DNA Structure, Replication, and Repair Flashcards
Nucleoside Analogs
Used in anti-viral and anti-cancer therapy
How do they work?
- analog is incorporated in the DNA during replication
- blocks further DNA synthesis (because it is missing an OH group)
- does not significantly affect host cell metabolism
Acyclovir
HSV deoxyguanosine analog (missing OH)
Azidothymidine (AZT)
HIV deoxythymidine analog (N3 instead of OH)
Nucleotide Polymerization Bond
3’,5’-phosophdiester bond
- between 3’ OH and the 5’ P on the next sugar
- results in chain POLARITY: free 5’-P and free 3’-OH
Nucleases
hydrolyze phosphodiester bonds
Exonucleases
cut at the end of a polynucleotide chain
Endonucleases
cleave internal phosphodiester bonds
Restriction Enzymes
site-specific cleavage
Secondary Structure Form of DNA
B-form (right handed double helix)
How many base pairs per helical turn?
10
Negative Supercoils
DNA double helix with FEWER HELICAL TURNS than the relaxed B-form DNA double helix
- allow for compaction of DNA
- facilitate DNA strand separation for DNA replication. transcription, and repair
DNA Topoisomerases
cut & paste to repair supercoiling form during replication
- change and relax DNA
- **important in order to remove POSITIVE SUPERCOILS AHEAD of the strand opening and “EXCESS” NEGATIVE SUPERCOILS BEHIND
DNA Topoisomerase Enzyme Activities
Nuclease and Ligase activities
- transiently break one or both DNA strands
- pass strands through the break
- rejoin strands
Topo I
cuts a single strand of the helix
Topo II
cuts both strands of the helix
DNA Gyrase
ONLY FOUND IN PROKARYOTES
Topo II
- removes + and - supercoils
Antibacterial Topoisomerase Inhibitors
block activity of bacterial DNA gyrase
- inhibit bacterial DNA synthesis
- safe because NO DNA GYRASE in eukaryotic cells
Quinolones
Antibacterial Topoisomerase Inhibitor
Anti-Cancer Topoisomerase Inhibitor Therapy
target eukaryotic topoisomerases
- inhibit ability of topoisomerases to join DNA
- convert topoisomerases into DNA break agents
- lead to cell death
- SIDE EFFECTS ASSOCIATED because no way to differentiate which topos it is attacking
Chromatin Structure
DNA associated with HISTONE proteins
Histones
small basic proteins (rich in Arg and Lys)
- five classes of histones
- arranged in repeating units called nucleosomes
Five Classes of Histones
H1, H2A, H2B, H3, H4
Nucleosome core
DNA double helix supercoils around a histone octamer
- two molecules of H2A, H2B, H3, H4 (not H1)
- +/- charge interactions
- H1 acts as spacer between nucleosome cores
DNA Spacer
histone H1 acts as spacer between nucleosome cores
Chromatin Condensation
H1 binds spacer DNA, promotes tight packing of nucleosomes
- chromatin winds into helical tubular coil
Solenoid
helical tubular coil of chromatin
- makes really large loops
Eukaryotic Chromosome Compaction
during Mitosis or Meiosis
- solenoid winds upon itself to form very large DNA loops
- DNA loops coil around a protein scaffold
- DNA loops radiate from scaffold
- CONDENSED METAPHASE CHROMOSOME FORMED (classic 4 arm structure with 2 chromatids joined by a centromere)
Condensed Metaphase Chromosome
classic 4-arm structure with 2 chromatids joined by a centromere
Replication mechanism in prokaryotes
- Initiation at origin
- DNA strand separation
- RNA primers
- DNA synthesis
- Chain elongation
- Proofreading
- RNA primer excision
- DNA ligation
What phase does replication take place during
S (synthesis) phase of the cell cycle
Prokaryote Origin
- single OfR
- rich in A:T base pairs
DNA helicase
catalyzes DNA strand separation
- binds near replication forks
- uses ATP to break the H bonds between DNA strands
Single Stranded Binding Proteins (SSBPs)
bind cooperatively
- keep DNA strands apart
- protects DNA from nucleases
Inhibitors of Herpes Simplex Virus (HSV) Helicase
makes zipper stuck so DNA can’t replicate
- stabilize interaction of helicase with viral DNA substrate
- inhibit progression on DNA replication
- effective against HSV strains resistant to nucleoside-based therapy
Primase directionality
ALWAYS 5’->3’
RNA primers
- needed to INITIATE DNA synthesis
- DNA polymerase cannot initiate on a single strand
- provide a free 3’-OH as the acceptor for the first deoxyribonucleotide
DNA synthesis is DISCONTINUOUS because:
DNA POLYMERASES CAN ONLY SYNTHESIZE DNA IN THE 5’->3’ DIRECTION
- leading strand synthesized continuous
- lagging strand discontinuous with OKAZAKI fragments
DNA Polymerase III
- primary replication polymerase in Prokaryotes
- has proofreading
Chain elongation
- catalyzed by DNA POLY III
- nucleophilic attack of 3’OH of growing chain and 5’P of incoming deoxyribonucleotide triphosphate
- formation of PHOSPHODIESTER bond
DNA Polymerase III Proofreading
3’->5’ exonuclease activity to remove nucleotides introduced in error
POST DNA Replication Proofreading
MMR Pathway
Remdesivir
Adenosine analog (SARS-coV2) - modified sugar groups prevent further DNA chain elongation and block DNA synthesis
DNA Polymerase I Proofreading
5’->3’ Exonuclease activity
- USED TO REMOVE RNA PRIMERS FROM OKAZAKI FRAGMENTS
(DNA then synthesized by DNA Poly I 5’->3’ Polymerase activity)
RNA Primer Excision and DNA Ligation
- DNA Poly III Synthesizes until blocked by RNA primer
- DNA POLY I removes RNA Primer and finishes replication
- DNA Ligase then joins Okazaki Fragments
Major differences between prokaryotic and eukaryotic replication
- Eukaryotic DNA has MANY origins
- Eukaryotic DNA has many more and different polymerases and proteins involved
Pol Alpha
PREP
- Eukaryotes
- primer synthesis for both LEADING and LAGGING strands
- PRIMASE activity synthesizes short stretches of RNA
- DNA POLYMERASE activity then extends RNA primers with DNA
- NO PROOFREADING
Pol Delta
LAGGING; removes RNA primers and strand is completed
- Eukaryotes
- SYNTHESIZES the bulk of the LAGGING STRAND DNA
- has PROOFREADING 3’->5’ EXONUCLEASE activity
Exonuclease FEN1
degrades 5’ primer ends
Pol Epsilon
LEADING (E lEading)
- SYNTHESIZES bulk of LEADING STRAND DNA
- has PROOFREADING 3’->5’ EXONUCLEASE activity
Processivity factor PCNA
associates with POL delta and epsilon
Eukaryotic DNA is packaged in nucleosomes
- DNA double helix is associated with histones (nuclosomes)
- nucleosomes are displaced as replication fork advances
- histones remain loosely associated with parental strand
- new histones synthesized simultaneous with DNA replication
- nucleosomes reform behind the advancing replication fork
Telomeres
- ends of linear chromosomes
- TAGGGGG telomeres it
- can form telomeric loops (T-loops)
Role of Telomeres
T-loops protect the ends of linear chromosomes from:
- recognition as broken DNA and degradation
- recombination
- end to end fusion
Prevent the loss of important coding sequences during replication
Telomerase
ribonuleoprotein complex (RNA + protein)
- has reverse transcriptase activity and makes DNA using RNA
- synthesizes short DNA repeats extended chromosome with TAGGGG
Telomerase is implicated in cell aging and cancer
- telomerase active in ALL CELLS before birth; remains active in STEM CELLS AND GERM CELLS AFTER BIRTH
- telomerase is INACTIVE in MOST SOMATIC cells AFTER birth (telomeres shorten with each cell division)
- if shorten too far senescence occurs
- IN HUMAN CANCERS telomerase is reactivated, and generally p53 activity is lost leading to unstable cell division and DNA elongation
Senescence
- occurs when telomere length declines to a critical point
- DNA-damage sensors (p53) notice
- induce cell growth arrest to prevent genomic instability
Dyskeratosis congenita
- inherited disease caused by REDUCED telomerase activity
- defects seen most often in tissues in which CELLS DIVIDE RAPIDLY AND OFTEN
- affects stem cells and germ cells
- can cause mutation in RNA component of telomerase
- patients generally die from bone marrow failure due to loss of hematopoietic renewal**
DNA Repair Pathways
- MMR
- BER (base excision repair)
- NER (global genomic nucleotide repair; transcription-coupled nucleotide excision repair)
- Single-Strand Break Repair
- Double-Strand Break Repair (non-homologous end joining; homologous recombination repair)
MMR
- Mismatched nucleotides; NO nucleotide damage
- incorrect via substitution, deletion, or insertion
- Recognize the mismatch and distinguish the newly synthesized strand from original
- recognize via DNA methylation (Prok) or Okazaki framents (Euk)
- Endonuclease cleaves strand on either side of mismatch
- Helicase and exonuclease remove error
- DNA Pol III fills the gap, followed by DNA ligase
Cancers from MMR
Lynch Syndrome (CRC +) - MSH2 MLH1 mutations
Base Excision Repair Pathway
DAMAGED BASE (caused by REACTIVE OXYGEN SPECIES; oxidation, deamination, depurination, alkylation, etc) occurs ~20,000x/day
- GLYCOSYLASE recognizes damaged base and cleaves N-glycosidic bond between base and deoxyribose (specific glycosylase/base)
- apurinic/apyrimidic ENDONUCLEASE cleaves sugar-phophate backbone
- deoxyribose phosphate LYASE removes sugar-phosphate residue
- DNA Pol I fills the gap, followed by DNA Ligase
- ** NEEDS 3’OH to connect and replace, which is why the sugar backbone is removed as well! ***
Defects in BER
mutation in gene encoding DNA glycosylase MYH leads to v high risk of CRC
UV Dimer formation
UV induces formation of dimers between adjacent pyrimidines in DNA of skin cells
- significant distortion of DNA helix
- causes DNA frameshifts
- without repair can result in skin cancer
- ** Lesions corrected by NER! **
ONLY way to extend DNA backbone and add a base
via BASE AND SUGAR BACKBONE
NEED free 3’OH for nucleotide attach between 3’OH and 5’ P
Cigarette Smoke Carcinogens
Once oxidized covalently bind to G residues in the DNA of lung cells
- interrupts normal H bonding, distorting the helix
- causes DNA framshifts
- without repair leads to lung cancer
- Lesions corrected by NER! ***
Nucleotide Excision Repair Pathways
BULKY FIX!
- can remove an INFINITE NUMBER OF LESIONS
- ONLY MECHANISM THAT REMOVES BULKY DNA ERRORS
2 Pathways
- Global Genomic NER (transcriptionally INACTIVE region of DNA)
- Transcription-coupled NER (transcriptionally ACTIVE region of DNA)
MMR BER NER (GG-NER; TC-NER) SSBR DSBR (NHEJ, HR)
MMR: wrong but not damaged
BER: single base damaged; need to remove sugar backbone to replace
NER: ONLY mechanism for multiple BULKY fix
- GG-NER: transcriptionally inactive; big cancer risk
- TC-NER: transcriptionally active; neuro disorder risk
SSBR: single missing nucleotide with frayed ends; ends processed by APTX
DSBR: Major NHEJ; Minor HR
- NHEJ: sloppy but gets the job done; occurs whenever
- HR: ONLY S and G2; PERFECT REPAIR; BRCAs/RADs
Global Genomic NER (GG-NER)
Transcriptionally INACTIVE region of DNA
- recognize issue of helix distortion
- ENDONUCLEASES separate on both sides of problem
- HELICASE unwinds DNA to release damaged oligmer
- DNA POL I (d/E) fills gap
- DNA Ligase seals
CANCER RISK
Xeroderma pigmenosum (XP)
Heriditary disorder from defects in GG-NER
- patients show extreme solar sensitivity
- highly increased risk of skin cancer and internal cancers
- MIGHT also show progressive neuronal degeneration depending on WHICH XP PROTEIN IS AFFECTED
Transcription Coupled NER (TC-NER)
Transcriptionally ACTIVE region of DNA
(DNA damage-induced helix disortion blocks progression of RNA pol II along template and halts gene transcription)
- proteins recognize stall of transcription
- RNA pol II displaced from lesion site
- recruitment of NER proteins
- HELICASE unwinds DNA to create a “bubble”
- EXCINUCLEASES make incisions on either side of lesion
- damaged oligomer is released
- DNA Pol I (d/E) fills gap
- DNA LIGASE seals chanin
- once repair is complete TRANSCRIPTION CAN RESUME
NEURO DISORDER RISK
Cockayne Syndrome
- hereditary developmental and neurological disorder associated with defects in TC-NER
- mutations affect recognition of stalled RNA pol II
Results in: - growth and mental retardation
- neurological deficiencies
- sun sensitivity
*** DO NOT have INCREASED CANCER RISK vs. XP - because transcription does not resume after RNA pol II is blocked
- damaged transcriptionally active cells likely under cell death via apoptosis
(cell dies rather than being transcribed so do not have the chance to proliferate)
Single-strand Break Repair Pathway causes
- breaks in one strand of the DNA double helix; commonly caused by reactive O2 species, BER issues, and TOPO I without resealance
- usually associated with loss of single nucleotide and damaged temini
SSB Repair Mechanism
- SSB recognized
- Recruits XRCC1 which recruits multiple repair proteins
- APTX processes the broken ends restoring proper 3’OH or 5’P groups
- DNA polB can insert missing nucleotide
- DNA ligation
Ataxia Oculomotor Apraxia (AOA1)
- autosomal uncoordinated eye movement disorder
- caused by mutation in the APRATAXIN gene (APTX) (DNA end processor)
Double-Strand Break Repair Pathway
- can be caused by a lot of stuff
- can severely compromise genome stability; lead to loss of chromosome fragments in mitosis; cause cancer due to joining of wrong ends; lead to chromosome translocations
2 repair pathways - Non-homologous end joining (NHEJ)
- Homologous recombination Repair (HR)
Non-homologous Endo Joining (NHEJ)
MAJOR DSB repair pathway
- can occur whenver
- ERROR Prone! rejoins random ends, HOWEVER repairs structural integrity so leads to some loss but not overwhelming loss
- KU70/KU80 sense and bind DNA broken end
- Artemis recruited and remove bad ends
DNA ligase rejoins ends
Homologous Recombination Repair
RESTRICTED to S and G2 PHASES (needs sister chromatid)
- undamaged homologous strand serves as template to transfer genetic information and repair broken DNA
NON MUTAGENIC
- RAD52 binds DNA ends
- RAD51 recombinase searches for SEQUENCE HOMOLOGY
** Human BRCA1 and BRCA2 regulate RAD 51 **
- NUCLEASE and HELICASE acitivites are involved, followed by joining of the strands
NO CHANGE IN DNA SEQUENCE
BRCA1 & BRCA2
Breast Cancer Susceptibility Genes
- Mutation in BRCA affects DSB HR
- tumors in BRCA carriers are more sensitive to ionizing radiation because of issues with DSB HR repair
Anti-cancer drugs which induce DSBs good chemotherapy options
- severe damage=cell death
Ataxia telangiectasia (AT)
- autosomal recessive disorder
- hypersensitivity to ionizing radiation
- associated with mutation in ATM protein which is activated by DSBs
- slows cell cycle to allow repair but increases chance of improper joining and cancer
