Chromosome Transmission Fidelity 4 (CTF4) plays an important role in maintaining replication fork stability by forming a homo-trimer that tethers the lagging strand DNA polymerase α (Polα) to the CMG helicase (Cdc45/Mcm2-7/GINS). CTF4 genetically interacts with more than 70 different genes annotated to DNA replication, recombination or repair. CTF4 deletion is especially sensitive to defects in DNA replication fork progression, and joint molecule resolution. Interestingly, CTF4 overexpression (CTF4OE) and ctf4Δ share many genetic interactions, and both of these genetic perturbations result in increased Rad52-YFP foci formation (an indicator of DNA repair centers), and increased a-faker frequency (an indicator of genomic instability). However, CTF4OE is uniquely sensitive to loss of 9-1-1 DNA damage checkpoint signaling (Rad17/Mec3/Ddc1), whereas ctf4Δ is uniquely sensitive to loss of the fork protection complex (Mrc1/Tof1/Csm3). Interestingly, mutation of the conserved Ctf4 binding site on either the CMG helicase, or Polα, fails to recapitulate CTF4's genetic interactions, which suggests that CTF4's role at the replication fork may be more complex than the current CMG-Ctf4-Polα tethering model. To elucidate CTF4's other possible roles in DNA replication and repair we have overexpressed an array of CTF4 mutations in combination with a small collection of 200 CTF4 genetic interaction network mutants. To improve the resolution of these experiments we have developed a system to measure arrayed colony growth over time in various sensitizing conditions. By comparing the resulting genetic interaction profiles of CTF4 mutations we have begun to separate the function of CTF4 domains. Preliminary results suggest that Ctf4 may physically interact with several other replication factors, and may also have a role in signaling the DNA replication checkpoint. These experiments highlight the utility of overexpression as a genetic perturbation that can complement a synthetic lethal network analysis to reveal separation of function and biological insight.