Flashcards in 432 Lecture 25 Deck (27):
What are the three fundamental rules of replication?
1) Replication is semi-conservative 2) Replication begins at an origin and proceeds, bidirectionally 3) Synthesis of new DNA occurs in the 5' to 3' direction
1) Strand Separation 2) New complementary strands synthesized using each separated strand as a template.
Requirements for DNA polymerase Activity (4)
1) Template (Basis for heredity) 2) dNTPs (building blocks) 3) Mg2+ ( promotes reactions) 4) Primer ( anneals to template DNA and is extended w/o DNA pol. can't start).
What are the three functions of DNA pol I?
1) 5' to 3' DNA polymerizing activity 2) 5' to 3' exonuclease activity 3) 3' to 5' exonuclease activity.
Mechanism of DNA polymerases (Active site)
1) Two conserved regions of Asp 2) Mg2+ causes 3' OH to become a nucleophile and negates the charges of the pyrophosphate on the incoming nucleotide 3) O attacks phosphate (a phosphate) and pyrophosphate leaving drives reaction.
Mechanism of DNA polymerase
1) Sampling of nucleotides 2) nucleotide base pairs with complementary base 3) removal of H from 3' OH 4) nucleophillic attack of 3' OH to a phosphate group of incoming nucleophile 5) displacement of pyrophosphate drives the reaction.
1) b-sheet 2) primary elements of catalytic site 3) binds two Mg2+ ions 4) Monitors accuracy.
1) bind to incoming dNTP 2) Fold over if correct base pair is made. (Lys, arg, and tyr rich) the tyrosine through Van der walls interactions places incoming base to growing chain.
How many active sites does DNA polymerase have?
one active site. Useful for ribonucleotide discrimination and nucleotide discrimination
How does DNA polymerase know an incorrect base pair has been made?
Distortions in the enzyme's active site. Enzyme can add up to five bases before they can no longer fix the mistake. Additions are made at a slower rate.
Net reaction of DNA polymerase
Based on the reaction between the separate sites of the active site of addition and the 3' to 5' exonuclease. Rate of nucleotide addition dominates the net reaction.
5' to 3' exonuclease activity; DNA repair and primer removal during replication; cleaves nucleotides in front of others; removes RNA primer
Which polymerase has 5' to 3' exonuclease activity?
DNA polymerase I
For DNA polymerase where and how do you start?
DNA-ATP binds to origins with help of helicase and SSB
For DNA polymerase, how do you get a single stranded template?
For DNA polymerase, how are instabilities in the SS template strand overcome?
Single stranded binding protein; RPA (replication protein A in eukaryotes)
Functions to form a primer
For DNA polymerase, how is the lack of a 3' to 5' polymerase overcome?
For DNA polymerase, how is DNA polymerases speed and processivity issues overcome
SSB and sliding clamp
Where does replication in E.Coli occur?
At OriC. DnaA recognizes OriC; promotes the loading of DnaB (helicase) by DnaC (helicase loader)
helicase loader ATP dependent
ATP dependent, to really get replication going; binds to DNA, causes positive super coiling, opens up DUE region, causes ssDNA formation. Compensating negative supercoils melt the replication bubble at the end
recruited by B clamp desposition; promotes ATP hydrolysis and progressively dissasembles DnaA
During replication, 1 strand is methylated and another strand is not methylated (The new strand is NOT methylated)
Binds to OriC, only when DNA is hemimethylated, (new DNA is NOT methylated) tethers DNA to membrane of a cell, which does not allow DnaA to interact with OriC