The linear plastid chromosomes of maize: Terminal sequences, structures, and implications for DNA replication.
Current Genetics. Oldenburg, D. J. and Bendich, A. J. University of Washington. email: [email protected]
Online Resource 3
Exonuclease digestion and PFGE of maize and tobacco ptDNAs
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Fig. S8 Exonuclease digestion and PFGE of maize and tobacco ptDNAs.
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The linear plastid chromosomes of maize: Terminal sequences, structures, and implications for DNA replication.
Current Genetics. Oldenburg, D. J. and Bendich, A. J. University of Washington. email: [email protected]
Maize (A and C) and tobacco (B and D) ptDNAs were prepared by in-gel lysis of
plastids, treated with or without exonucleases, and fractionated by PFGE. The blue arrow
indicates the 140-kb maize monomer and red arrow the 155-kb tobacco monomer. Lysis was
performed using 1% sarkosyl, either low EDTA (5 mM, pH 8) (A and B) or high EDTA (470
mM, pH 8) (C and D), and with or without 200 µg/mL proteinase K (PK). The PK was
inactivated with PMSF and gel slices washed extensively in TE (1 mM Tris, pH 8, 1mM EDTA).
Exonuclease treatment was for 60 min at 37°C with 10 units of λ exonuclease (λ exo) or 20 units
of Exonuclease III (ExoIII). λ exo digestion proceeds 5’-to-3’ and requires a 5’-PO4 at the DNA
end. ExoIII digestion proceeds 3’-to-5’ with both blunt ends and 5’-overhangs, but not 3’overhangs. All four gel images have been adjusted using Adobe Photoshop to increase the
brightness and contrast of the ethidium-DNA fluorescence uniformly throughout the entire gel in
order to enhance the visualization of the ptDNA unit-genome-sized monomer and concatemer
bands.
Since maize ptDNA, prepared under all four lysis conditions, was digested by both
exonucleases, there are no structural features blocking access of the enzymes to the ends. In
contrast, tobacco ptDNA prepared with low EDTA without PK was digested both by λ exo and
ExoIII, but was not digested when prepared with low EDTA and PK or with high EDTA and
with or without PK. Note that with ExoIII and low EDTA without PK (B Lane 3) the band
intensity of the tobacco ptDNA monomer was reduced (partial digestion), but with higher
enzyme concentration or longer incubation times the monomer band disappeared completely.
These results suggest a covalently bound 5’-protein at the end of tobacco pDNA, like that
proposed for liverwort mitochondrial DNA (Oldenburg and Bendich 2001). Under mild lysis
conditions (5 mM EDTA without PK), hydrolysis of the linkage between the protein and DNA
would release the protein, leaving a 5’-PO4 end that would then be susceptible to λ exo activity.
Under similar conditions, reversible binding of topoisomerase to a 5’-end has been reported
(Christiansen et al. 1994; Wang 1996); thus a topoisomerase-like protein may be present at the
5’-end of tobacco ptDNA. However, after PK treatment, a peptide remnant remains covalently
attached to the 5’-end blocking access of λ exo.
References for Online Resource 3
Christiansen, K., Knudsen, B. R., Westergaard, O. (1994). "The covalent eukaryotic
topoisomerase I-DNA intermediate catalyzes pH-dependent hydrolysis and alcoholysis." J Biol
Chem 269: 11367-11373.
Oldenburg, D. J., Bendich, A. J. (2001). "Mitochondrial DNA from the liverwort Marchantia
polymorpha: Circularly permuted linear molecules, head-to-tail concatemers, and a 5' protein."
Journal of Molecular Biology 310: 549-562.
Wang, J. C. (1996). "DNA topoisomerases." Ann Rev Biochem 65: 635-692.
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