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. 2005 Mar 31;24(8):1502–1511. doi: 10.1038/sj.emboj.7600634

Figure 3.

Figure 3

Filament formation is essential for SeqA function in vivo. (A) In a wild-type strain DNA replication is synchronous (WT), whereas in the ΔseqA∷tet strain it is asynchronous (ΔseqA). An empty pET11a plasmid does not affect replication synchrony of the ΔseqA∷tet strain (+pET11a). A pET11a plasmid encoding wild-type SeqA (+pSS1) restores replication synchrony of the ΔseqA∷tet strain (+SeqA), whereas a pET11a plasmid encoding the DNA-binding mutant SeqA-N150A/N152A (pWY1420) does not (+N150A/N152A). Effects of IPTG addition (25 μM) are shown on the bottom panels. The amounts of DNA equivalent to two chromosomes are indicated with arrowheads. (B) pET11a plasmids encoding SeqA mutants with defects in filament formation (SeqA-I21R, SeqA-T18E and SeqA-A25R) do not restore DNA replication synchrony of the ΔseqA∷tet strain (+I21R, +T18E, +A25R). Overexpression of these mutants induced by addition of 25 μM IPTG restores replication synchrony (bottom panels). Mutation of Glu9 to Ala (+E9A) shows a wild-type phenotype in the absence of IPTG but loses synchrony when protein is overexpressed, whereas the SeqA-E9R mutant plasmid (+E9R) behaves similarly to the filament defective mutants. (C) EMSAs of an oligonucleotide containing two hemimethylated GATC sites separated by 12 bp when incubated with increasing quantities (10, 50, 100 and 500 ng) of wild-type SeqA, SeqA-N150A/N152A, SeqA-I21R, SeqA-T18E and SeqA-A25R.