Edward J. Steele

Genomic Interactions Group
Research School of Biological Sciences
GPO Box 475
Canberra ACT 2601
ph: +61 (0)2 42 673 518
fax: +61 (0)2 42 673 518

email: edward.steele@anu.edu.au

Information for prospective students

Current position

Visiting Fellow, Genomic Interactions Group

Research interests

Our current research concerns the antigen-driven somatic generation of antibody diversity. Results of companion studies on the origin of germline antibody diversity can be found in selected publication numbers 6,7,11-15.

Somatic hypermutation (SHM) of rearranged immunoglobulin (Ig) variable genes (V[D]J) is the molecular mechanism underpinning the process of the affinity maturation of antibodies during an immune response.

Published data from several laboratories have shed much light on the SHM mechanism which unfolds in two phases. Phase I requires activation-induced cytidine deaminase (AID)-mediated deamination of deoxy-cytosine to deoxy-uracil ( C-to-U) at C-sites mainly in RGYW/WRCY* hotspot motifs.   The resulting G-U mispairs in the DNA trigger Phase II, a presumed error-prone short-patch DNA repair mechanism involving DNA polymerase - η (eta) that causes mutations mainly at A-T base pairs where As are preferentially mutated at WA motifs which are established SHM hotspots.

A key feature of SHM is the strand biased mutation signature focused on A-T base pairs. Whereas AID-induced phase I mutations at C-G base pairs occur with similar frequency on both the transcribed (TS) and non-transcribed (NTS) strands those at A-T base pairs are strand imbalanced. Thus mutations at A-sites recorded on the NTS exceed mutations at T-sites by at least 2-fold (17). However we have shown that Pol-η is a reverse transcriptase (16) which raises the possibility that A-T targeted Phase II could involve error-prone DNA repair copying off RNA as well as DNA templates. Given the key role Pol-η is known to play in generating A-T mutations this result has increased the plausibility of the reverse transcriptase (RT-) model of SHM (1,8,14,16) and provided a rational explanation for the A>>T strand bias mutation signature (17).

We have investigated a role for an IgV mRNA template intermediate by analysing the somatic mutation data of the V k Ox1 transgene (published by the Milstein-Neuberger group). We compared quantitative patterns of double stranded (ds)RNA secondary structures (stem loops or hairpins) with the prominent A-to-G component of the SHM mutation spectrum. In an RNA-based pathway it is conceivable that adenosine-to-inosine (A-to-I) RNA editing causes A-to-G transitions since I like G pairs with C. Adenosine deaminases which act on RNA (ADARs) are known to preferentially deaminate A nucleotides that are preceded by an A or U (W) in imperfect dsRNA substrates. On this assumption and using a bioinformatics approach we have shown that a significant and specific correlation ( P <0.002) exists between the frequency of WA-to-WG mutations and the number of mRNA hairpins that could potentially form at the mutation site. This result implies that an RNA editing step coupled to reverse transcription is involved in somatic hypermutation of rearranged Ig genes (21).

Thus A-to-I RNA editing, long known to be of broad biological significance for both viral and cellular genes, is now strongly implicated as a key step in the generation of point mutations during SHM. The updated version of the RT-model of SHM therefore invokes both transcription-coupled DNA and RNA deamination plus reverse transcription as an explanation for somatic hypermutation (see figure).

* R = A or G, Y = T or C and W = A or T(U)

Selected Publications

1. Steele, E.J. and J.W. Pollard (1987). Hypothesis: Somatic Hypermutation by gene conversion via the error prone DNA-to-RNA-to-DNA information loop.  Molec. Immunol.   24: 667-673.
2. Both, G.W., Taylor,L.,J.W.Pollard and E.J.Steele (1990) Distribution of mutations around rearranged heavy-chain antibody variable-region genes. Mol. Cell. Biol. 10: 5187-5196.
3. Rothenfluh, H.S., Taylor, L., Bothwell, A.L.M., Both, G.W. and Steele, E.J. (1993) Somatic hypermutation in 5' flanking regions of heavy chain antibody variable genes.   Eur. J. Immunol. 23: 2152-2159.
4. Steele E.J., Pollard, J.W., Taylor, L. and Both, G.W. (1990). Evaluation of possible mutator mechanisms active on mammalian variable region genes. In: Somatic hyper-mutation in V-regions. Ed. E. J. Steele. CRC Press. Boca Raton, Fl. 1991. pp 137-148.
5. Steele. E.J., Rothenfluh,H.S. and Both, G.W. (1992). Defining the nucleic acid substrate for somatic hypermutation. Immunol. Cell Biol. 70: 129-144.
6. Steele, E.J., Rothenfluh, H.S., Ada, G.L. and Blanden, R.V. (1993) Affinity maturation of lymphocyte receptors and positive selection of T cells in the thymus.  Immunol. Rev. 135: 5-49.
7. Rothenfluh, H.S., Blanden, R.V. and Steele, E.J. (1995) Evolution of V genes: DNA sequence structure of functional germ-line genes and pseudogene. Immunogenetics 42: 159-171.
8. Steele, E.J., Rothenfluh, H.S. and Blanden, R.V. (1997) Mechanism of antigen-driven somatic hypermutation of rearranged immunoglobulin V(D)J genes in the mouse. Immunol. Cell Biol. 75: 82-95.
9. Blanden, R.V., Rothenfluh, H.S. and Steele, E.J. (1998)  On the possible role of natural reverse genetics in the V gene loci. In:   Kelsoe, G. and Flajnik, M. (Eds ) Curr. Top. Micro. & Immunol. 229: 21-32.
10. Blanden, R.V. and Steele, E.J. (1998) A unifying hypothesis for the molecular mechanism of somatic mutation and gene conversion in rearranged immunoglobulin variable genes. Immunol. Cell Biol. 76: 288-293.
11. Weiller, G.F., Rothenfluh, H.S., Zylstra, P., Gay, L., Averdunk, H., Steele, E.J. and Blanden, R.V. (1998) Recombination signature of germline immunoglobulin variable genes. Immunol. Cell Biol. 76: 179-185.
12. Blanden, R.V., Rothenfluh, H.S., Zylstra, P., Weiller, G.F., and Steele, E.J. (1998 )The signature of somatic hypermutation appears to be written into the germline IgV segment repertoire. Immunol. Rev. 162: 117-132.
13. Steele, E.J. and Blanden, R.V. (2000) Lamarck and Antibody Genes. Science 288: 2318 -2319.
14. Steele, E.J. and Blanden, R.V. (2001) The reverse transcriptase model of somatic hypermutation. Philosophical Transactions of the Royal Society (Series B): Biological Sciences.  356: 61-66.
15. Steele, E.J., Hapel, A.J. and Blanden, R.V. (2002) How can DNA patterns of somatically acquired immunity be imprinted on the germline of immunoglobulin variable (V) genes? IUBMB Life 54: 305-307.
16. Franklin, A., Milburn, P.J., Blanden, R.V. and Steele, E.J. (2004) Human DNA polymerase-η, an A-T mutator in somatic hypermutation of rearranged immunoglobulin genes, is a reverse transcriptase. Immunol. Cell Biol. 82: 219-225.
17. Steele, E.J., Franklin, A. and Blanden, R.V. (2004) Genesis of the strand biased signature in somatic hypermutation of rearranged immunoglobulin variable genes. Immunol. Cell Biol. 82: 208-218.
18. Blanden, R.V., Franklin, A. and Steele, E.J. (2004) The boundaries of the distribution of somatic hypermutation of rearranged immunoglobulin variable genes. Immunol. Cell Biol. 82: 205-208.
19. Steele, E.J. (2004) DNA Polymerase-η as a reverse transcriptase: implications for mechanisms of hypermutation in innate anti-retroviral defences and antibody SHM systems. DNA Repair 3: 687-692.
20. Steele, E.J., (2004) Processed switch-region transcripts and retrotranscripts as possible generators of heteroduplex ‘R-loop’ targets for AID deamination. Nat. Rev. Immunol. Published as online correspondence 1 Nov 2004 vol 4 (11). doi:10.1038/nri1395-c1 .
21. Steele, E.J., Lindley, R.A., Wen, J. and Weiller, G.F. (2006) Computational analyses show A-to-G mutations correlate with nascent mRNA hairpins at somatic hypermutation hotspots. DNA Repair 5: 1346-1363.
22. Steele, E.J. (2008) Reflections on the state of play in somatic hypermutation. Molec. Immunol. 45: 2723-2726.

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