Inhibiting thymidylate synthase lowers dTTP without lowering the other deoxynucleotides. The imbalance, not just the shortage, is what damages DNA: dUTP is misincorporated, uracil-DNA glycosylase excises it, and the resulting abasic sites and strand breaks accumulate. Replication forks stall at these lesions and can collapse into double-strand breaks, driving the cell toward apoptosis.
The lethal consequence depends on replication. Cells in G1, G2, M, or G0 are not actively copying DNA, so they are largely spared during the exposure window. A tumor with a high growth fraction continuously feeds cells into S phase, which is why phase-specific antimetabolites require prolonged or repeated exposure to catch successive cohorts.
Leucovorin rescue
Leucovorin is a reduced folate that bypasses the methotrexate block on dihydrofolate reductase and restores thymidylate synthesis. After a high-dose methotrexate infusion, it is given to rescue normal tissues, which have accumulated less drug than the tumor. The tumor remains blocked long enough to die. Timing is critical: rescue given too early or too generously can protect the tumor as well.
Antimetabolite selectivity is not absolute; it is a consequence of the S-phase restriction of the lethal lesion. The therapeutic window depends on the tumor having a higher fraction of cycling cells than the dose-limiting normal tissues, and on the drug remaining present long enough to catch those cells in S phase.