Fig. 3: Orc4 α-helix mutants change the pattern of origin firing and MCM binding.

Genome-wide origin firing profile from yeast that were α-factor blocked and released into S phase in 200 mM hydroxyurea (HU) for 90 mins. a and b Orange arrows and dotted lines indicate the de novo origin locations. a shows a new origin at location ChrXII: 95,827-117,318 in the orc4^{F485I, Y486Q} strain. b shows the completely new origin location ChrV: 248,069–251,910 in orc4^{F485A, Y486A} strain. c Whole genome replication profiles. Chromosome IV (ChrIV) is shown as a representation. Strains are in the order of shorter to longer doubling time (Supplementary Table 3) from top to bottom. d–i ChIP profile of MCM (anti-Mcm2) of NTAP-Orc4 integrated strains at ChrIV in G1 phase and it’s comparison to replication origins profile. orc4^{F485I, Y486Q} strain is used as an example of NTAP-Orc4 mutant strains to compare with Orc4^WT strain in whole-chromosome view d and zoomed-in views e–i. Green arrows indicate the example locations of origins with firing pattern changes in the orc4^{F485I, Y486Q} strain. At these locations, similar changes were also observed in other mutants (see c). Black arrows indicate the example locations of origins with firing patterns that remained the same in orc4^{F485I, Y486Q} strain. At these locations, origins with firing pattern also remained the same in other mutants (see c). Red arrows indicate the example locations of origin firing pattern changes that are unique in the orc4^{F485I, Y486Q} strain but not in other mutant strains (see c). Blue arrows indicate the examples of inactive late origins in HU in WT that have lower or no Mcm2-ChIP signal in the orc4^{F485I, Y486Q} mutant strain and are not active origins in the mutant strain.