Determination of human DNA polymerase utilization for the repair of a model ionizing radiation-induced DNA strand break lesion in a defined vector substrate

Human DNA polymerase and DNA ligase utilization for the repair of a major c lass of ionizing radiation-induced DNA lesion [DNA single-strand breaks con taining 3'-phosphoglycolate (3'-PG)] was examined using a novel, chemically defined vector substrate containing a single, site-specific 3'-PG single-s trand break lesion. In addition, the major human AP endonuclease, HAP1 (als o known as APE1, APEX, Ref-1), was tested to determine if it was involved i n initiating repair of 3'-PG-containing single-strand break lesions. DNA po lymerase beta was found to be the primary polymerase responsible for nucleo tide incorporation at the lesion site following excision of the 3'-PG block ing group. However, DNA polymerase delta/epsilon was also capable of nucleo tide incorporation at the lesion site following 3'-PG excision, In addition , repair reactions catalyzed by DNA polymerase beta were found to be most e ffective in the presence of DNA ligase III, while those catalyzed by DNA po lymerase delta/epsilon appeared to be more effective in the presence of DNA ligase I. Also, it was demonstrated that the repair initiating 3'-PG excis ion reaction was not dependent upon HAP1 activity, as judged by inhibition of HAP1 with neutralizing HAP1-specific polyclonal antibody.