A Dam1-based artificial kinetochore is sufficient to promote chromosome segregation in budding yeast.
Kinetochores are large multiprotein complexes that mediate chromosome segregation in all eukaryotes by dynamically connecting specialized chromosome regions, termed centromeres, to the plus-ends of spindle microtubules. Even the relatively simple kinetochores of the budding yeast Saccharomyces cerevisiae consist of more than 80 proteins, making analysis of their respective roles a daunting task. Here, we have developed a system that allows us to artificially recruit proteins to DNA sequences and determine whether they can provide any aspect of kinetochore function in vivo. We show that artificial recruitment of the microtubule-binding Dam1 complex to a plasmid lacking any centromere DNA is sufficient to confer mitotic stabilization. The Dam1-based artificial kinetochores are able to attach, bi-orient and segregate mini-chromosomes on the mitotic spindle, and they bypass the requirement for essential DNA-binding components of natural kinetochores. Thus, we have built a simplified chromosome segregation system by directly recruiting a microtubule force-transducing component to DNA.
Kiermaier
Eva
E
Research Institute of Molecular Pathology, Vienna, Austria.
Woehrer
Sophie
S
Peng
Yutian
Y
Mechtler
Karl
K
Westermann
Stefan
S
D018797:Cell Cycle Proteins / Q000378:metabolism*; D020090:Chromosome Segregation; D015825:Chromosomes, Fungal / Q000378:metabolism; D018386:Kinetochores / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
C447843:ASK1 protein, S cerevisiae; D018797:Cell Cycle Proteins; C116003:DAM1 protein, S cerevisiae; D008869:Microtubule-Associated Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; C034811:tetracycline resistance-encoding transposon repressor protein
J Cell Biol. 2001 Jan 8;152(1):197-212
11149931
J Cell Biol. 2007 Jul 16;178(2):269-81
17620411
Proc Natl Acad Sci U S A. 2007 Sep 11;104(37):14706-11
17804787
Cell. 2006 Dec 1;127(5):983-97
17129783
Curr Opin Cell Biol. 2008 Feb;20(1):53-63
18182282
Cell. 2005 Sep 23;122(6):849-60
16179255
Nat Struct Mol Biol. 2007 Aug;14(8):721-6
17643123
Cell. 2001 Jul 27;106(2):195-206
11511347
Proc Natl Acad Sci U S A. 2006 Jun 27;103(26):9873-8
16777964
Curr Biol. 1996 Dec 1;6(12):1599-608
8994824
Cell. 2008 May 2;133(3):427-39
18455984
Cell. 1998 Sep 4;94(5):607-13
9741625
Nature. 2008 Aug 7;454(7205):728-34
18660802
Yeast. 1997 Jul;13(9):837-48
9234672
Cell. 2006 Dec 15;127(6):1179-91
17174893
Nature. 1980 Oct 9;287(5782):504-9
6999364
Proc Natl Acad Sci U S A. 2008 Oct 7;105(40):15423-8
18824692
Nature. 2004 Mar 4;428(6978):93-7
14961024
Yeast. 1998 Jul;14(10):953-61
9717241
Annu Rev Biochem. 2007;76:563-91
17362199
Cell. 2002 Oct 18;111(2):163-72
12408861
Nat Rev Mol Cell Biol. 2008 Jan;9(1):33-46
18097444
Nat Struct Mol Biol. 2005 Feb;12(2):138-43
15640796
Nature. 2006 Mar 23;440(7083):565-9
16415853
Science. 2004 Nov 5;306(5698):1021-5
15528442
Mol Cell. 2005 Jan 21;17(2):277-90
15664196
Science. 1999 Jul 9;285(5425):254-7
10398602
J Cell Biol. 2001 Dec 24;155(7):1137-45
11756468