Edward J. Steele

Genomic Interactions Group
Research School of Biological Sciences
GPO Box 475
Canberra ACT 2601
ph: +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. Until recently the concensus of the SHM field has been that whereas AID-induced phase I mutations at C-G base pairs appear to 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 VkOx1 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).

In a critical analysis of somatic hypermutation data published by the field since 1984 (23) data sets were classified as to whether or not they contained significant contamination with polymerase-chain reaction recombinant artifacts (PCR hybrids): such hybrid DNA molecules lead to misleading DNA sequence data and suppress strand biased mutation patterns (23). Three major strand biased mutation signatures are evident during full blown somatic hypermutation in vivo in normal and genetically deficient mice. The first concerns the mutations generated at G-C base pairs in mice genetically deficient in uracil-DNA glycosylase and MSH2-MSH6-mediated mismatch repair. Such mice display the AID deaminase footprint and here C mutations exceed G mutations at least 1.5 fold. This supports earlier and more recent studies of others claiming that C-to-U deaminations occur preferentially in the single stranded DNA regions of the displaced nontranscribed strand (NTS) during transcription. The second concerns the signature generated in immunised mice where G mutations exceed C mutations by at least 1.7 fold. This is a newly identified strand bias which has previously gone undetected because of significant PCR hybrid contamination. It is consistent with the polynucleotide polymerisation signature of RNA polymerase II copying the template DNA strand carrying AID-mediated lesions generated at C bases viz. uracils and abasic sites (Figure). A reverse transcription step would then need to intervene to fix this mutation pattern in DNA. The third concerns the long recognised strand biased signature at A-T bases (above) generated in normal aged or actively immunised mice whereby A mutations exceed T mutations by at least 2 fold. As before (21) it is argued that this pattern is best understood as a combination of adenosine-to-inosine (A-to-I) RNA editing followed by a reverse transcription step fixing the A-to-G, as well as the transversions A-to-T and A-to-C, as strand biased mutation signatures in DNA. The most economical SHM mechanism therefore involves both DNA and RNA deaminations coupled to a reverse transcription process, most likely involving DNA polymerase-η acting in its reverse transcriptase mode (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., Pollard, J.W. 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.
23. Steele, E.J. (2009) Mechanism of somatic hypermutation; Critical analysis of strand biased mutation signatures at A:T and G:C base pairs. Molec. Immunol. 46: 305-320.

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