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Human DNA polymerase‐η, an A‐T mutator in somatic hypermutation of rearranged immunoglobulin genes, is a reverse transcriptase
Author(s) -
Franklin Andrew,
Milburn Peter J,
Blanden Robert V,
Steele Edward J
Publication year - 2004
Publication title -
immunology and cell biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.999
H-Index - 104
eISSN - 1440-1711
pISSN - 0818-9641
DOI - 10.1046/j.0818-9641.2004.01221.x
Subject(s) - somatic hypermutation , reverse transcriptase , dna polymerase , biology , polymerase , gene , immunoglobulin gene , genetics , virology , polymerase chain reaction , antibody , b cell
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.