Structure of the replication regulator Sap1 reveals functionally important interfaces



Figure S1 :

 S1 Figure S1: (A) Sequence alignment of Schizosaccharomyces Sap1 proteins. The secondary structure annotation is derived from the Sap1 crystal structure. (B) MALDI peak of the thermolysin limited proteolysis fragment in Fig. 1C. 


Figure S2 :

 S2 Figure S2: The Sap1 DNA binding domain resembles telomere-binding proteins. (A) Structure of the native Sap1 DNA binding domain with putative position of DNA modeled based on structural homology to Trf1 as in Fig. 2. The deep pocket is colored in red. This pocket is filled by the dimethylarsinoyl adduct to C81 in the derivative crystal. (B) Superposition of Sap1 DBD onto NGTRF1 (PDBID:2CKX). (C) Mapping of amino acids essential to Sap1-DNA interaction in vitro onto the crystal structure. Amino acid substitutions leading to reduced (purple dots) or unaffected (black dots) DNA interaction 1 are shown on a linear representation of Sap1. (D) The amino acids whose substitution leads to loss of DNA binding 1 are shown as green sticks in a cartoon representation of the Sap1 structure. 


Figure S3 :

 S3 Figure S3: Analysis of Sap1 suppressors. (A) Tetrad dissection of diploids of the indicated strains retesting suppression of the abp1D cbh1D phenotype by mutations in sap1. (B) Electrophoretic mobility shift assay with radiolabeled Ter1 fragment for Sap1 and Sap1-c with corresponding fit of binding isotherm to gel data. (C) EMSA as (D) for radiolabeled Tf2 DNA fragment. 


Table S1 . Strain genotypes

 S1 
	Table S2. Oligonucleotides
	Identifier	Sequence
	Strain GTO-563	Genotype ACAGAGAACAGATTGGTGGAGCCAAGGCACAGCGTACTCAC
	MJ15 GTO-565 MJ17 GTO-569 MJ18 GTO-570 GTO-815	GAGCTCGATTCGATCCTATTAGAGACGTTCATTAATTTCATCATTGC h 90 sap + [sap1-C81M loop-in] ura4-D18 ade6-DN/N TCCACCAATCTGTTCTCTGTGAGC h 90 sap + [sap1-K132A loop-in] ura4-D18 ade6-DN/N TAATAGGATCGAATCGAGCTCCGTCGACAAGC h 90 sap + [sap1-F133A Y129 loop-in] ura4-D18 ade6-DN/N GTCTTTCCCGGGTCATCGCTATCGCACGTTCCAGATGTG
	MJ26 GTO-816	h 90 sap1-K132A ura4-D18 ade6-DN/N CAGGGCTGCAGATACCTACGAGGAAGGAAGTAACCAGC
	MJ28 GTO-844 MJ30 GTO-845 MJ31 GTO-848 GTO-849	TTCGCGATACTGTTGGAGCTCGCTG h 90 sap + [ sap1-L34A L112A loop-in] ura4-D18 ade6-DN/N GCTTTCTTGGTTGGCAAGCGCAAGTGTG h 90 sap + [ sap1-C81R loop-in] ura4-D18 ade6-DN/N GCCTTCCATTCATCGCAAAGTTGGTCGAGCTCATCTTTTTCGGGAG h 90 sap + [ sap1-K26A-R29A loop-in] ura4-D18 ade6-DN/N CCGTCTCAGCGAGCTCCAACAGTATCGCGAAGAGTTCTTGGTTGGC
	GTO-850 MJ42 GTO-851 MJ62 GTO-852 MJ77 GTO-853 GTO-854	h + sap1-F133L abp1Δ::KanMX6 cbh1Δ::Nat his3Δ leu1-32 ura4-DS/E ade6-CGAGCCTGTTGGAGCTCGCTGAGACG CGAAAAAGCTTTGGTTGGCAAGCGC 210 GGCGGACTCGGTATGAATAGCGAGG h + sap1-F133L abp1Δ::LEU2 cbh1Δ::LEU2 leu1-32 ura4-DS/E ade6-216 CGCCGCAATCAAATCATCTCCATCATG h 90 sap1-D53G-5FLAG-hph1 leu1-32 ura4-D18 ade6-216 ATGCGCAATCAAATCATCTCCATCATG
	MJ75 GTO-855	h 90 sap1-5FLAG-hph1 leu1-32 ura4-D18 ade6-216 CGGACAATCGAATCCAAATTCCACC
	MJ88 GTO-906 MJ89 GTO-907 GTO-1362 PG3764 h 90 ura4-D18 ade6-DN/N GCAATCCACATGACGCCACCATCG h 90 sap1-E36K-5FLAG-hph1 leu1-32 ura4-D18 ade6-216 GGCGTAATACGACTCACTATAGGG h 90 sap1-R122H-E131K-5FLAG-hph1 leu1-32 ura4-D18 ade6-216 CACCATGCCAAAAATTACACAAGATAGAATGG5 GTO-1376 GAAGCAACCCAACTAGCGCACATACAG
	TV418 GTO-1377	h + abp1Δ::LEU2 cbh1Δ::LEU2 leu1-32 ura4-DS/E ade6-216 GAGGTTGTGTGTTCACAGGTAGGAGG
	ZB371 O.10A ZB724 Ter-1-fw Ter-1-rev Tf-2-fw ZB762 Tf-2-rev	ACAGAGAACAGATTGGTGGATTATCTCCCAGTTCCTCTCCC h + sap1-E109D abp1Δ::LEU2 cbh1Δ::LEU2 leu1-32 ura4-DS/E ade6-216 AACCAGGGATTCAGTGCAGCTATCTTG h + abp1Δ::KanMX6 cbh1Δ::Nat his3Δ leu1-32 ura4-DS/E ade6-210 CAAGATAGCTGCACTGAATCCCTGGTT h + sap1-E36K abp1Δ::KanMX6 cbh1Δ::Nat his3Δ leu1-32 ura4-DS/E ade6-AGCAATACTACACTACGCTATAATACACTACGTTGAGTATCACTATATGT 216 ACATATAGTGATACTCAACGTAGTGTATTATAGCGTAGTGTAGTATTGCT
	O.109 ZB786	h + sap1-D53G abp1Δ::KanMX6 cbh1Δ::Nat his3Δ leu1-32 ura4-DS/E ade6-GAGCTCGATTCGATCCTATTAGAATTTTTCGCGATACTGTTGG 216 CGAATCCAAA TTCCACCTAT AAATTATTCA AGACGGATCC
	ZB787	CCTATTTTCT ATGGAAGCTC h + sap1-R122H-E131K abp1Δ::KanMX6 cbh1Δ::Nat his3Δ leu1-32 ura4-CCAAGATGGA ACTGAAGAGC TATAAACGAA AGAATGCTTC DS/E ade6-216 GTTATCTCCC AGTTCCTCTC
		CCGCCAAGGC ACAGCGTACT CACTTGTCCG CTGAGGAAAA
		AATCAAGCTC ATGCGCTTAG
		TCGTGCGTCA CAAGCACGAA CTCGTCGACC GTAAAACTAG
	gGT4	TGAGTTTTAC GCCAAGATCG CCCGTATCGG TTATGAGGAC GAGGGCCTCG CTATTCATAC
		CGAGTCCGCC TGTCGCAATC
		AAATCATCTC CATCATGCGC GTCTACGAAC AGCGTTTGGC
		CCATCGTCAA CCCGGCATGA
		AGACCACTCC CGAAGAGGAT GAGGCTGACC AACTTTGCGA
		TGAATGGAAG GCCCGTCTCA
		GCGAGCTCCA ACAGTATCGC GAAAAATTCT TGGTTGGCAA
		GCGCAAGTGT GATTGC
Maria M Jørgensen, Babatunde Ekundayo, Mikel Zaratiegui, Karen Skriver, Geneviève Thon, Thomas Schalch
Structure of the replication regulator Sap1 reveals functionally important interfaces