DNA Repair and Replication Flashcards
DNA replication
- duplication of hereditary material
- semiconservative
- occurs once in cell cycle
- begins at replication origins
- forks and replisome
DNA repair
- correction of mistakes in the hereditary material
- DNA damage vs. mutation
- sources and types of DNA damage
- DNA repair mech-BER, NER, MMR
- clinical relevance of DNA repair defects
degrees of genome instabilities
- base pair changes
- small scale insertions, deletions, or inversions
- large scale segmental duplications, translocations
- large scale repeat expansion- fragile X, huntingtons
- whole chromosome loss or gain- down, klinefelter’s syndrome (XXY), patau- trisomy 13, triple X
- cancer genome contains all of above changes (oncogene induced DNA damage)
DNA replication
- 5’-3’
- semiconservative (M & S- heavy and light DNA half and half)
- multiple ORCs-early and late
- leading goes 5’-3’, lagging needs Okazaki fragments (100-200 bp)
helicase
unzips DNA to expose templates for H bonding interactions with newly synthesized complementary strands
RPA (ssDNA binding protein)
-prevents exposed DNA strands from re-annealing
primase
RNA polymerase, lays down RNA primer
-leading and lagging strand (single vs multiple)
DNA pol E
- leading strand
- synthesizes DNA
DNA pol delta
-lagging strand
sliding clamp
- PCNA
- keels DNA pol on template
clamp loader
- RF-C
- keeps clamp on to keep pol on
RNAse
removes RNA primers
DNA ligase
closes gaps b/n Okazaki fragments
DNA pol and self-correcting
- 5’-3’ is polymerizing
- 3’-5’ is exonuclease
- both on palm part of pol
- 1 error per 10 million nucleotides
exonucleases
-chew in one direction- can be either
endonucleases
-cut like scissors within DNA- not involved in replication
error prevention
- DNA mismatch repair
- proteins scan newly replicated DNA for mismatches, removes is, and synthesizes a new strand
- makes mistake 1 in 10^9
mismatch repair mistakes
- seven genes encoding for mismatch repair proteins
- predisposition for cancer- hereditary non-polyposis colin cancer
- accumulate mutations rapidly
- mutator phenotype
- hMSH2 and hMLH1
error prone DNA pol
- damaged template needs low fidelity polymerases
- lesion avoidance vs translesion synthesis
- see picture
DNA pol alpha
-primase
DNA pol delta
3-5 exo
lagging
DNA pol epsilon
-3’-5’ exo
leading
family X and Y pols
-error prone
replication stress response
- forks vulnerable to DNA damage or nucleotide starvation
- ckpts deal with stress
- if they don’t work-ssDNA, reversed forks, ds breaks
replication stress response 2
- lesions activate ckpts that stabilize forks
- fork restart mediated by proteins (originally found in homologous recombination repair of ds breaks)
- DNA-PKcs, Ku, DNA ligase IV-XRCC4, Artemis, XLF and Metnase
- signaling or DNA end joining?
- cancer therapies create fork stress
- see picture
- revolves around stabilized fork
Ataxia Telangiectasia
- susceptible to lymphomas
- ataxia (abnormalities of balance)
- telangiectasias in skin and eyes
- chromosome abberations
- immune dysfunction
- mutations in ATM, protein kinase important for replication stress respons and regulates p53- accumulations
Bloom syndrome
- susceptible to carcinomas, leukemias, and lymphomas
- facial telangiectases, chromosome alterations
- sensitive to mild alkylating agents
- caused by mutation in BLM, RecQ helicase that functions in replication stress response
- can also get progeria
TDP1 mutation
- Tyrosyl DNA phosphodiesterase
- SCAN1- spinocerebellar ataxia with axonal neuropathy 1
APTX mutation
- nucleotide hydrolase/transferase
- Ataxia oculomotor apraxia 1 (AOA1)
DNA ligase I mutation
-immunodeficiency, defects in repair of DNA breaks induced by UV
DNA ligase IV mutation
-Lig4 syndrome (immunodeficiency, radiosensitivity, pancytopenia, developmental abnormalities, microcephaly
FEN1 mutation
- cancer susceptibility
- autoimmune problems (Okazakis accumulate and act as antigens
pre-replication complex mutation (ORC1, ORC4, ORC6, CDT1 CDC6)
-Meier-Gorlin syndrome- dwarfism
anti-cancer: replication inhibitors
- nucleotide analog chain terminators- AZT, ddC, ddl
- enzyme inhibitors- hydroxyurea (RNR_, camptothecin (topo I), 5-FU (thymidylate synthesis)
DNA damage
- spontaneous events
- exposure to environmental mutagens
- DNA replication errors or stress
DNA repair mechanisms:template independent damage-direct reversal
- pyrimidine dimers-photoreactivation by photolyase (t-t dimers, no longer functional in humans)
- alkylation- O-methylguanine methytransferase
ssDNA damage repair (one intact copy)
- base excision repair
- nucleotide excision repair
- mismatch repair
dsDNA break repair
- homologous recombination
- single strand annealing
- non-homologous end joining
BER, NER, MMR
- altered portion of DNA strand is recognized and removed by enzymes called DNA repair nucleases- hydrolyze phosphodiester bonds- small gap left behind
- DNA pol binds to the 3’-OH end of the cut DNA and fills in the gap by making a complementary copy of the information stored in the good strand
- break or nick left when DNA pol leaves is sealed by ligase
*first step differs by what enzyme does the removing
ds break repair
-HR and NHEJ
Homologous recombination
- dsDNA exonucleolytically processed to form 3’ ssDNA tails, which invade homologous intact sequences. DNA strand exchange generates a joint molecule b/n damaged and undamaged duplex DNAs
- sequence info that is missing is restored by DNA synthesis
- interlinked molecules processed by branch migration, holliday junction resolution and DNA ligation
single strand annealing
- at DSB, 3’ ssDNA tails (direct repeats) generated
- they are aligned and the intervening sequences as well as protruding 3’ ends are removed
NHEJ
- broken DNA ends are processed to yield appropriate substrates for direct ligation
- no homology necessary
- breaks can be joined accurately but sometimes some insertions or deletions are created
- preferred by humans
- DSB= Ku protein–cascade–look at picture
-xeroderma pigmentosum
- severe predisposition to skin cancers
- seven genetic complementation groups- XP-A to XP-G have been identified and associated with defective nucleotide excision repair
- loss of function in XPV also results in XP, but not associated with NER but defects in lesion bypass of UV induced damage
Nijmegen breakage syndrome
- gamma iradiation sensitivity
- DSB repair problem
- lymphomas, microcephaly, short stature, distinct facial appearance, immunodeficiency, radiation sensitivity
cockayne syndrome
- UV light sensitive
- NER problem
- dwarfism, retinal atrophy, photosensitivity, progeria, deafness, trisomy 10
fanconi anemia
- cross linking agent sensitivity
- FANCD1, 2 mutation
- leukemias, hypoplastic pancytopenia
breast and ovarian cancer
- ionizing radiation and other genotoxin sensitivity
- BRCA1 and 2
- hereditary breast and ovarian cancer
