MCM helicases

jambomber's version from 2015-05-07 15:19

Section 1

Question Answer
What are the two steps of eukarotic rep initiation?Liscencing and initation- seperation ensures once and only once
What is the liscencing reactionFormation of the pre replicative complex
Who prepared PreRC in vitro? What did they show?Remus et al 1) Showed that passively slide without unwinding and ATP required for loading process, Cdc6+ ORC involved 2) Double hexamers in head to heead config are loaded
What did Remus et al do? How did they show passively sliding?If passively sliding, MCMs would be topologically linked to DNA, Showed that half life of MCMs on circular DNA templates is greater than linear 1kb templates 60 mins a/o 10 mins
How was it shown that ATP is required for loading?ADP-y-S incubation, In the absence of ATP MCM sub units are removed by a low strength salt wash
What is the domain structure of MCMs?CTD= AAA+ ATPase domain, N term domains are distinct. Forms a two lobed structure ( c term outside)
What occurs during the transistion from inactive to active helicase?Melting, Ring opening in absence of cdc6 and ejection of the non engaged strand
What systen did Remus et al conduct exp in?EM in S.cerevisae protiens
Where do Rep forks stall in the Fu and Walter exp? What exp system?-40 from the site of Cross linking. Xenopus extracts w cross linked plasmids
Why is MCM inactive in PreRC?1) Wrong Substrate 2) Forms a holohelicase requ
What is the evidence that a holohelicase is required for unwinding?A perfect substrate with Radiolabelled oligonucleotide tail, Can be displaced by melting to biol. Cannot by purified MCMs
How do CD45 and GINS activate the helicase?Bridge the gap between 2-5 , Forming a topologically closed assembly
What structures do MCMS adopt?lock-washer-shaped spiral state and a planar, gapped-ring form—in which Mcm2 and Mcm5 flank a breach in the helicase perimeter
How does DNA replication inhibit unfired orgins?Passive replication destroys PreRC activity by pushing along

Section 2

Question Answer
What evidence suggests that SLd2+3 are minimal substrates for Cdk for rep?Synchronised cells in G1with a mating factor and release in high levels of Sic1( under control of Gal). In double mutants rerep occurs
What elements of CMG are bound by BRCT sub unitsCdc45 and MCMS -by Sld2 GINs by Sld 3 and Pol E by Dpb11
What does DDK target in human cells?Treselin + RecQ4?
How is labour divided between DDK and CDK?DDK activates the replicative helicase, CDK recruits other components of replicsome
What is the dual role of CDK in cell cycle?Inhibition of Liscencing and Origin activation
What occurs at low CDKPreRC assembly + origin firing prohibited
At high CDK?Origin firing activated and PreRC assembly prohibited
Who demonstrated the dual role of CDK? How?Paul Nurse 1991 Fission yeast FACS experiments
What experiment did Nurse et al conduct?Nocodozole to prevent cell division- arrest at G2/M phase boundry (2C).. Transient pulse of cdk inhibition with Gal Sic. Off- observe perfect round of re rep