Deoxyribonucleic Acid Repair Synthesis in Permeable Human Fibroblasts Exposed to Ultraviolet Radiation and N-Acetoxy-2-(acetylamino)fluorene

We have studied deoxyribonucleic acid (DNA) repair synthesis in permeable, confluent normal and repairdeficient human fibroblasts using [³H]dCTP, BrdUTP, dATP, and dGTP as substrates. In this system repair synthesis occurred as judged by the following criteria: (1) labeled nucleotides were incorporated into parental DNA (p = 1.73 g/cm³ in alkaline CsCl); (2) incorporation into parental DNA was negligible in the absence of damage and increased 10-20-fold in normal cells following exposure to ultraviolet radiation (UV) or the direct-acting chemical carcinogen N-acetoxy-2-(acetylamino)fluorene; and (3) damage-dependent DNA synthesis was absent in preparations made from excision repair-deficient human diploid fibroblasts (xeroderma pigmentosum cells, complementation group A). The reaction was linear for 10 min and continued for at least 1 h. Repair synthesis was stimulated at least fivefold by the addition of 5 mM ATP. It was strongly Mg²⁺ dependent, inhibited by NaCl, and only partially dependent upon the addition of exogenous dNTPs. The pH optimum in Tris-HCl buffer was 7.6. Following damage (UV), labeled deoxycytidine and dCMP were incorporated into parental DNA but at reduced levels (38 and 88%, respectively) compared to dCTP. The addition of β-NAD⁺ (the naturally occurring isomer) or α-NAD⁺ (a competitive inhibitor) had little effect on repair synthesis in this system. By saturating the system with dNTPs and using published estimates of patch size, we calculated that in this system a normal cell can put in a minimum of 100-900 repair patches/min.