncorrected?
Nucleotide excision repair (NER)
Frameshift
What enzymes are involved in NER? Whats the difference between Global genomic and TC?
- Helicase
- Excinuclease
- Polymerase
- Ligase
Only mechanism that removes bulky adducts.
GG-NER => transcriptionally inactive region
TC-NER => active region
What pathogensis does GG-NER defect lead to? TC-NER? Why? Can it be both?
GG-NER: cancer w/o CNS disorder
TC-NER: CNS w/o cancer (blocks progression of RNA pol along transcribed gene)
GG-NER = Xeroderma Pigmentosum (XP)
-mutations in XPC/XPE/XPD/XPA, affecting damage recognition activity. Cancers increased, can be part of common pathway which would also lead to neuro deficits.
TC-NER = Cockayne syndrome (CS)
Hereditary developmental and neurological disorder associated mutations in CSA/CSB (affect recognition of stalled RNA pol II)
Growth and mental retardation
Why is cisplatin useful for NER deficient tumor cells?
MOA: forms bulky intra-strand adducts with DNA.
What enzymes are involved in SSB repair?
Recognition by PARP1 & recruitment of XRCC1 to serve as scaffold to recruit multiple repair proteins. Ex. Aprataxin APTX
- Hydrolase/transferase
- Polymerase
- Ligase
What diz is deficient SSB repair associated with?
Ataxia Oculomotor Apraxia (AOA1)
-autosomal recessive spinocerebellar ataxia
Uncoordinated gait, peripheral neuropathy,
Resembles Ataxia telangiectasia (AT) w/o immunodeficient featues
Caused by mutation in APTX gene
What enzymes are needed for DSB repair? Proteins? Recognition?
- NHEJ - major pathway, mutagenic
A. Broken DNA sensor
(Ku70/80 binds DNA ends, recruits DNAPKcs-Artemis)
B. Nucleases (artemis has exonuclease activity)
C. Polymerases
D. Ligases - Homologous recomination - minor, non-mutagenic
Restricted to S/G2 phases
A. Recombinases (RAD52 binds DNA ends, rad51 looks for homology (mediated by BRCA1/2)
B. Endonucleases
What is Ataxia telangiectasia?
Autosomal recessive with mutation in ATM protein (that is normally activated by DSBs)
Increased chromosomal abnormalities (t+B cells)
Develop lymphoid cancer.
How does a mutation in an allele of BRCA1/2 affect lifetime risk of developing cancer?`
Increases by 80%
