DNA Replication Flashcards
Meselson and Stahl (1958)
E Coli cells were grown in medium with heavy nitrogen 15 as the only source of nitrogen for many generations
Cells transferred to medium containing nitrogen 14. After cells divided, sample was collected and the DNA purified
Cells divided second time, sample collected and DNA purified
Centrifuge the three samples.and compare the location of the bands
Differ models of DNA replication
Semiconservative- parental strands go to one parental strand and one daughter strand
Conservative- one helix full parental, one full daughter
Dispersive- a mix of both
Results of meleson and stahl’s experiment
Fist gen- heavy band
Second gen- heavy and light
Third gen- mostly light
BrdU
Combo of fluorescent dye and giesma stain distinguishes between chromosomes with one strand containing BrdU and those with two stands of BrdU
DNA replication
Catalyzed by DNA polymerases, none of which can initiate DNA chains in either direction, can’t unwind DNA
Direction of synthesis is 5’ to 3’
Both strands are duplicated
What is needed to start DNA replication?
Need Available 3’ hydroxyl group for base to come in on- 3’ OH attacks phosphate on incoming nucleoside triphosphate
Initiation requires a primer with a 3’ OH, Primer made from RNA because DNA polymerase a cannot add on bases by themselves
Many accessory proteins required
Replication forks
Replication forks are where there is unwinding of the parental DNA and the nucleotides are being incorporated into new complimentary strands
DNA replication in prokaryotes
Replication is bidirectional
One replicon
Where does replication begin? (Pro)
Begins at a specific point called the origin oriC
Proteins (DnaA-ATP) bind the origin and initiate replication (actually 30 proteins involved)
Prokaryotic replisome
In vivo, it’s thought that DNA polymerase III (holoenzyme dimer), the primosome, and DNA helicases are associated in a replisome that synthesizes DNA at 900bp per second
Initiation (pro)
OriC contains four 9bp binding sites for the initiator protein DnaA
Eventually it will form a group of 30-40 molecules each bound to ATP (negatively supercoiled)
Three 13 bp AT regions regions “melt” open and that allows DnaB (helicase)to bind. Helicases use ATP to move into and “melt” open the double stranded DNA
Separating the DNA strands (pro)
Causes many problems with the topology- DNA gets overwound, so there’s positive supercoiling in the unreplicated portion of the DNA
DNA gyrase is a topoisomerase (II) that helps relieve the tension caused by supercoiling further from the origin
DNA Topoisomerase
Type one- Nick one strand of DNA, let it roll, then reattach. Relieves strain.
Type two- Cuts both strands.
Binds one strand of the replicated DNA, close around it and cut it, and next strand will be passed through, then seal the cut.
Unwinding (pro)
DNA helicases must move along the template strands to open it for copying
Other proteins (single stranded binding proteins) also promote further unwinding by stabilizing the single stranded unwound DNA
Positive supercoiling is relaxed by DNA gyrase
Elongation overview (pro)
Two strands of DNA are anti-parallel, replication is anti parallel
One strand, the leading strand, is replicated from 5’-3’, one primer
Lagging strand replicated in 3’-5’ but it is done in small fragments from 5’ to 3’ that are joined together as a new fragment is begun at the replication fork by DNA ligase
Okazaki fragments begin with RNA primer put down by enzyme primase
Elongation steps (pro)
Primase and helicases form a primosome. Periodic binding of the primase gives short RNA primers that generate the Okazaki fragment
Both strands elongated by DNA polymeraseIII
Lagging strand primers are removed and the gaps are filled by DNA polymerase I
Final phosphodiester bond between fragments is formed by DNA ligase- joins fragments so there is a continuous strand of DNA on the lagging strand
DNA polymerase III
This multiunit holoenzyme is a dimer- all subunits are required for it to work
One half synthesizes the leading strand and one half synthesizes the lagging strand
Two polymerases in the same strand is good- Speed efficiency, lagging and leading at the same time