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Hypothesis: Scm3 is a histone chaperone that favors the right handed wrap for Cse4 octamers

Explore how Scm3 acts as a histone chaperone favoring the right-handed wrap for Cse4 octamers. Investigate DNA wrapping by Scm3 and Nap1 in differing directions. Conduct one-dimensional supercoiling assays with plasmid constructs.

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Hypothesis: Scm3 is a histone chaperone that favors the right handed wrap for Cse4 octamers

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  1. Hypothesis: Scm3 is a histone chaperone that favors the right handed wrap for Cse4 octamers

  2. Removal of histones Topo I 0 (relaxed) 0 (relaxed) +1 -1 Mix R S P P+1 nuc P+2 nuc P+5 nuc P+6 nuc P+8 nuc P+9 nuc (0) (-6) Relaxed 0 0 0 -1 1 -2 2 3 4 5 -5 6 Supercoiled -6 -6 -6 -6 - choloroquine +6 0 Relaxed +1 0 +1 1 -2 2 -3 3 4 +4 5 +5 Supercoiled +6 +6 6 +choloroquine One Dimensional Supercoiling Assay Nakata et al.

  3. Plasmid constructs used for supercoiling assay BamHI 5S 5S EcoRI XhoI BamHI NdeI pG5E4-5S pCEN1-10X pG5E4-5S

  4. Do Scm3 and Nap1 wrap DNA in different directions? Scm3 Nap1 DNA. Only DNA. Only No Topo I No Topo I No Topo I DNA. Only DNA. Only No Topo I Oct. only Oct. only Oct. only Oct. only DNA:: Cse4 Octamer ratio DNA:: Cse4 Octamer ratio DNA:: Cse4 Octamer ratio DNA:: Cse4 Octamer ratio - Chloroquine pCEN1-10X pG5E4 pCEN1-10X pG5E4 + Chloroquine Shivaraju

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