Lysines in the lyase active site of DNA polymerase b destabilize nonspecific DNA binding, facilitating searching and DNA gap recognition

Abstract

DNA polymerase (pol) b catalyzes two reactions at DNA gaps generated during base excision repair, gap-filling DNA synthesis and lyase-dependent 5´-end deoxyribose phosphate removal. The lyase domain of pol b has been proposed to function in DNA gap recognition and to facilitate DNA scanning during substrate search. However, the mechanisms and molecular interactions used by pol b for substrate search and recognition are not clear. To provide insight into this process, a comparison was made of the DNA binding affinities of WT pol b, pol l, and pol m, and several variants of pol b, for 1-nt-gap-containing and undamaged DNA. Surprisingly, this analysis revealed that mutation of three lysine residues in the lyase active site of pol b, 35, 68, and 72, to alanine (pol b KD3A) increased the binding affinity for nonspecific DNA ~11-fold compared with that of the WT. WT pol m, lacking homologous lysines, displayed nonspecific DNA binding behavior similar to that of pol b KD3A, in line with previous data demonstrating both enzymes were deficient in processive searching. In fluorescent microscopy experiments using mouse fibroblasts deficient in PARP-1, the ability of pol b KD3A to localize to sites of laser-induced DNA damage was strongly decreased compared with that of WT pol b. These data suggest that the three lysines in the lyase active site destabilize pol b when bound to DNA nonspecifically, promoting DNA scanning and providing binding specificity for gapped DNA.