Evolution of the RecA Protein: from Systematics to Structure 1995 talk for CALPEG
Evolution of the RecA Protein:from Systematics to Structure Jonathan A. Eisen Department of Biological Sciences Stanford Univeristy
E. coli RecA General Information - 352 amino-acids. - High resolution crystal structure available. - 100s of mutants characterized and sequenced. Genetic Roles - Homologous recombination. - Recombinational repair of DNA damage. - Induction of the SOS response. - DNA damage induced mutagenesis. Biochemical Activities - DNA binding (single- and double-stranded). - Pairing and exchange of homologous DNA. - ATP hydrolysis. - Coproteolytic cleavage of LexA, UmuD, and cI.
RecA Homologs and Analogs Bacterial RecA Homologs - Proteobacteria ( and subgroups). - Gram-positives (low-GC and high-GC). - Cyanobacteria and Chloroplasts. - Spirochaetes, Chlamydia and Bacteroides. - Deinococcus-Thermus Group. - Thermotogales and thermophilic oxygen reducers. Eukaryotic RecA-like Proteins - RAD51, RAD57, DMC1. - Functional and 3D structural similarity to RecA. - Sequence similarity to RecA is low. Archaeal RecA-like Proteins - RadA from S. solfataricus. - Sequence similarity to RecA is low.
The Use of SS-rRNA for Bacterial Systematics Advantages - Conserved sequence and structure in all organisms. - Relatively easy to clone and sequence. - Variable substitution rates within molecule. - Lateral transfers unlikely. - Can be used for in-situ hybridizations. - 1000s of sequences available. Potential Problems - Nucleotide frequency bias between species. - Non-independence of substitution patterns. - Variable substitution rates between species. - Alignments can be highly ambiguous. - Duplications, concerted evolution, and paralogy.
Other Molecules for MolecularSystematic Studies of Bacteria EF-Tu (e.g., Delwiche et al. 1995) ATPase- (e.g., Ludwig et al. 1994) GroEL (e.g., Viale et al. 1994) HSP70 (e.g., Gupta et al. 1994) RNA pol B (e.g., Klenk & Zillig 1994) 23S rRNA(e.g., Ludwig et al. 1992) 70 (e.g., Lonetto et al. 1992) GS (e.g., Brown et al. 1994) RecA (e.g., Lloyd & Sharp 1993)
RecA Evolution Lloyd & Sharp (1993) - Trees of 25 recA genes. - For Proteobacteria, branching patterns similar to those for SS-rRNA. - Low resolution for deep branches in RecA tree. - recA genes not as highly GC biased as SS-rRNA genes. - Only 6 genes from species outside the Proteobacteria. 1995 - 65 complete recA sequences available. - 25 genes from outside the Proteobacteria. - 3D crystal structure available.
The Use of RecA for Systematics Advantages - Relatively easy to clone for a protein. - Conserved function among bacteria. - Sequence conservation varies across molecule. - Alignments are unambiguous. - 3D structure available. - Sequence can be used to create rec- mutants. - RecA is cool (not shown). Disadvantages - Relatively small (352 aa). - Similarity to eukaryotic & Archaeal proteins is low. - "Only" 65 sequences available. - Multiple divergent genes in at least one species.
Methods Choose a SS-rRNA to represent each complete RecA. Generate RecA trees ( FM, NJ, DeSoete, PHYLIP protpars, PAUP). Generate SS-rRNA trees ( FM, NJ, DeSoete, PHYLIP dnapars). Compare RecA and SS-rRNA trees of same technique. Compare all RecA trees to each other (consensus tree). Compare all SS-rRNA trees to each other (consensus tree). Compare consensus trees and bootstrap values. Compare to trees of other molecules and to trees of all SS-rRNAs.
Methods Choose a SS-rRNA for each complete RecA. Generate RecA trees. - FM, NJ, DeSoete, PHYLIP protpars, PAUP, consensus. - 100 Bootstraps (FM, NJ, protpars). Generate SS-rRNA trees. - FM, NJ, DeSoete, PHYLIP dnapars, consensus. - 100 Bootstraps (FM, NJ, dnapars). Compare SS-rRNA and RecA trees. - Consensus groups. - Bootstrap values. - Trees of same technique. Compare to trees of other molecules and to
"Replacement" SequencesRecA Sequence rRNA ReplacementAcetobacter polyoxogenes A. pasteurianusAzotobacter vinelandii Flavobacterium lutescensMethylomonas clara M. methylovoraMyxococcus xanthus 2 Cystobacter fuscusProteus mirabilis Arsenophonus nasoniaePseudomonas fluorescens P. flavescensThiobacillus ferrooxidans T. caldusStreptococcus pneumoniae S. salivariusStreptomyces violaceus S. coelicolorArabidopsis thaliana Nicotiana tabacum CPSTAnabaena variabilis A. sp. PCC7120Synechococcus sp. PCC7942 Phormidium minutumSynechococcus sp. PCC7002 S. sp. PCC6301
RecA vs. SS-rRNA Trees Overall topology highly similar. Similar robustness and resolution. - Nearly identical consensus clades. - Similar branching among and within clades. - Similar LOW resolution for poorly representedgroups. - RecA resolves some relationships: •Deinococcus- Thermus group. • Monophyly of Proteobacteria. - SS-rRNA resolves others: • Monophyly of low-GC gram-positives. • L. pneumophilia in gammas. Consistent differences: - Acidiphilium facilis. - Cyanobacteria with high-GC gram-positives for RecAs. - Thermotoga maritima.
Features of RecA Evolution Few insertions/deletions over time. Large variation in rates between sites. Protein introns found in Mycobacteria. Conserved indels within cyanobacteria. Myxococcus xanthus has two highly diverged recA genes. Gram-positives not monophyletic. Cyanobacteria group with high-GC gram-positives. Arabidopsis thaliana nuclear encoded gene probably transfered from chloroplast. Patterns of amino-acid substitutions help identify structural constraints not identified by "normal" sequence comparisons.
Conclusions Inferred trees of RecAs and SS-rRNAs from the same species are highly congruent. RecA and SS-rRNA trees have similar degrees of resolution. RecA comparisons are useful for studies of molecular systematics of bacteria. Studies of aa substitutions help understand structure-function relationships of RecA. Studying RecA is cool (not shown).
Acknowledgements Philip Hanawalt Alberto Roca Marc Feldman Mitch Sogin Michael Eisen Dan Distel Jeff PalmerW. Finch, W. Huang, S. Mongkolsuk, J. Coleman, & A. Clark for unpublished recA sequences.Steve Smith and Joe Felsenstein for free computer programs. National Science Foundation National Institutes of Health