Chromosome biology Extra Flashcards
Prokaryote DNA replication regulation
- Replicate as fast as possible
- Circular, single origin of replication
- 9 bp where DnaA binds, DnaB/C then bind w/ PolIII
- Key regulation = before helicase loads
Prokaryotic helicase loading control
- Levels + activity of DnaA
- Inactivation of DnaA (RIDA) (complex of clamp, ADP-Hda promotes hydrolysis of DnaA bound ATP → inactive DnaA) - DnaA binding to origin
- DatA locus (by OriC, hierarchy of affinity for DnaA, datA has highest binding (8x more), as is replicated ↓ [DnaA}
- OriC methylation (DNA transiently hemimethyl, SeqA binds + sequesters OriC, DnaA promotes initiation of ‘old’ methylated origins - Conformation of DNA (DnaA binding to datA stimulated by IHF + datA-IHF stimulates hydrolysis of DnaA (DDAH), DDAH is reg by supercoiling
Prokaryotic DNA replication termination
- Occurs at Ter sites, trap 2 forks to meet each other
- Have polarity so stop replication in 1 direction
- Coord removal of 1 fork or 2 complexes (allow another Pol to be recruited)
Eukaryotic DNA replication regulation
- DNA linear molecules, ↑ in size, need to be coordinated, early, late + dormant origins
Eukaryotic DNA replication origin firing
- loading helicase in inactive state
(ORC binds origin, Cdt1 recruited + cdc6 then MCM2-7, regulated by CDK, regulated in after to prevent re-replication) - Origin firing
(S phase, DDK recogn. NTD of MCM + phosph → bs for Sld3, Cdc45 binds, Dbp11 recruits GINS + polE, regulated) - Regulating epigenetic code
(Recycling or de novo assembly, histone chaperones e.g. CAF-1 by pCNA, 1/2 normal no., histone PTM diluted = regulatory marker, HTA1-H3/H4, HIR represses histone gene expression)
Eukaryotic DNA replication termination
- Thought forks converge
- Converging CMGs bypass each other + translocate until reach Okazaki fragment
- Leading strand extends to Okazaki fragment + processed by recruitment of Pol gamma
- Removal of CMG
Recognition of DNA damage
MMR
( base mismatches/smaller insertions/deletions, identify template strand, prokaryotes = hemimethyl (MutH nicks unmeth strand, eukaryotes = PCNA (RFC loads w/ defined orientation, when MutLa interacts in this orientation ensures cleavage = new strand, HMSH2/6 heterodimer, bends to find mismatch)
NER
(detects bulky lesions, GGR vs TCR, XPC recog, + binds strand opposite, Rad4 = recognised through DBD at ss DNA)
BER
( recognises chemical features of DNA bases, DNA glycosylases, specific, bind minor groove, kink DNA + flip bases out, mono functional vs bifunctional)
Verification of DNA damage
MMR
(overlaying 2 crystal structures → MutS binds mismatch w/ Phe-X-Glu, heteroduplex kinked → URC (unbent), The disrupts base, Glu recogn misfired bases
NER
(TFIIH = key, XPB opens helix + anchors proteins, XPD has iron-sulfur cluster, forms tunnel through ssDNA fits through)
BER
(bases fit into glycosylase e.g. uracil glycosylase, A+G too big, T has bulky C5 methyl)
DNA damage removal
MMR
(coordinate nicked DNA to mutS, MutS similar to Smc thought extrude DNA through loop, after MutH activated, helicase (UvrD) unwinds, MutLa nicks DNA in euk, exo1)
NER
(2 incisions made, XPG 3’ end, XPF 5’ after ERCC1-XPF joins)
BER
(short vs long-patch BER, mono functional vs bifunctional glycosylases)
Re-synthesis
- Relies of DNA pol, RFC, PCNA
- Pol uses undamaged ssDNA as template
Repair of ds break overview
- DSB arise from sources like ROS, when DNA replication fork encounters DNA ss break
- Danger of DSB:
- If x detect, damaged cells x die + → progeny that have genomic instability
- DNA breaks → chromosome breakage 0> chromosome fragments unequally distributed → genomic heterogeneity
- Chromsome translocation (promoter of other gene)
- Telomerase makes telomere from DSB
Repair of ds break
NHEJ
- G1 where x homologous chromosome
- DSB recog. by Ku70/80, stops floating away, DNA-PKC recruited, Artemis phosph (exonuc), joined by PolX + ligase IV
- ‘Microhomology’ (just XRCC4 + ligase)
- Insertions, deletions + erroneous joining of DSBs → translocation, loss of chromosome material
Repair of ds break
HR
- Uses homologous sequences
- Broken ends resected (MRX/MRN then exo1, RPA coats ssDNA, Rad51 replaces + invades, min 8 bp, D loop, capture 2nd DSB → 2HJ, RuvC resolvase, dissolution)
SSA
(resection reveals compl. strands that recomb like NHEJ, ERCC1 assoc w/ XPF removes sequences btw direct repeats)
SDSA
(Invading strand displaced + anneals w/ 2nd resected DSB, x HJ formed)
BIR
(during S phase at telomeres or broken forks, 3’ end of D loop extended, x have 2nd end to anneal w/ extended invading strand)
Repair of ds break
HR vs NHEJ
- HR ↑ efficient, NHEJ always on
- Resection = 1st major difference, Ct1p interacts w/ MRN, activated by cdc28 during S phase
Sister chromatid?
(epigenetic code btw new vs old, H4K20 methyl, H2AK15 recruits machinery to look for H4K20, TONSL-MMS22L reads H4KM20 in HR via ARD)
Is ds DNA most dangerous damage?
- Purposefully used in VDJ recomb + meiosis
- Controlled e.g. meiotic hotspot
- 50,000 ss dna a day, mismatches likely to go un-noticed
