we know that all VNA polymerase is catalyzed five prime to three prime strand growth and that no DNA polymerase can synthesize DNA three prime to five prime since this is true how can we rationalize replication of both strands against their templates at a replication fork.
The only reasonable hypothesis was that while one strand could be synthesized continuously as the replication fork keeps unwinding at least one DNA strand would have to be synthesized in pieces or fragments that would later be stitched together here is our replication fork modeling continuous synthesis of one strand of DNA in the five prime to three prime direction towards a replication fork and the replication of fragments of DNA also five prime to three prime but extending away from the replication fork the strand made continuously is called the leading strand while the strands made in pieces is called the lagging strand in part because it's synthesis is slowed down by the need to put the pieces together the enzyme that stitches the fragments together is a DNA ligase DNA ligase was known for a different function before it was shown to be essential in replication during a viral infection in bacteria.
The DNA that enters the cell is a linear molecule with sticky or complementary ends such as those shown here once in the bacterial cytoplasm the linear DNA circular Rises because of self complimentary ends then a DNA ligase encoded by the viral genome is produced in the infected cell two covalently close the circle the ligated circular viral DNA molecule then uses bacterial DNA polymerase to replicate itself eventually producing new viruses this slide illustrates events after placing a dilute viral suspension on a lawn of bacterial cells on a petri dish each virus infects a single cell which makes more viruses that then infect neighboring cells on the lawn as more and more cells lyse and release new infectious viruses small clearings develop on the bacterial lawn where the bacteria have lysed these clearings are called plaques okazaki knew the role of DNA ligase enclosing bacteriophage DNA circles and he had isolated several mutant strains of the phage called t4 that were deficient in making DNA ligase these phase multiplied more slowly than wild-type phage as shown by the growth curves on this slide because the mutant phage were slow to close their DNA circles after infecting ecoli cells replication of these mutant phage was also slower than in the wild-type but Okazaki also proposed that phage DNA replication itself might be using DNA ligase to stitch together fragments of DNA made.
Discontinuously on the lagging template strand at replication forks and that these mutants were doing this process slowly so Okazaki did experiments to test his hypothesis he predicted that during an infection by ligase deficient t4 phage replication of new phage DNA would result in the accumulation of shortened DNA fragments that would not be efficiently stitched together here's what he did he incubated ligates deficient t4 phage with e.coli cells he then added radioactive tritium thymine to the infected cells at two to five second intervals after infection in each case a few seconds after adding the radioactive nucleotide he extracted total DNA from the infected cells and used physical methods to shear the DNA down to an average of about 10,000 base pairs in length finally he put the sheared DNA isolates through a process called sucrose density gradient centrifugation to separate DNA fragments by size in other words by length here's what one might expect in this experiment if both strands were somehow synthesized continuously and not in fragments are not in pieces after two seconds only short lengths of DNA would have replicated and the added radioactive nucleotides would be put on the ends of these fragments as shown in red here after four seconds of replication the replicated DNA would be longer and the radioactive bits would be added to these longer pieces and after longer and longer times aphasia infection the labeled radioactive nucleotides would be added to longer and longer if replicated DNA but that's not what Okazaki found in his experiments this graph plots the sizes of radioactive DNA on the x axis against the amount of radioactivity of the DNA on the y axis looking at the larger curves on the right side of the graph as expected during an infection you could see longer and longer DNA being made radioactive up to the last 120 second time point tested of course few fragments ever exceeded the average ten thousand base pairs since the DNA had been intentionally cut to that size before separation but look at the left side of the graph that small portion circled here at every time even as the overall length of DNA molecules continue to increase short fragments are accumulating in cells infected with the ligase deficient t4 phage when Okazaki did the same experiment with wild-type phage adding hot thymine only tagged longer and longer DNA as indicated by the dark blue curves in this graph and that's because ligase was so efficient that the short pieces that were made were quickly ligated to make long strands of new DNA Okazaki had demonstrated discontinuous synthesis of short pieces of DNA using the T for deficient ligase phage which explained how one strand must behave at the replication fork in his honor these pieces of DNA were later named Okazaki fragments we know that new DNA made along the lagging strand template is made in pieces that are later stitched together by DNA ligase and that this occurs in all species and all cells we also know that all species have genes for their own DNA ligase and can therefore make their own enzyme when they replicate their own DNA and not for quite what happened to this slide but we will live with it
