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

Abstract

SummaryWe 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.

Genesis of ancestral haplotypes: RNA modifications and reverse transcription–mediated polymorphisms

“…In isolated systems in vitro the processivity of DNA Pol-η is thought to be low but the role of sliding clamps (e.g. PCNA, proliferating cell nuclear antigen) in its RT mode [49] has not been investigated either in vitro or in vivo. In anycase, as already discussed,"processivity" is a biochemical concept describing the affinity of a DNA polymerase for its template determined in isolated in vitro systems.…”

“…Frozen Block haplotypes, as just discussed, and (2) The accumulated knowledge gained at the 'microgenomic' level from the work of Steele and associates [36,37,41,46,49] and others [43][44][45]47,48] on the molecular mechanism of SHM of rearranged immunoglobulin V(D)J genes expressed in Germinal Center B lymphocytes.…”

“…associates, DNA Pol-η, DNA Pol-k, and DNA Pol-i [49]. DNA polymerase eta (-η) is the sole error-prone DNA polymerase involved in somatic hypermutation [48] and is thus the most likely cellular RT involved in the fixing of the RNA mutation patterns in the DNA of hypermutating B lymphocytes [36,37,49]. In isolated systems in vitro the processivity of DNA Pol-η is thought to be low but the role of sliding clamps (e.g.…”

“…Aberrant control of the activity or substrate specificity of this DNA polymerase may cause it to be deployed in non-mitochondrial reverse transcription as envisaged in the hypothesized genomic RT-LDC process. associates, DNA Pol-η, DNA Pol-k, and DNA Pol-i [49]. DNA polymerase eta (-η) is the sole error-prone DNA polymerase involved in somatic hypermutation [48] and is thus the most likely cellular RT involved in the fixing of the RNA mutation patterns in the DNA of hypermutating B lymphocytes [36,37,49].…”

“…Once again, as already discussed, these striking strand-biased mutation spectra are best understood as occurring first in RNA molecules which are then copied back into DNA. It is most likely that this occurs by a cellular reverse transcription (RT) process [36,37,41] carried out by the sole error-prone DNA polymerase known to be involved in SHM, DNA polymerase-η (eta) [47,48] which has been shown by in vitro experiments to be an efficient reverse transcriptase [49]. The significance of these findings is that SHM-induced strand biased mutation signatures can be potentially generated in non-Ig loci across the genome in many different genes expressed in different tissue types.…”