DNA Replication And Repair Flashcards
Covalent structure of DNA
. Double stranded polymer of deoxyribonucleotides joined by 3’-5 phosphodiester bonds
Hydrogen bonding between base pairs in DNA
. 3 hydrogen bonds with ____
. 2 H bonds w/ ______
. Stabilizes double helix in non-polar interior
Is phsophodiester connection btw deoxynucleotides directional or not?
. Directional
. Connects 5’ phosphate of 1 nucleotide w/ 3’ hydroxyl group of next nucleotide
DNA replication occurs in what phase of cell cycle?
S phase
Substrates for DNA replication
??
Product of DNA replication
2 identical strands???
DNA polymerase
. Select nucleotide that is added to 3-OH end of chain
. Catalyzes formation of phosphodiester bond
. Substrates are dATP, dCTP, dGTP, and dTTP and single stranded template DNA
T/F there is one place for replication to begin
F, there are multiple sites that start at different times
. When it nears completion the bubbles of newly replicated DNA meet and fuse forming 2 new molecules
DNA chain elongation
. DNA polymerase adds deoxyribonucleotides to 3’ end of growing chain
. Dictated by base sequence of template (coding) strand
New DNA strand is synthesized in what direction?
5’ to 3’ because it is antiparallel
Leading strand DNA replication
. 5’-3’
. In same direction as replication fork movement
.
Landing strand DNA replication
. 5’ to 3’
. Synthesis in direction opposite of fork movement
. Short (100-200 nucleotides) fragments (Okazaki fragments) made
. Lipase joins fragments together
DNA helicase
. Unwinds short segment of parental duplex DNA by catalyzing ATP dependent strand separation
. Forms replication fork
DNA primase
. Initiates synthesis of RNA molecule that primes DNA synthesis on leading and lagging strand
. First few nucleotides are ribonucleotides and then subsequent ones are ribo or deoxyribonucleotides
Singles stranded DNA binding proteins during replication
. Bind to single stranded DNA
. Prevent premature annealing of single stranded DNA to double stranded
Proof reading in DNA replication
. DNA polymerase w/ 3’-5’ exonuclease activity
. Remove nucleotides that aren’t part of double helix and are mismatched
. Do not has 5’-3’ exonuclease activity
DNA ligase
. Seals nick in DNA after DNA polymerase fills gaps left by RNA primers
Topoisomerases
. Facilitates unwinding of double helix super twisting to achieve negative supercoils for transcription
. As replication fork moves, daughter molecules rotate around each other and become overcoiled
. Relieve torsional stress by inducing reversible single stranded breaks in DNA at phosphodiester bond
Topoisomerase I
. Catalyzes break in only 1 strand of DNA
. Allows unwinding of broken strand
. Rejoin broken ends by catalyzing formation of new phosphodiester bonds
Topoisomerase II
. Catalyzes break in both strands of DNA
. Both strands unwind
. Catalyzes formation of new phosphodiester bonds
Classes of DNA synthesis inhibitors
. Prevent/reduce purine/pyrimidine synthesis (_____)
. Drugs affect template/priming ability of growing strand (intercalating agent doxorubicin for cancer)
. Topoisomerase inhibitors
DNA gyrase
. Bacterial type II topoisomerase
. Introduces neg. supercoils into DNA molecules via ATP dependent reaction ahead of replication fork
quinolone antibacterials
. 1st and 2nd generation: Norfloxacin and ciprofloxacin
. 3rd generation: levoflaxacin and moxifloxacin
. DNA gyrase inhibitor via inhibiting strand cutting reaction
Camptothecins
. Anti cancer agents
. Inhibit topoisomerase I
Doxorubicin, etoposide, and teniposide
. Inhibit human topoisomerase II
. Anti-cancer drugs
. Enhances rate at which topoisomerase II cleaves or reduces rate at which the breaks are resealed
Telomere
. Tandem array of simple sequence (TTAGGG)
. DNA replication leaves short gap where RNA primers were removed
. Linear chromosome shortened w/ each cell division, telomeres prevent actual genes form being lost in that
Telomere maintenance
. RNA dependent DNA polymerase
. Adds TTAGGG repeats to end of chromosomes
. Telomerase ribonucleoprotein complex contains RNA template
. DNA repeats added to leading strand
. Lagging strand completed by DNA polymerase as normal
Basal mutations ate
. Errors during DNA replication in absence of environmental mutagens
. Spontaneous tautomeric shifts contribute to this (rare)
Exogenous agents that damage DNA
. Radiation: energy rich enough to react w/ DNA, penetrates whole body causing somatic and germ line mutations
. UV radiation: can’t penetrate beyond outer layer of skin
. Chemical in environment: hydrocarbons
. Chemo chemical: carcinogenic
Base analogs
. Can cause mutations
. Erroneously incorporated into DNA
. Bromouracil incorporated in place of thymine
Alkylation agents
. Alkylate N Or O atoms in bases
. Found in food, Tobacco smoke, endogenous metabolic products
. O-methyl guanine is major carcinogenic lesion on DNA
. Methylguanine DNA methyl transferase important for genomic stability to repair these lesions
Dominating agents
. Turn adenine to hypoxanthine
. Guanine to xanthine
. Cytosine to uracil
. Lead to errors in replication
Categories of DNA mutations
. Point mutations
. Length mutations
Point mutations
. Change occurring in single nucleotide
. Transitions of transversions
Transition mutation
.pyrimidine changed to another pyrimidine
. Purine changes to another purine
Transversions
. Purine to pyrimidine
. Pyrimidine to purine
. Less common than transitions showing mutations are non random
Missense mutation
. Amino acid substitution
Nonsense mutation
. Mutation causes stop codon to be introduced to coding sequence
Sense/silent mutation
. Mutation but no change in amino acid sequence
Cystic fibrosis mutation
. No single mutation is responsible
. Single point, deletion, and insertions found
Types of length mutations
. Insertion
. Deletion
. Affect reading frame depending on nu,bear of nucleotides inserted or deleted
Frame shift mutation
. Not a multiple of 3 nucleotides are inserted in sequence
. Resulting amino acid may be radically different from that point onward
Inframe mutation
. If 3 nucleotides added or deleted, an AA is added or lost, but it does not effect sequence further down the line
Translocation mutation
. Transfer of genetic material from one chromosomal location to another
. Large chunk or whole arm of chromosome
. Expressed normally as long as regulatory site is not severed
Mutation in intron-exon boundary
. Results in abnormal splicing
. Nonfunctional protein product
Mutation in promoter or enhancer
. Leaves polypeptide structure intact
. Inc. or dec. rate of synthesis
Gene amplification
. Portions of chromosomes amplify resulting in large excess of the protein product
Sickle cell gene mutation
. Missense mutation
. GAG to GTG
T/F larger genes show mutations more often
T
Mutational hot spots
. CG region accounts for over 30% of all single nucleotide substitutions
. More common due to issues with converting cytosine to methylcytosine because spontaneous hydrolysis of methylcytosine makes thymine
How age of parent affects gene mutation rate
. Some chromosomal abnormalities inc. w/ age of parent
DNA damage response (DDR)
. DNA damage sensors that detect damage and launch activation of stress response pathways
. Temporary arrest in cell cycle for DNA damage to be repaired
. Enzymes can recognize, remove, repair, and relegate strand
Types of DNA repair mechanisms
. Mismatch repair
. Base excision
. Nucleotide excision
. Double-stranded break
Mismatch repair
. Occurs w/ error in DNA polymerase
. Corrects mismatches of normal bases
. Repair proteins coded by MSH2, MLH1, MSH6, PMS-1, and PMS2 genes
. Mutations in these repair genes cause person more likely to get colon cancer and other cancers
Microsatellite instability
. Happens when mismatch enzymes are dysfunctional
. Can cause cancers
Base excision repair
. Fixes issues w/ spontaneous depurination of cytosine to uracil and deamination
. Removes nucleotides that have lost a base as result of either of these
. Endonuclease recognizes space from lost base, DNA polymerase and ligase fill in space and seal nick in strand
Nucleotide excision repair
. Fixes issues from environmental damage
. Forms pyrimidine-pyrimidine dimers from adjacent pyrimidine bases in DNA
. Smoking causes formation of benzopyrene that interacts w/ DNA
. Transcription-coupled repair or global genomic repair
Transcription-coupled nucleotide excision repair
. DNA damaged recognized when transcription is stalled at DNA-damage site
Global genomic nucleotide excision repair
. DNA lesion detected on scanning of genome by enzymes (xeroderma pigmentosum group of proteins)
Xeroderma pigmentosum
. Disease that suggests 10 proteins required for excision of damaged bases in DNA by single repair system
. Autosomal recessive
. Defective repair of thymine dimers
. Prone to developing skin cancers
Cockayne syndrome
. Loss of transcription-coupled repair
. Cachexia, growth retardation
. No predisposition to skin cancer
Double strand break repair
. Fixes environmental damage
. Homologous recombination: gets info from homologous chromosome for break repairs, sister chromatid is template to repair
. Non-homologous end joining: permits joining of ends if there is no sequence similarity btw them
. May proteins involved in this
BRCA 1 and 2 proteins
. Play role in homologous recombination repair
. Inc. breast and ovarian cancer risk if dysfunctional
. Homozygous mutations in BRCA 2 cause Fanconi’s anemia
