Human DNA polymerase-η, an A-T mutator in somatic hypermutation of rearranged immunoglobulin genes, is a reverse transcriptase

We have proposed previously that error-prone reverse transcription using pre-mRNA of rearranged immunoglobulin variable (IgV) regions as templates is involved in the antibody diversifying mechanism of somatic hypermutation (SHM). As patients deficient in DNA polymerase-η exhibit an abnormal spectrum of SHM, we postulated that this recently discovered Y-family polymerase is a reverse transcriptase (RT). This possibility was tested using a product-enhanced RT (PERT) assay that uses a real time PCR step with a fluorescent probe to detect cDNA products of at least 27-37 nucleotides. Human pol-η and two other Y-family enzymes that are dispensable for SHM, human pols-ι and -κ, copied a heteropolymeric DNA-primed RNA template in vitro under conditions with substantial excesses of template. Repeated experiments gave highly reproducible results. The RT activity detected using one aliquot of human pol-η was confirmed using a second sample from an independent source. Human DNA pols-β and -μ, and T4 DNA polymerase repeatedly demonstrated no RT activity. Pol-η was the most efficient RT of the Y-family enzymes assayed but was much less efficient than an HIV-RT standard in vitro. It is thus feasible that pol-η acts as both a RNA- and a DNA-dependent DNA polymerase in SHM in vivo, and that Y-family RT activity participates in other mechanisms of physiological importance.