Slides from a talk I gave in 1995 at the CALPEG meeting.

1. Evolution of the RecA Protein:
from Systematics to Structure
Jonathan A. Eisen
Department of Biological Sciences
Stanford Univeristy

2. 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.

3. 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.

4. 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.

5. Other Molecules for Molecular
Systematic 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)

6. 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.

7. 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.

8. 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.

9. 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

10. "Replacement" Sequences
RecA Sequence rRNA Replacement
Acetobacter polyoxogenes A. pasteurianus
Azotobacter vinelandii Flavobacterium lutescens
Methylomonas clara M. methylovora
Myxococcus xanthus 2 Cystobacter fuscus
Proteus mirabilis Arsenophonus nasoniae
Pseudomonas fluorescens P. flavescens
Thiobacillus ferrooxidans T. caldus
Streptococcus pneumoniae S. salivarius
Streptomyces violaceus S. coelicolor
Arabidopsis thaliana Nicotiana tabacum CPST
Anabaena variabilis A. sp. PCC7120
Synechococcus sp. PCC7942 Phormidium minutum
Synechococcus sp. PCC7002 S. sp. PCC6301

11. 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.

12. Consensus Phylogenetic Groups
RecA Clade ComparableBoots% RecABoots ssRNA
SS-RNA cladePPNJFMDPNJFM
Proteobacteria - g1 Yes 78 91 100 100 100 100
Proteobacteria - g2 Yes 100 100 100 100 100 100
Proteobacteria - g Yes (+ Lp) 33 63 75 48 85 92
Proteobacteria - b1 Yes (+ Ng) 74 84 88 100 100 100
Proteobacteria - b2 No 100 100 100 0 0 0
Proteobacteria - bg Yes (- Af) 53 86 95 90 94 95
Proteobacteria - a Yes (+Af) 14 68 72 100 100 100
Proteobacteria - abg Yes 10 57 58 93 96 96
Proteobacteria - d Yes 43 71 42 * * *
Proteobacteria - e Yes 100 100 100 100 100 100
Proteobacteria No 14 38 49 0 0
36
Gram "+" High GC Yes 97 100 100 100 100 100
Gram "+" Low GC Yes (+ Mycs) 27 59 63 50 56 80
Mycoplasmas Yes (+ Al) 88 100 98 71 88 84
Cyanobacteria Yes 100 96 91 100 100 100
Deinococcus-Thermus No 95 96 95 0 0

13. 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.

14. 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).

15. Acknowledgements
Philip Hanawalt
Alberto Roca
Marc Feldman
Mitch Sogin
Michael Eisen
Dan Distel
Jeff Palmer
W. 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