REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Flag M2	Sigma-Aldrich	Cat# F3165; RRID: AB_259529
V5 E10/V4RR	Thermo Fisher Scientific	Cat# MA5-15253; RRID: AB_10977225
GFP JL-8	Living Colors	Cat# 632380; RRID: AB_10013427
Myc 9E10	Thermo Fisher Scientific	Cat# 13-2500; RRID: AB_2533008
(Polyclonal) Dam1 complex	Pacific Immunology	N/A
Chemicals, Peptides, and Recombinant Proteins
1-NM-PP1	Toronto Research Chemical	Cat# A603003
Lambda Protein Phosphatase	New England Biolabs	Cat# P0753L
SNAP-Surface 647 dye	New England Biolabs	Cat# S9136S
CLIP-Surface 547 dye	New England Biolabs	Cat# S9233S
Recombinant Dam1 and Ndc80 complex	[15, 18, 51, 57]	N/A
Experimental Models: Organisms/Strains
S. cerevisiae; Strain background: W303	This study	N/A
SBY3 (W303); MATa ura3-1 leu2-3,112 his3-11 trp1-1 can1-100 ade2-1 bar1-1	This study	N/A
SBY8253; SBY3, DSN1-6His-3Flag:URA3	This study	N/A
SBY8944; SBY3, DSN1-6His-3Flag dad1-1:KanMx	This study	N/A
SBY12464; SBY3, DAD1-3Flag:TRP1	This study	N/A
SBY13507; SBY3, DAD1-3Flag:TRP1 cdc28-13	This study [58]	N/A
SBY13509; SBY3, DAD1-3Flag:TRP1 cdc28-as1	This study [58]	N/A
SBY13538; SBY3, DAD1-3V5:HIS	This study	N/A
SBY16766; SBY3, DAD1-SNAP-3V5:KanMx	This study	N/A
SBY16826; SBY3, MTW1-CLIP:KanMx dad1-1:KanMx	This study	N/A
SBY16828; SBY3, DSN1-His-Flag:URA3 MTW1-CLIP:KanMx dad1-1:KanMx	This study	N/A
SBY17831; SBY3, DAD1-3Flag:TRP1 ask1::KanMx leu2-3,112::ask1-S216A, S250A:LEU2	This study	N/A
SBY17833; SBY3, DAD1-3Flag:TRP1 ask1::KanMx leu2-3,112::ask1-S216D, S250D:LEU2	This study	N/A
SBY18086; SBY3, ask1::KanMx leu2-3,112::ASK1:LEU2	This study	N/A
SBY18088; SBY3, ask1::KanMx leu2-3,112::ask1-S216A, S250A:LEU2	This study	N/A
SBY18466; SBY3, DSN1-6His-3Flag:URA3 dam1(S20D):KanMx ask1::KanMx leu2-3,112::ASK1:LEU2	This study [25, 28]	N/A
SBY18468; SBY3, DSN1-6His-3Flag:URA3 dam1(S20D):KanNMx ask1::KanMx leu2-3,112::ask1-S216A, S250A:LEU2	This study	N/A
SBY18469; SBY3, DSN1-6His-3Flag:URA3 dam1(S20D):KanMx ask1::KanMx leu2-3,112::ask1-S216D, S250D:LEU2	This study	N/A
SBY18606; SBY3, CDC28-3V5:KanMx	This study	N/A
SBY18722; SBY3, CFIII (CEN3.L.YPH278)URA3-SUP11 ask1::KanMx leu2-3,112::ASK1:LEU2	This study [59]	N/A
SBY18724; SBY3, CFIII (CEN3.L.YPH278)URA3-SUP11 ask1::KanMx leu2-3,112::ask1-S216A, S250A:LEU2	This study [59]	N/A
SBY18726; SBY3, CFIII (CEN3.L.YPH278)URA3-SUP11 ask1::KanMx leu2-3,112::ask1-S216D, S250D:LEU2	This study [59]	N/A
SBY19132; SBY3, dam1-9:kanMx ask1::KanMx leu2-3,112::ASK1:LEU2	This study	N/A
SBY19136; SBY3, dam1-9:kanMx ask1::KanMx leu2-3,112::ask1-S216A, S250A:LEU2	This study	N/A
SBY19138; SBY3; dam1-9::kanMX ask1::KanMx6 leu2-3,112::Ask1-S216D, S250D:LEU2	This study	N/A
SBY19314; SBY3, DSN1-6His-3Flag: URA3 ask1::KanMx leu2-3,112::ask1-S216A, S250A:LEU2	This study	N/A
SBY19319; SBY3, DSN1-6His-3Flag: URA3 ask1::KanMx leu2-3,112::ask1-S216D, S250D:LEU2	This study	N/A
SBY19361; SBY3, DSN1-6His-3Flag:URA3 ask1::KanMx leu2-3,112::ASK1:LEU2	This study	N/A
SBY20049; SBY3, pMET-CDC20:TRP1 cenIII-lacO128:TRP1 ask1::KanMx6 leu2-3,112::ASK1:LEU2 his3-11:pCUP1-GFP12-LacI12	This study	N/A
SBY20050; SBY3, pMET-CDC20:TRP1 cenIII-lacO128:TRP1 ask1::KanMx6 leu2-3,112::ASK1:LEU2 his3-11:pCUP1-GFP12-LacI12 dam1-9	This study	N/A
SBY20052; SBY3, pMET-CDC20:TRP1 cenIII-lacO128:TRP1 ask1::KanMx6 leu2-3,112::ASK1-S216A, S250A:LEU2 his3-11:pCUP1-GFP12-LacI12 dam1-9	This study	N/A
Oligonucleotides
See Table S3 for primer sequences	This study	N/A
Software and Algorithms
Igor Pro	Wavemetrics	https://www.wavemetrics.com/; RRID: SCR_000325
LabVIEW	National Instruments	https://www.ni.com/; RRID: SCR_014325
ImageJ	NIH image	https://imagej.net/; RRID: SCR_003070
Fiji	[60]	http://fiji.sc; RRID: SCR_002285
PRISM	GraphPad Software	https://www.graphpad.com/scientific-software/prism/; RRID: SCR_002798
NIS-Elements Software	Nikon	https://www.microscope.healthcare.nikon.com/products/software/; RRID: SCR_014329