Conserved Active Site Loop Residues of E. coli DinB and Human DNA Polymerase Kappa Govern Activity.

DNA damage is ubiquitous and can arise from numerous sources. To mitigate the potential effects of DNA damage, cells possess varied DNA repair and damage tolerance mechanisms. Conserved throughout evolution, specialized DNA damage-bypass DNA polymerases from the Y family provide DNA damage tolerance. E. coli harbors two Y-family polymerases whereas humans have four members of the Y family. E. coli DinB and human DNA polymerase kappa have shown similar damage bypass profiles in that they both are specific for minor groove adducts and are inhibited by major groove adducts. These two proteins share a similar active site loop that is adjacent to the nascent base pair. These active site loops were analyzed by alanine scanning mutagenesis with the resulting proteins characterized in primer extension assays and for their thermal stability. Most variants show similar activity to the respective wild-type proteins, with a few mutations resulting in dramatic losses of activity and changes in stability. The effects of the mutations are remarkably similar in DinB and polymerase kappa, with mutation of specific aligned residues showing decreased activity and/or stability. Most of the variants have similar thermal stability as the respective wild-type proteins and show the characteristic increase in stability in the presence of substrates, with the less active variants in general showing limited stabilization by DNA or DNA and incoming nucleotides.