Unit 2 - Cell Cycle & DNA Repair Flashcards
What do DNA Polymerases require for activity?
3’-OH group so need a primer they can extend (5’->3’)
What is the overview of DNA replication?
- DNA unwound +RNA primer molecules bound are synthesized by Primase
- primers extended by replication polymerases (δ or ε)in 5’->3’
- lagging strand is discontinuously synthesized
- also leads to ‘end replication problem’
What solves the ‘end replication problem’?
Telomerase (ensures intact replication of DNA ends) (needed because of primer)
What are the steps in DNA synthesis (lagging strand)?
- Helicase unwinds DNA, RPA loads
- Pol α-primase synthesizes a short primer
- Pol α displaced + Pol ε or δ loaded
- Pol ε or δ extend the primer
- downstream primer is removed (nuclease)
- okazaki fragments are ligated = continuous strand of new DNA
What happens during Prophase of Mitosis?
Chr. Condense
Centrosomes separate
Histones modified
What happens during Prometaphase of Mitosis?
Microtubles + chr’s kinetochores
Nuclear envelope breakdown
What happens during Metaphase of Mitosis?
Chrs align @ metaphase plate
What happens during Anaphase of Mitosis?
Chromatin separate & move -> opp. Spindle poles
Nuclear envelope reassembly
What happens during Telophase of Mitosis?
Nuclear envelope reassembles
Poleward movement
Cleavage plane
What happens during Cytokinesis of Mitosis?
Separation
Clevage furrow 2/ actin filaments ring
Chrs. Decondense
Nuclear structures reform
What are the key regulatory steps of the cell cycle?
Making sure DNA replicates completely + only once (S Phase control)
Making sure that DNA is intact before mitosis begins (G2 phase delays)
Making sure all chrs. Are segregated equally (spindle checkpt.)
What is involved during S Phase control?
Prereplicative complex + origins of replication (licensing) during late M/G1
Licensed origins of replication are bound by initiator proteins
What detects DNA lesions in G2 Phase control?
Checkpt. Kinase proteins (ATR, ATM) =signal -> effector kinases (ChK1, ChK2)
What does the centrosomes Pericentriolar Material contain?
γ-tubulin ring complex that nucleates microtubles
How are centrosomes duplicated? The
G1 = disorientation of centrioles
S= procentriole formation
G2= elongation & maturation
M= separation & activation
What separates the spindle poles?
Interpolar microtuble motors
not attached to centrosomes
What helps establish the overall cytoskeleton structure?
Astral microtubles
What is required for satisfaction of the Spindle Assembly Checkpt.?
Both occupancy of kinetochores by microtubles & inter-kinetochore tension
=>anaphase
What are the key regulators of the cell cycle?
Cyclin dependent kinases (CDKs)
What are CDKs controlled by?
Cyclin levels
What determines the directionality of cell cycle?
Transcriptional regulators
How do cycling & CDKs combine?
G1/S cyclin
- CDK4, CDK6 => cyclin D1, 2, 3
- CDK2 => cyclin E
S cyclin
- CDK1 => cyclin A
- CDK2 => cyclin E & A
M cyclin
-CDK1 => cyclin B
What are the key target sites in regulated activation of CDK1?
Phosphorylation sites
- inhibitory sites = T14 Y15
- activating sites= T161
What will the inhibit activity of CDK1?
Wee1/Myt1 phosphorylate inhibitory sites T14 Y15
What will activate activity of CDK1?
Cdc25 dephosphorylates activating site T161
CAK phosphorylates activating site T161
What are the targets in cell cycle in cancer therapy?
M= Antimitotics (target microtubles- vincristine, vinblastine, taxol)
G1->S= Antimetabolites (block nucleic acid synthesis)
S & G2 = (DNA damage) alkylating agents/Pt drugs (radiotherapy type II inhibition)
What mutation which changes gene fnx. Is found in 20% of tumors?
Activating pt. Mutation in RAS
=prevents hydrolysis of GTP on RAS = RAS accumulates in GTP bound active form
How is Base Damage repaired?
Base excision repair
How is incorporation of mismatched base repaired?
Mismatch repair
How is the formation of UV photoproduct: 6-4 thymidine dimmer repaired?
Nucleotide excision repair
How is DSB repaired?
Homologous recombo repair
Non-homologous end joining
What are the causes of DNA damage that can be repaired by base excision?
Gen. Of abasic sites through hydrolytic clevage
Deamination
Ox. Damage
Alkylation damage
What recognizes & removes the damaged base in BER?
DNA glycosylase
=abasic site
What cleaves the DNA @ the AP site in BER?
AP endonuclease
What carries out repair synthesis in BER?
DNA polymerase
What rejoins the sugar-phosphate backbone in BER?
DNA ligase
What recognizes DNA distortion from damaged bases in NER?
Complex containing XPA & XPC
What can the stalled RNA polymerase act as in transcribed DNA?
Recognition signal
What unwinds the DNA helix in NER?
XPB/XPD
What nicks the DNA 5’ & 3’ of the lesion in NER?
XPF/Erccl
& XPG removes it
What synthesizes the excised sequence in NER?
DNA polymerase δ or ε
What seals the nicks in NER?
DNA ligase
=restoration of integrity of DNA sequence
What is required to correct these DNA copying errors?
Mismatch repair (MMR)
What detects the error in MMR?
Mismatch recognition proteins (MSH2/MSH6)
What happens to the DNA until the mismatch in MMR?
Exonucleotically degraded (remove a lot of sequence & mismatch)
What performs the repair synthesis in MMR?
DNA polymerase δ or ε
What seals the nicks in MMR?
DNA ligase
What does defective MMR cause?
Micro satellite instability
What is an eg. of defective MMR’s link to cancer?
Hereditary non-polyposis colon cancer (mutation in MSH2 & MLH1)
What is the telomere?
Special protective nucleoprotein complex @ the ends of linear chrs. to ensure that they are not treated as broken ends
What does the rapid repair of SSB in DNA require?
poly (ADP-ribose) polymerase (PARP)
What initiates repair by non-homologous end joining?
Breaks bond by Ku70/Ku80 dimer = recruit kinase = activate repair
What religates the broken ends in non-homologous end joining?
DNA ligase IV
or
XRCC4
When is non-homologous end joining repair of DSB critical?
In V(D)J recombo during immune system dev.
What initiates homologous recombo repair of DBS?
Resection @ break to expose a tract of ssDNA
What carries out a homology search in homologous recombo repair?
Rad51 recombinase forms a nucleoprotein filament on ssDNA =searches
What allows templates DNA synthesis in homologous recombo repair occur?
Strand invasion
=holliday jnx.
What happens to the resulting Holliday jnx. In homologous recombo repair?
Cleaved to = repaired sequence
What is a key component of homologous recombo repair?
BRCA2 (modulates Rad51)
What repairs spontaneous breaks that occur during normal DNA replication?
Homologous repair & Parp I- dependent ssb repair
What repairs do BRCA2-defective cells use?
Rely only on Parp I
