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Molecular and Cellular Biology, November 2008, p. 6844-6857, Vol. 28, No. 22
0270-7306/08/$08.00+0     doi:10.1128/MCB.01235-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Mechanistic Insights into Replication Termination as Revealed by Investigations of the Reb1-Ter3 Complex of Schizosaccharomyces pombe{triangledown} ,{dagger}

Subhrajit Biswas and Deepak Bastia*

Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425

Received 5 August 2008/ Returned for modification 27 August 2008/ Accepted 2 September 2008

Relatively little is known about the interaction of eukaryotic replication terminator proteins with the cognate termini and the replication termination mechanism. Here, we report a biochemical analysis of the interaction of the Reb1 terminator protein of Schizosaccharomyces pombe, which binds to the Ter3 site present in the nontranscribed spacers of ribosomal DNA, located in chromosome III. We show that Reb1 is a dimeric protein and that the N-terminal dimerization domain of the protein is dispensable for replication termination. Unlike its mammalian counterpart Ttf1, Reb1 did not need an accessory protein to bind to Ter3. The two myb/SANT domains and an adjacent, N-terminal 154-amino-acid-long segment (called the myb-associated domain) were both necessary and sufficient for optimal DNA binding in vitro and fork arrest in vivo. The protein and its binding site Ter3 were unable to arrest forks initiated in vivo from ars of Saccharomyces cerevisiae in the cell milieu of the latter despite the facts that the protein retained the proper affinity of binding, was located in vivo at the Ter site, and apparently was not displaced by the "sweepase" Rrm3. These observations suggest that replication fork arrest is not an intrinsic property of the Reb1-Ter3 complex.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 174 Ashley Ave., Charleston, SC 29425. Phone: (843) 792-0491. Fax: (843) 792-8568. E-mail: bastia{at}musc.edu

{triangledown} Published ahead of print on 15 September 2008.

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/.

Molecular and Cellular Biology, November 2008, p. 6844-6857, Vol. 28, No. 22
0270-7306/08/$08.00+0     doi:10.1128/MCB.01235-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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