Nanobiotechnology
Peter Pfeiffer
Demonstration of discontinuous DNA synthesis
Caption Nascent DNA molecules are labeled in E. coli with a short pulse of 3H-deoxythymidine. The cells
are lysed, the DNA is isolated, and single strands are separated by size. The labeled DNA molecules fall
into two classes: long molecules arising from continuous synthesis of the leading strand and short
fragments arising from discontinuous synthesis of the lagging strand.
Notes The overall conclusion from the demonstration is that DNA is being added to either long or short
segments of daughter strands during replication. The short ones are called "Okasaki fragments" and are
found on the lagging strand.
Diagram of lagging-strand synthesis
Caption A short piece of RNA (brown) serves as a primer for the synthesis of each Okazaki fragment. The
length of the Okazaki fragment is determined by the distance between successive RNA primers.
Notes One primer is made about every second (about 1000nucleotides) in E. coli, such that each Okasaki
fragment is about 1000 nucleotides long.
Joining of Okazaki fragments
Caption Joining of Okazaki fragments requires the combined action of DNA polymerase I and DNA ligase.
After the synthesis of an Okasaki fragment, there is a gap between the ends. Also, the 5' end of the newly
synthesized strand contains an RNA primer segment. This segment is removed and the gap filled-in by
polymerase I. The final phosphodiester bond between Okasaki fragements is formed by DNA ligase.
Notes The process carried out by polymerase 1 is called "nick translation." The mechanism of DNA ligase
from E. coli is shown in Figure 20.14.
Simultaneous synthesis of leading and lagging strands at a replication fork
Caption The replisome contains the DNA polymerase III holoenzyme (only the core complexes are
shown); a primosome containing primase, a helicase, and other subunits; and additional components
including single-strand binding protein (SSB). One core complex of the holoenzyme synthesizes the
leading strand, while the other core complex synthesizes the lagging strand. The lagging-strand template
is looped back through the replisome so that the leading and lagging strands can be synthesized in the
same direction as fork movement.
Notes This is part a-b of a four-part figure. These two figures show (a) the initiation of synthesis on an
RNA primer on the lagging strand and continued synthesis of the leading strand and (b) the initiation, on
the lagging strand, of a new RNA primer by the primosome. On the lagging strand it can be seen that
there is a long (~1000nucleotide) single-stranded segment of DNA.
Simultaneous synthesis of leading and lagging strands at a replication fork.
Caption The replisome contains the DNA polymerase III holoenzyme (only the core complexes are
shown); a primosome containing primase, a helicase, and other subunits; and additional components
including single-strand binding protein (SSB). One core complex of the holoenzyme synthesizes the
leading strand, while the other core complex synthesizes the lagging strand. The lagging-strand template
is looped back through the replisome so that the leading and lagging strands can be synthesized in the
same direction as fork movement.
Notes This is parts c-d of a four part figure. The lagging strand is released (c) until a new Okasaki
fragment is initiated on the next RNA primer. (d) Single-strand binding proteins are displaced as the
single strand is copied.