show Abstracthide AbstractChromosomal translocations result from the joining of DNA double-strand breaks (DSBs) and frequentlycause cancer. However, the steps linking DSB formation to DSB ligation remain undeciphered. Wereport that DNA replication timing (RT) directly regulates lymphomagenicMyctranslocations duringantibody maturation in B cells downstream of DSBs and independently of DSB frequency. Depletion ofminichromosome maintenance complexes alters replication origin activity, decreases translocations,and deregulates global RT. Ablating a single origin atMyccauses an early-to-late RT switch, loss oftranslocations, and reduced proximity with the immunoglobulin heavy chain (Igh) gene, its majortranslocation partner. These phenotypes were reversed by restoring early RT. Disruption of early RT alsoreduced tumorigenic translocations in human leukemic cells. Thus, RT constitutes a general mechanismin translocation biogenesis linking DSB formation to DSB ligation. Overall design: LAM-HTGTS in LacZ and Mcm infected CH12 cells, C13 deltaori and C23 deltaori clones, C13 restored and C2 restored clones using a Myc-bait; LacZ and Mcm infected CH12 cells, C13 deltaori and C13 restored clones using a Irf2bp2-bait