Human DNA polymerase-eta, 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-eta 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-eta and two other Y-family enzymes that are dispensable for SHM, human pols-iota and -kappa, 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-eta was confirmed using a second sample from an independent source. Human DNA pols-beta and -mu, and T4 DNA polymerase repeatedly demonstrated no RT activity. Pol-eta 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-eta 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.
Franklin
Andrew
A
Division of Immunology and Genetics, John Curtin School of Medical Research, Australian National University, GPO Box 334, Canberra, ACT 2601, Australia. Andrew.Franklin@anu.edu.au
Milburn
Peter J
PJ
Blanden
Robert V
RV
Steele
Edward J
EJ
D001483:Base Sequence; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism; D015321:Gene Rearrangement; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D000094769:DNA Polymerase iota; D000099270:Y-Family DNA Polymerases
D004259:DNA-Directed DNA Polymerase; D007135:Immunoglobulin Variable Region; D012194:RNA-Directed DNA Polymerase; D000094769:DNA Polymerase iota; C404302:POLK protein, human; D000099270:Y-Family DNA Polymerases; C112338:Rad30 protein; C000722070:POLI protein, human