Mechanistic insights into replication termination as revealed by investigations of the Reb1-Ter3 complex of Schizosaccharomyces pombe.
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.
Biswas
Subhrajit
S
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 174 Ashley Ave., Charleston, SC 29425, USA.
Bastia
Deepak
D
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D005816:Genetic Complementation Test; D008958:Models, Molecular; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000378:metabolism; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics* / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014364:Tryptophan / Q000378:metabolism
D004268:DNA-Binding Proteins; D046912:Multiprotein Complexes; D029702:Schizosaccharomyces pombe Proteins; D014157:Transcription Factors; C512907:reb1 protein, S pombe; D014364:Tryptophan
Gene. 1989 Dec 21;85(1):15-23
2533576
Proc Natl Acad Sci U S A. 2006 Jan 24;103(4):897-902
16418273
J Biol Chem. 1995 Dec 8;270(49):29138-44
7493939
J Biol Chem. 2004 Jan 16;279(3):1932-41
14576157
Cell. 1988 Nov 18;55(4):637-43
3052854
Genes Dev. 2006 Nov 15;20(22):3104-16
17114583
Cell. 2006 Jun 30;125(7):1309-19
16814717
Proc Natl Acad Sci U S A. 1994 Nov 8;91(23):11143-7
7972025
Proc Natl Acad Sci U S A. 2001 Aug 14;98(17):9569-74
11493686
Trends Biochem Sci. 1996 Mar;21(3):87-8
8882580
Cell. 1985 Nov;43(1):189-97
3000600
Mol Cell. 2003 Dec;12(6):1525-36
14690605
Proc Natl Acad Sci U S A. 1992 Jul 15;89(14):6428-32
1631139
Anal Biochem. 1976 May 7;72:248-54
942051
Mol Cell Biol. 2004 Oct;24(19):8342-55
15367656
Genes Dev. 2005 Aug 15;19(16):1905-19
16103218
J Biol Chem. 1998 Jan 30;273(5):3051-9
9446621
Proc Natl Acad Sci U S A. 2006 Jul 5;103(27):10236-10241
16798881
J Biol Chem. 1990 Nov 15;265(32):19990-5
2246275
Cell. 1987 Nov 6;51(3):463-71
2822257
Proc Natl Acad Sci U S A. 2004 Sep 28;101(39):14085-90
15371597
J Biol Chem. 2001 Jun 29;276(26):23471-9
11313334
Genes Dev. 2003 May 1;17(9):1153-65
12730134
J Biol Chem. 2001 Mar 23;276(12):8771-7
11124956
J Biol Chem. 2000 Jun 23;275(25):18871-8
10770926
Mol Cell Biol. 2004 Jan;24(1):398-406
14673172
Nucleic Acids Res. 1997 Feb 15;25(4):904-10
9016645
Cell. 1989 Nov 17;59(4):667-74
2684415
Nucleic Acids Res. 1996 Oct 1;24(19):3677-84
8871544
Cell. 1996 Nov 29;87(5):881-91
8945515
Mol Cell Biol. 1993 Feb;13(2):1173-82
8423784
Nature. 1999 Jul 8;400(6740):181-4
10408447
EMBO J. 1991 Oct;10(10):3025-32
1915277
EMBO J. 1997 Feb 17;16(4):760-8
9049305
Mol Cell Biol. 2006 Nov;26(21):8061-74
16940176
J Mol Biol. 2007 Jul 13;370(3):481-91
17521668
J Biol Chem. 1992 Sep 15;267(26):18612-22
1326530
Proc Natl Acad Sci U S A. 1991 Apr 1;88(7):2618-22
1849268
Int J Biochem Cell Biol. 2002 Sep;34(9):1031-4
12009298
Mol Cell. 2005 Sep 2;19(5):699-706
16137625
Cell. 1983 Aug;34(1):125-34
6224568
Proc Natl Acad Sci U S A. 2008 Sep 2;105(35):12831-6
18708526
Mol Microbiol. 1999 Mar;31(6):1611-8
10209736
Exp Cell Res. 1998 Jan 10;238(1):220-30
9457075
Seikagaku. 1996 Feb;68(2):111-28
8721791
Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):10647-52
8855233
EMBO J. 2003 Jul 1;22(13):3431-40
12840005
Cell. 2000 Sep 15;102(6):745-51
11030618
EMBO J. 1996 Jun 17;15(12):3164-73
8670817
Proc Natl Acad Sci U S A. 1987 Oct;84(19):6673-6
2958845
Nat Rev Mol Cell Biol. 2004 Feb;5(2):158-63
15040448
Genes Dev. 2005 Oct 15;19(20):2447-57
16195416
Mol Cell Biol. 2005 Oct;25(19):8755-61
16166653
J Biol Chem. 2005 Nov 25;280(47):39135-42
16195226
Methods Enzymol. 1991;208:118-46
1779833
Nucleic Acids Res. 2004 Jun 10;32(10):3148-55
15192117
Nucleic Acids Symp Ser. 1993;(29):201-2
8247769