Reconstitution of a eukaryotic replisome reveals suppression mechanisms that define leading/lagging strand operation

Abstract
Article and author information
Metrics

Abstract

We have reconstituted a eukaryotic leading/lagging strand replisome comprising 31 distinct polypeptides. This study identifies a process unprecedented in bacterial replisomes. While bacteria and phage simply recruit polymerases to the fork, we find that suppression mechanisms are used to position the distinct eukaryotic polymerases on their respective strands. Hence, Pol ε is active with CMG on the leading strand, but it is unable to function on the lagging strand, even when Pol δ is not present. Conversely, Pol δ-PCNA is the only enzyme capable of extending Okazaki fragments in the presence of Pols ε and α. We have shown earlier that Pol δ-PCNA is suppressed on the leading strand with CMG (Georgescu, Langston et al. 2014). We propose that CMG, the 11-subunit helicase, is responsible for one or both of these suppression mechanisms that spatially control polymerase occupancy at the fork.

Article and author information

Author details

Roxana E Georgescu

DNA Replication Laboratory, Howard Hughes Medical Institute, Rockefeller University, New York, United States

Competing interests
The authors declare that no competing interests exist.
Grant D Schauer

DNA Replication Laboratory, Howard Hughes Medical Institute, Rockefeller University, New York, United States

Competing interests
The authors declare that no competing interests exist.
Nina Y Yao

DNA Replication Laboratory, Howard Hughes Medical Institute, Rockefeller University, New York, United States

Competing interests
The authors declare that no competing interests exist.
Lance D Langston

DNA Replication Laboratory, Howard Hughes Medical Institute, Rockefeller University, New York, United States

Competing interests
The authors declare that no competing interests exist.
Olga Yurieva

DNA Replication Laboratory, Howard Hughes Medical Institute, Rockefeller University, New York, United States

Competing interests
The authors declare that no competing interests exist.
Dan Zhang

DNA Replication Laboratory, Howard Hughes Medical Institute, Rockefeller University, New York, United States

Competing interests
The authors declare that no competing interests exist.
Jeff Finkelstein

DNA Replication Laboratory, Howard Hughes Medical Institute, Rockefeller University, New York, United States

Competing interests
The authors declare that no competing interests exist.
Mike E O'Donnell

DNA Replication Laboratory, Howard Hughes Medical Institute, Rockefeller University, New York, United States

For correspondence
odonnel@mail.rockefeller.edu

Competing interests
The authors declare that no competing interests exist.

Copyright

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.