|
Originally published In Press as doi:10.1074/jbc.M508996200 on September 28, 2005
J. Biol. Chem., Vol. 280, Issue 47, 39135-39142, November 25, 2005
Sap1p Binds to Ter1 at the Ribosomal DNA of Schizosaccharomyces pombe and Causes Polar Replication Fork Arrest*
Gregor Krings and
Deepak Bastia1
From the
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425
Eukaryotic DNA replication forks stall at natural replication fork barriers or Ter sites located within the ribosomal DNA (rDNA) intergenic spacer regions during unperturbed DNA replication. The rDNA intergenic spacer of the fission yeast Schizosaccharomyces pombe contains four polar or orientation-specific fork barriers, Ter13 and RFP4. Whereas the transcription terminator Reb1p binds Ter2 and Ter3 to arrest replication, the factor(s) responsible for fork arrest at Ter1 and RFP4 remain unknown. Using linker scanning mutagenesis, we have narrowed down minimal Ter1 to 21 bp. Sequence analysis revealed the presence of a consensus binding motif for the essential switch-activating and genome-stabilizing protein Sap1p within this region. Recombinant Sap1p bound Ter1 with high specificity, and endogenous Ter1 binding activity contained Sap1p and comigrated with the Sap1p-Ter1 complex. Circular permutation analysis suggested that Sap1p bends Ter1 and SAS1 upon binding. Targeted mutational analysis revealed that Ter1 mutations, which prevent Sap1p binding in vitro, are defective for replication fork arrest in vivo, whereas mutations that do not affect Sap1p binding remain competent to arrest replication. The results confirm the hypothesis that the chromatin organizer Sap1p binds site-specifically to genomic regions other than SAS1 and support the notion that Sap1p binds the rDNA fork barrier Ter1 to cause polar replication fork arrest at this site but not at SAS1.
Received for publication, August 15, 2005
, and in revised form, September 28, 2005.
* This work was supported by grants from NIAID and NIGMS, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, 173 Ashley Ave., Charleston, SC 29425. Tel.: 843-792-0491; Fax: 843-792-8568; E-mail: bastia{at}musc.edu.

CiteULike Complore Connotea Del.icio.us Digg Reddit Technorati What's this?
This article has been cited by other articles:

|
 |

|
 |
 
S. Biswas and D. Bastia
Mechanistic Insights into Replication Termination as Revealed by Investigations of the Reb1-Ter3 Complex of Schizosaccharomyces pombe
Mol. Cell. Biol.,
November 15, 2008;
28(22):
6844 - 6857.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
T. Eydmann, E. Sommariva, T. Inagawa, S. Mian, A. J. S. Klar, and J. Z. Dalgaard
Rtf1-Mediated Eukaryotic Site-Specific Replication Termination
Genetics,
September 1, 2008;
180(1):
27 - 39.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
A. K. Hyvarinen, J. L. O. Pohjoismaki, A. Reyes, S. Wanrooij, T. Yasukawa, P. J. Karhunen, J. N. Spelbrink, I. J. Holt, and H. T. Jacobs
The mitochondrial transcription termination factor mTERF modulates replication pausing in human mitochondrial DNA
Nucleic Acids Res.,
October 8, 2007;
35(19):
6458 - 6474.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
E. V. Mirkin and S. M. Mirkin
Replication Fork Stalling at Natural Impediments
Microbiol. Mol. Biol. Rev.,
March 1, 2007;
71(1):
13 - 35.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
C. Noguchi and E. Noguchi
Sap1 Promotes the Association of the Replication Fork Protection Complex With Chromatin and Is Involved in the Replication Checkpoint in Schizosaccharomyces pombe
Genetics,
February 1, 2007;
175(2):
553 - 566.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
J. L. O. Pohjoismaki, S. Wanrooij, A. K. Hyvarinen, S. Goffart, I. J. Holt, J. N. Spelbrink, and H. T. Jacobs
Alterations to the expression level of mitochondrial transcription factor A, TFAM, modify the mode of mitochondrial DNA replication in cultured human cells
Nucleic Acids Res.,
November 6, 2006;
34(20):
5815 - 5828.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
G. Krings and D. Bastia
Molecular Architecture of a Eukaryotic DNA Replication Terminus-Terminator Protein Complex
Mol. Cell. Biol.,
November 1, 2006;
26(21):
8061 - 8074.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
S. Coulon, E. Noguchi, C. Noguchi, L.-L. Du, T. M. Nakamura, and P. Russell
Rad22Rad52-dependent Repair of Ribosomal DNA Repeats Cleaved by Slx1-Slx4 Endonuclease
Mol. Biol. Cell,
April 1, 2006;
17(4):
2081 - 2090.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
B. K. Mohanty, N. K. Bairwa, and D. Bastia
The Tof1p-Csm3p protein complex counteracts the Rrm3p helicase to control replication termination of Saccharomyces cerevisiae
PNAS,
January 24, 2006;
103(4):
897 - 902.
[Abstract]
[Full Text]
[PDF]
|
 |
|
Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.
|
Advertisement
Advertisement
|