Lec5-7 Flashcards
what was Arthur Kornberg’s goal
study DNA replication in vitro
describe Kornberg’s experiment steps
- develop an in vitro assay for DNA synthesis
- grind up e.coli cells to get cell extract containing all cellular proteins (in theory DNA pol.)
- add radioactive thymine (C14), all 4 dNTPs, and DNA
- incubate to allow DNA synthesis
- add perchloric acid to stop rxn and precipitate DNA, found some radioactive nt in the pellet therefore successful in vitro synthesized DNA
- add DNase enzyme to break up DNA and radioactive nt, precipitated again, and found DNA in supernatant, realized something in crude cell extract is synthesizing DNA - purify DNA synthesizing “activity” from crude protein fraction
- separate crude protein into fractions and test each one for DNA synthesizing ability
how did Kornberg discover DNA synthesis is 5’ to 3’
- he started with DNA, all 4 dNTPs and radioactive thymine again
- allowed the rxn to go in vitro without the radioactive nt then he added it briefly (pulse)
- then continued with the addition of perchloric acid and a pulse of 3’-5’ exonuclease
- discovered radioactivity is lost from DNA, becomes acid soluble
-added pulse of 5’-3’ exonuclease and discovered radioactivity is retained on DNA - therefore nts are added to 3’ end of DNA
how did Kornberg prove DNA involves the release of pyrophosphate from dNTPs
- he started with a template, primer, all 4 dNTPs, and radioactive carbon
- shot gamma rays at terminal phosphate group
- saw radioactive carbon goes into precipitate (incorporated in DNA) but radioactive phosphate goes in supernatant
- therefore phosphate gets removed
what would occur if DNA synthesis was random
radioactivity would be found in both pellet and supernatant for both exonuclease treatments
what direction is continuous DNA synthesis
5’-3’
what direction is discontinuous DNA synthesis
5’-3’
what experiment provided evidence for lagging strand synthesis
- a pulse of radioactive thymidine was used to label DNA in vivo with E.coli
- alkali denaturation to separate the strands
- sucrose gradient separated DNA according to size
- measured radioactivity in each fraction
- when pulsed shortly (5-10secs), almost all radioactivity was found at the top (small pieces of ssDNA) but when pulsed longer (60 sec), they found some radioactivity in bigger pieces as ligase eventually joined fragments together but they didn’t know this
how did they discover ligase joins fragments?
used temp. sensitive e.coli mutants for ligase gene and found e.coli raised in permissive temp, their ligase worked but when temp was raised (restrictive), ligase denatured and they found lots of radioactivity in small DNA pieces due to ligase not able to join them in longer pulse
describe lagging strand synthesis
- DNA primase synthesizes a short RNA primer on the lagging strand as it’s exposed
- DNA pol extends until it meets the older primer
- old RNA primer is replaced by DNA
- ligase joins the Okazaki fragments on the growing chain
how did Okazaki discover DNA synthesis on the lagging strand requires RNA
- added RNase to the rxn which degrades the primer so no synthesis of the lagging strand and only saw radioactivity in fractions corresponding to large DNA pieces
- added radioactive uridine, which is only incorporated in RNA, instead of H3-dT and saw radioactivity corresponding to small DNA pieces
DNA replication machinery
- Mcm helicase: opens up double helix at replication fork by denaturing DNA as it moves
- RPA: stabilizes ssDNA on the lagging strand to prevent ssDNA tendency to make hairpins by complementarity
- PCNA: encircles DNA to keep DNA pol from sliding off by forming a ring around DNA, ATP binding to clamp loader opens PCNA which binds to DNA duplex and ATP hydrolysis locks PCNA around DNA and releases the clamp loader
- DNA polymerase, binds to PCNA
DNA replication mechanism
- clamp loader binds to PCNA to open PCNA ring
- PCNA ring encircles DNA/RNA hybrid
- primase adds primers to ssDNA
- PCNA-clamp loader moves along DNA util it finds 3’ end
- PCNA closes around DNA
- PCNA binds to DNA pol
- DNA pol now stably associated with DNA
what are the 3 mechanisms that ensure DNA replication accuracy?
- bp precedes covalent attachment of nt
- 3’-5’ exonuclease activity, if the wrong nt is incorporated into the primer strand, it changes the configuration at the replication fork and DNA pol stalls, chews back the wrong nt on 3’OH of primer strand and resumes to add the correct nt
- mismatch repair, MSH2, and MLH1 scan newly synthesized DNA, detect mismatches, make nicks on the new DNA strand, the strand is removed by DNA pol I, the large chunk of ssDNA left is used for repairing and families with mutations in these genes have high cancer incidence
true or false: polymerase and exonuclease activity are found in different parts of DNA pol
true
what bacteriophage first identified exonuclease function
T7, mutations in this DNA pol produce a mutator phenotype
how many replication origins do proks and euks have
one, many