Fig. 16-2 Living S cells (control) Living R cells (control) Heat-killed S cells (control) Mixture of heat-killed S cells and living R cells Mouse dies Mouse healthy Living S cells RESULTS Griffith’s experiment
In 1944, Oswald Avery, Maclyn McCarty, and Colin MacLeod announced that Griffith’s transforming substance was DNA ! Their conclusion was based on experimental evidence that only DNA worked in transforming harmless bacteria into pathogenic (S- cell)bacteria in vitro (in a petri plate), protein and carbohydrate were inactive in this regard. Many biologists remained skeptical, mainly because little was known about DNA
Oswald Avery, Maclyn McCarty, and Colin MacLeod Avery McCarty with Watson and Crick MacLeod
Bacteriophages produce areas of dead cells or “plaques” on bacterial “lawns.” When bacteria are mixed into to an agar medium the cells grow into a uniform “lawn” (center and right) instead of as individual colonies on the agar surface (left). Sometimes “holes” or plaques were seen in these lawns where something seemed to be “eating” the bacteria. The bacteria “eater” proved to be bacterial viruses or bacteriophage. The plaques are limited in size (diameter) because the bacteria must be actively growing in order for the viruses to use their enzymes to reproduce and kill the bacteria. When the plate is covered, bacterial growth stops and so does the ability of the phage to kill bacterial cells.
Fig. 16-3 100 nm Bacterial cell Phage head Tail sheath Tail fiber DNA
Fig. 16-9-3 A T G C TA TA G C (a) Parent molecule AT GC T A T A GC (c) “Daughter” DNA molecules, each consisting of one parental strand and one new strand (b) Separation of strands A T G C TA TA G C A T G C T A T A G C
Fig. 16-11 EXPERIMENT RESULTS CONCLUSION 1 2 4 3 Conservative model Semiconservative model Dispersive model Bacteria cultured in medium containing 15 N Bacteria transferred to medium containing 14 N DNA sample centrifuged after 20 min (after first application) DNA sample centrifuged after 40 min (after second replication) More dense Less dense Second replicationFirst replication
Fig. 16-12a Origin of replication Parental (template) strand Daughter (new) strand Replication fork Replication bubble Double- stranded DNA molecule Two daughter DNA molecules (a) Origins of replication in E. coli 0.5 µm
Fig. 16-12b 0.25 µm Origin of replicationDouble-stranded DNA molecule Parental (template) strand Daughter (new) strand Bubble Replication fork Two daughter DNA molecules (b) Origins of replication in eukaryotes
Helicase ds DNA opening of a replication bubble cases the DNA on either side to overwind and supercoil. This illustrates the problem with bacterial closed loop DNA, but similar problems also occur as the multiple replication bubbles form in a linear chromosome also. This “untangling” activity is so important, that preventing it can cause cell death. Anti-topoisomerase materials can be used as anti-cancer drugs.
Topoisomerases serve to “untangle” DNA by allowing the super coiling produced by helicase activity to be relieved. Topoisomerase I enzymes “nick” one side of a DNA, allow supercoiling strain to unwind, then re-join the opened strand Topoisomerase II enzymes use ATP to grab one double stranded DNA (G), cut it, allow another double strand of DNA (T) to pass through (see 3a), then re-join the opened strand.
Topoisomerase I poisons are used as anti-cancer drugs. A. Topoisomerase I introduces a nick in the DNA backbone allowing the rotation of one strand around the other. This releases the torsional strain which otherwise accumulates in front of the advancing replication fork (large arrow). The DNA break is extremely transient and is re-ligated almost immediately at the same time that the topoisomerase I releases the other strand. B. When a drug such as irinotecan is present (black oval with C), it binds to the topoisomerase I-nicked DNA complex. This prevents the re-ligation of the nicked strand and the release of the enzyme. Eventually, the replication fork collides with the complex, causing the formation of a double-strand break.
Fig. 16-15 Leading strand Overview Origin of replication Lagging strand Leading strandLagging strand Primer Overall directions of replication Origin of replication RNA primer “Sliding clamp” DNA poll III Parental DNA 5 3 3 3 3 5 5 5 5 5
Fig. 16-17 Overview Origin of replication Leading strand Lagging strand Overall directions of replication Leading strand Lagging strand Helicase Parental DNA DNA pol III PrimerPrimase DNA ligase DNA pol III DNA pol I Single-strand binding protein 5 3 5 5 5 5 3 3 3 3 1 3 2 4
Fig. 16-19 Ends of parental DNA strands Leading strand Lagging strand Last fragment Previous fragment Parental strand RNA primer Removal of primers and replacement with DNA where a 3 end is available Second round of replication New leading strand New lagging strand Further rounds of replication Shorter and shorter daughter molecules 5 3 3 3 3 3 5 5 5 5