Rodgers 2-5 to 2-10 Flashcards
Where does energy come from to produce the energetically unfavorable addition of complements to a growing DNA strand
Hydrolysis of PPi from incoming NTPs
How large is E. Coli’s genome
~4.6 million b.p.’s
What 2 characteristics define the OriC region of E. coli
~3 A-T-rich-13bp tandem repeat segments, and ~5 9bp DnaA boxes
How does DnaA affect the DNA Lk?
Positively supercoils (increases Lk)
What are the domains of DnaA?
Helix-turn-helix motif, ATPase domain
How does DnaA interact with DNA?
One helix provides specificity by sitting in the major groove, One helix provides affinity by interacting with the neg. charge backbone
What 2 characteristics define yeast replication origin sites
Autonomously Replicating Sequences (ARS) Consensus Sequences (ACS), B-elements (containing DNA Unwinding Elements- DUEs)
What is the eukaryotic equivalent of DnaA?
ORC
How is ORC different from DnaA
Made of 6 different proteins
How do chromosomes appear during replication
Stretched out
How many replicons in human genome
~50,000
What are groups of clustered replicons called
foci
How are fission yeast origin sites different from budding yeast?
They have Origin Binding Recognition Sites (OBRs) that are very large and vary variable
What is the function of Cdc6 and Cdt1in yeast
They work with ORC to recruit MCM’s (mini-chromosome maintenance helicases)
Why are some replisomes not activated in Eukaryotes
presence of CpG islands, methylation, or other transcription-regulating machinery - sometimes this is an environmental effect
What is the main difference between MCM and DnaB helicase
MCM goes around double stranded DNA, while DnaB is toroid hexamer around one strand
What are the 2 distinct domains are found in Orc1 of ORC?
winged helix (identifies bp’s in the major groove) , and AAA+ ATPase (interacts with minor groove)
Why is DNA deformed from its original bDNA helix when bound by DNA recognizing proteins
Because the protein creates an induced-fit conformation on the DNA (more energetically conducive to binding)
What shape does the ORC make on DNA? What probable purpose is there for this?
a ‘kink,’ perhaps to help load the helicase
What is the ‘processivity number’
the number of base pairs a polymerase can traverse before falling off
What loads the helicase to the DNA
an ATPase, like ORC or DnaA
What increases processivity for polymerases
the bonding interaction with a clamp (helicase)
What 2 things comprises the checklist for what must occur before replication starts
- Cell must be large enough 2. Must not have just opened the DNA for replication (DnaA-ATP:DnaA-ADP ratio of 1)
What 3 things prevent re-initiating replication in E. Coli
- SeqA binds at OriC, 2. DatA locus soaks up free DnaA proteins near the OriC, 3. Less DnaA-ATP is available
What is the action of SeqA in E. coli
SeqA binds hemi-Me GATC i(Dam sites) n the OriC to prevent DnaA binding
Which is more abundant in the cell: ATP or ADP?
ATP
Why is cell size important for regulating initiation of replication in E. coli?
faster growth means faster protein production, means quicker resumption of 1:1 ratio of DnaA-ATP/ADP - may re-initiate replication faster
Who was the first to characterize Ploymerases of E.coli?
Arthur Kornberg
What is the most abundant , most well-studied polymerase in E. coli?
Pol. I
Which polymerase of E. coli does the most work? Why?
Pol. III- it is the most processsive
Where are polymerases’ active sites located on the complex?
In the ‘palm’ domain
What do the fingers and thumb domain do in Polymerase?
Interact with the backbone to generate affinity to the DNA
Which polymerase does most of the work in replication for Eukaryotes?
Pol. alpha
What are the features of all polymerase active sites?
central beta sheets, flanking helices, and catalytic aspartate residues
What is special about aspartate? What function does it have?
Neg charge. It coordinates divalent ions (Mg) bonding with DNA backbone
What 3 things do the divalent metal ions do during polymerization?
- Encourage the ionized (deprotonated) form of the 3’OH to make it act as a better nucleophile 2. Coordinates the in-line exit of the PPi leaving group (LG) 3. Stabilizes the charge of the PPi LG
What makes the core of Pol. III in E. coli?
Three enzymes (alpha, epsilon, theta)
What process makes the Pol III holoenzyme processive
the atp-bound gamma complex attracts the beta clamp subunit (forces open conformation when binds), then when DNA enters the clamp, hydrolysis is stimulated in gamma which closes beta. gamma and beta lose affinity for one another.
