Lecture 5 - Chromosomal Replication Flashcards
What are the 3 Phases of Chromosomal DNA Replication
- Initiation/Priming - to assemble replisome
- Elongation - to faithfully replicate chromosome
- Termination - to stop replication and disassemble the replisome
Define DnaA in terms of:
(i) Function
(ii) Binding Sites
(iii) Nucleotide Binding
(5 Points)
(i) Master Replication Initiator protein, that orchestrates the initiation of replication
(ii) Binds to specific 9bp known as DnaA Boxes (R-sites) and I-sites
(iii) DnaA is a ATPase, with its nucleotide binding status influencing its association with sites
* ATP - can bind to low affinity sites (e.g., ATP-DnaA boxes)
* ADP - can bind to high affinity sites only
What is the DUE?
DNA Unwinding Element (DUE) is a region adjacent to R and I sites, which is rich in A/T residues to facilitate DNA unwinding at replication initiation
Is oriC (Chromosomal Origin) Architecture consistent across bacterial species?
Different across bacteria, with in some cases additional nucleoid-associated proteins (NAPs) like IHF binding to the oriC to co-operatively assist DnaA to remodel DNA
How does DnaA-binding change throughout the cell cycle?
- Cell cycle - DnaA is bound to several high affinity sites
- Initiation - additional ATP-DnaA molecules bind to low affinity sites, oligomerising into largo nucleoprotein complex that remodels DNA to facilitate DUE melting
What happens to the DUE after melting?
- Helicase DnaB (in complex with DnaC) is loaded onto exposed ssDNA strands via an ATP-driven reaction
- Energy - required to open helicase ring, allowing threading of DNA through central hole before closing of ring (Traps DNA)
Describe the multi-domain architecture of DnaA
(4 Points)
- N-Terminal Domain (I) - mediates interaction with DnaB, and promotes self-association
- Domain II - linker connecting Domains I and III
- Domain III - has AAA ATPase activity, and main oligomerisation activity
- Domain IV - DNA-binding subdomain
How does Domain IV recognise and bind to the DNA?
(2 Points)
- HTH element - inserts helix into major groove of DNA
- Basic Loop + R399 - critical for binding adjacent minor groove of DNA
Describe the Mechanism of ATP-dependent DnaA Oligomerisation
- ADP-bound (Closed Conformation) - lid subdomain of DnaA monomer prevents binding of second monomer due to steric hindrance from Arg in Sensor II
- ATP-bound (Open Conformation) - Arg in Sensor II is engaged by y-phosphate of ATP, repositioning lid subdomain and allowing Arginine in box VII of other monomer to dock into active site
How does DnaA indirectly induce melting of DUE?
- ATP-DnaA oligomerises into RH helical filaments, with DNA wrapped around outside to form positive toroidal wrap
- Due to Chromosome being closed topological system, positive toroidal wrap introduces compensatory -ve supercoiling, facilitating DNA melting within nearby DUE
How can DnaA directly induce melting of the DUE?
DnaA/DNA complex produced in indirect melting serves as a nucleation center, allowing DnaA protomers to engage and melt the DUE directly via AAA+ domains
* i.e. Filament extends into opening DUE, causing further DNA melting
In what two ways can DnaA bind to the DNA?
- State 1 - uses Domain IV dsDNA-binding activity
- State 2 - uses ssDNA-binding activity of Domain III (AAA ATPase), but also N-terminal Domain
(i) How do the Secondary DNA-binding sites recognise and bind to the DNA?
(ii) How does this induce DUE melting?
(i) ssDNA binding occurs via four helices (a3,a4,a5,a6) that form a continuous staircase lining the inside of the filament, with each AAA+ domain binding 3 nucleotides
(ii) Each AAA+ domain is separated by 10Å gap, causing the ssDNA to extend relative to regular B-form DNA (42Å to 65Å). This stretching helps to melt the DUE
Describe the Mechanism of Origin Sequestration, explaining how it regulates Origin Firing
(2 Points)
- 11 GATC sites are interspersed through oriC, which are the target sites for Dam Methylase (A = methylated)
- However, Immediatly after passage of replication forks these sites a transiently hemimethylated, which allows protein SeqA to bind and prevent DnaA binding
Describe the process of Regulatory Inactivation of DnaA (RIDA)
- Sliding clamp and protein known as Homologous to DnaA (Hda) are involved in sequentially stimulating ATP-hydrolysis of DnaA monomers after DUE is melted
- This occurs via the Arg330 of Hda, which inserts into active site of 1st monomer, stimulating ATP hydrolysis, which will then trigger ATP hydrolysis in 2nd monomer etc.
