To watch a strand of DNA replicating

The van Oijen lab (here at HMS) had a nice paper in Nature where they indirectly observe single T7 DNA polymerase complexes copying DNA.

How do they do it? One end of the DNA is fixed while the other side is coupled to a bead that can be visualized. A laminar flow is applied to the reaction causing the DNA strand to stretch out. Thus the bead becomes a marker for the length of the DNA.

As the T7 complex duplicates the leading strand, the uncopied lagging strand agglomerates. Since the lagging strand is fixed to the cover slip, the DNA shortens and this reels in the bead like a fish on a line. Once in a while leading strand synthesis pauses as the complex lays down an RNA primer to initiate lagging strand synthesis (aka the synthesis of Okazaki fragments). When this happens, the bead's movement momentarily halts. Then as the enzyme proceeds to copy the leading and lagging strands, a lagging strand loop is formed (see below) and the reeling in of the bead resumes. At a certain point the Okazaki fragment is filled in causing the complex to release the lagging strand loop. This increases the length of the DNA causing the bead to fall back (kind of like releasing the fishing line).

http://bcmp.med.harvard.edu/images/stories/faculty_fig/richardson_01.jpg

By visualizing the conjugated bead, the van Oijen lab can mausure the frequency and duration of all these growth, pause and bobble events and thus deduce the properties displayed by single T7 complexes. The speed of the replication is dictated by the speed of DNA unwinding (by the helicase). The transient pausing is a good sign that when a new lagging strand primer is laid-down, the helicase is temporarily inactivated. To read the article (subscription req) click here.

Ref: Jong-Bong Lee, Richard K. Hite, Samir M. Hamdan, X. Sunney Xie, Charles C. Richardson and Antoine M. van Oijen DNA primase acts as a molecular brake in DNA replication. Nature (06) 439:621-624.

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