MBB 267 Week 4: Barnes 7 Flashcards
Compare origin of replication in humans and E. coli.
A single origin of replication in the E. coli genome, vs
tens of thousands of origins of replication in the human genome
a. They all have bidirectional replication forks in common
What are the early stages of eukaryotic application?
Binding of Origin Recognition Complex
a. Assembly of pre- replication complex = MCM proteins, CDC6, etc
b. Initiation of replication
What is an Autonomously replicating sequence?
A sequence that contains the origin of replication in yeast.
How does Autonomously replicating sequence work in S. cerevisiae?
So:
a. There are 4 domains that are seen ; B3, B2, B1 and A
i. B1 has the 11-bp “autonomous consensus sequence”
b. 250-400 replication origins in each round of replication
c. Only some of the hundreds of ARS consensus sequences actually initiate replication: essential but not sufficient for origin activity
d. Transcriptionally silent areas are more likely to be bound by replication proteins
How does Autonomously replicating sequence work in S. pombe?
AT-rich intergenic regions – at least half of intergenic regions have the capacity to serve as origins of replication
How to find origin of replication, for simple origins?
eg, ARS
a. Clone pieces of S. cerevisiae genome into recombinant plasmids to test whether they can guide replication.
b. Test many different sequences to find the ones that allow plasmids to be replicated
c. Grow the cell under selective conditions and read sequnces in survived cells
What is immunoprecipition?
Immunoprecipitation is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein. This process can be used to isolate and concentrate a particular protein from a sample containing many thousands of different proteins.
How to find origin of replication, for Animals?
In animals – eg, studies on nascent strand abundance
a. Can isolate newly synthesised DNA using BrdU
i. BrdU is an analogue of thymidine; can be integrated instead of T during replication. Can be immunoprecipitated.
b. Add BrdU to medium instead of thymidine. This allows you to pull down nascent DNA.
i. Then – microarrays or high-throughput sequencing to identify which parts of the genome those are
What features seen in animal replication origins?
No consensus sequence found however common features include:
a. AT-rich DUE
b. CpG islands
c. DNA topology
d. Loop MAR
e. Nucleosome
f. DNase I-sensitive site
Different types of flexibility of different origins of replication?
Most origins are not used in any given cell cycle Some origins are only used under specific conditions
a. eg following DNA damage
i. Constitutive: used always – a minority of origins
ii. Inactive: never used under normal conditions – can be used eg in stressful conditions
iii. Flexible: used sometimes – stochastically )
Origins of replications can be fired at different times, why is this important?
Different replication origins fire at different stages of S phase
a. Late origins tend to be more “sequence- dependent”
