REPAIR OF O-6-ALKYLGUANINE DURING DNA-SYNTHESIS IN MURINE BONE-MARROW HEMATOPOIETIC PRECURSORS

Abstract

O6-Alkylguanine, a DNA adduct formed by nitrosoureas, becomes the site of a point mutation during DNA synthesis by preferentially base mispairing with thymine rather than correctly base pairing with cytosine. To repair this adduct, cells contain a limited amount of O6-alkylguanine-DNA alkyltransferase (alkyltransferase), a protein which removes the alkyl group in a stoichiometric reaction. To prevent mutations, repair must occur before DNA replication takes place. Consequently, formation of point mutations is related inversely to the number of alkyltransferase molecules and directly to the rate of DNA synthesis. Bone marrow hematopoietic precursors, the target for nitrosourea-induced leukemia, are deficient in alkyltransferase activity. We questioned whether regenerating bone marrow is more susceptible to nitrosoureas than other organs due to persistently low levels of alkyltransferase activity during periods of increased cell proliferation and DNA synthesis. Following syngeneic bone marrow transplantation, murine hematopoietic cells underwent rapid cell proliferation but alkyltransferase activity remained well below the activity in liver. After N-nitrosomethylurea exposure, [3H]thymidine incorporation in rat bone marrow increased 3-fold and stem cell proliferation over 10-fold within 2 days of exposure, but alkyltransferase activity remained low. The relative susceptibility of bone marrow to mutagenic damage from O6-alkylguanine adducts was determined by comparing the ratio of alkyltransferase activity to [3H]thymidine incorporation in marrow, kidney, and liver. In untreated animals, the ratio was lowest in bone marrow and decreased further 48 h after N-nitrosomethylurea exposure to only 21% that of kidney and 1% that of liver. Thus, proliferating hematopoietic precursors appear more likely to form point mutations following nitrosourea exposure than other rodent tissues because they undergo rapid proliferation soon after DNA damage and before O6-alkylguanine adducts can be repaired. The combination of rapid cell proliferation and low DNA repair capacity may be the mechanism of nitrosourea induced leukemic transformation of the bone marrow.