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Alessandro Costa : Structure and dynamics of the eukaryotic replisome

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The replisome coordinates DNA unwinding and synthesis. The CMG helicase is the organizing centre of the replication machinery and has been reported to form a stable complex with replicative polymerases Pol alpha and epsilon, although not Pol delta.

Pol alpha primes both the leading strand at the onset of replication and the Okazaki fragments on the lagging strand. According to the consensus view, Pol epsilon extends the leading and Pol delta the lagging strand, however recent studies indicate that the division of labor between replicative polymerases might be more promiscuous than originally thought. We use cryo-EM to study the structure of the replisome.

We are reconstituting the replisome and its subcomplexes for single-particle cryo-EM studies. For example, we have reconstituted a complex containing the CMG helicase bound to Pol epsilon and found that the catalytic domain of Pol epsilon is free to move with respect to the rest of the complex.

We also found that the replisome component Ctf4 is a homo-trimer that can bind up to three different client proteins at the same time, including CMG and Pol alpha replisome components, but also sister chromatid cohesion protein Chl1.

We now want to establish what role the catalytically defunct components of leading strand polymerase Pol epsilon have in the replisome, and how efficient and processive leading strand synthesis can be catalyzed.

Structure of the CMG-Pol epsilon assembly

Figure 1:Structure of the CMG-Pol epsilon assembly reveals that the DNA-synthesis domain (light purple) of Pol epsilon is free to move, with respect to the core complex. The non-catalytic portion of Pol epsilon (dark purple) anchors the polymerase to the CMG helicase.

Ctf4

Figure 2: Ctf4 can link up to three client proteins concurrently. Ctf4 (blue and yellow) forms a homo-trimer, here bound to one, two or three copies of the helicase activator, GINS (orange).