Chromosome biology lecture 2 Flashcards
Defining proteins binding to ARS
Experiment (yeast replication origin in absence of protein, expose to DNAse1 → hypersensitive site where DNA slightly bent, footprint where DNA sat on DNA (x show fragment of DNA)
Mutate different regions of origin, mutated A = no protein footprint, mutate B1
ORC
- 6 subunits, binds ACS
- Subunits are ↑ conserved
- Needs ATP to bind DNA
ORC structure
- Winged helix domain, DNA binding HTH, B sheet wing, AAA+ ATPase
ATP binding + hydrolysis by ORC
- ↑ ATP binding to complex in presence of origin DNA
- Mutate different subunits e.g. ORC5 mutant x bind ATP
- ORC binds ATP in presence of ARS, endogenous ATPase activity inhibited by ARS
ORC binding to ARS
- DNAse I footprinting
- Mapped binding of individual subunits of ORC to ARS DNA
- ORC binds centrally within ARS consensus sequence
- ORC binds nucleosome deleted region
- ORC binding distorts ARS DNA helical axis by 35o
S pome replication origin
- S pome ARS cloned w/ shotgun method
- Unlike S cerevisiae, x have ARS consensus sequence
- Also ATP rich, clusters of AT regions
- Also promoter but x need transcription initiation for firing
- Longer
- Specific origins of replication but x contain specific sequences
How do ORC recognise origins of replication in S pome
- Recognise certain motifs
- Also hexameric recognised complex (4 in Pombe, bind specifically to origin via NTD)
- Orp4 has 9 repeats of AT hook DBD
- Spacing important to allow Orp to interact w/ DNA
- Quasi-random distribution
Metazoan origin
- Lack consensus origin sequence
- No sequence specificity requirement for replication of exogenous DNA
- Indicates low origin specificity in early embryos
- Human = shotgun plasmid 2D mapping
Origin plasticity related to transcription
- E.g. mammalian DHFR gene can amplify ↑ times by selecting methotrexate
- B globin is ↑ transcribed, v repetitive loci, replicate early
- non-B cells replicated passively through passage through a fork initiated at a ds origin
- Pre-B cells that are transcripting at that locus replicate entire locus early
- Xenopus have temporal developmental pattern
- In early embryo x transcription
Mammalian origins are found mainly in promoters
- 46% replication origins in Ch3
- Generally promoters have ↑ controlled chromatin organisation, open + accessible
Metazoan origins
- Can identify by sequence or individual origins
- Chromatin state = important
- Histone acetylation can promote initiation
- ORC can be recruited at distinct sites but ORC-interacting factors
- MCM coats chromatin
- Chromatin structure may contain DNA loop, inter-origin distance
- Replication factories
Replicon model
- Initiator ORC binds cis acting replicator
- If take all yeast + look at ORC + MCM bs = ↑ sites
- ↑ replication origins
- e.g. S cerevisiae has 2000kb stretch, 14 Ars, only 5 act as OBR so context important
Why replicate once
- If fails to prevent, get 4 copies of genome not 2
- Oncogenes can be over-copied
How to regulate replication = only once
- Experiment w/ hola cells at diff stage of cell cycle, G2 nucleus x replicate
- Experiment 2 w/ Xenopus cell extract, analysed DNA On CsCl gradient, DNA undergo 1 round of replication (1 heavy, 1 light chain)
Replication licensing factor model
- Gained access to nuclei only when envelope breaks down
- Once binds chromatin = stable
- So, in G1 nuclei have this factor bound to chromosomes that entered during M
- Licenses DNA for replication
- Factor destroyed by DNA replication in S phase
- In G2, x active licensing factor
