Isolation of a New Hemimethylated DNA Binding Protein Which Regulates dnaA Gene Expression

Abstract

In this report, we show that yccV, a gene of unknown function, encodes a protein having an affinity for a hemimethylated oriC DNA and that the protein negatively controls dnaA gene expression in vivo.The minimum replication origin of Escherichia coli (oriC) has an elevated number of DNA adenine methylation (Dam) sites (18) that become hemimethylated immediately after initiation of replication and stay in this state for one-quarter of the generation time; these sites differ from most Dam sites located elsewhere on the chromosome (6). The finding that the hemimethylated oriC interacts with an outer membrane preparation of E. coli (20) prompted a search for hemimethylated DNA binding proteins in the membrane fraction. The existence of such a category of proteins in the membrane has been reported previously; in fact, Southwestern blot analysis of the membrane preparation demonstrated the presence of 25-and 16-kDa peptides reacting with a hemimethylated oriC probe (9). These proteins might participate in the hemimethylated oriC membrane sequestration that prevents initiation of chromosome replication in E. coli (16). Lu et al. (17) isolated the seqA gene that codes for a 21-kDa peptide and has an affinity for the hemimethylated oriC DNA (24). In a seqA mutant, the duration of the hemimethylation period of oriC is shortened, and the reinitiation of replication occurs at oriC repeatedly in a single replication cycle (17). These phenotypes may be explained by a lack of hemimethylated oriC sequestration by either SeqA or a SeqA membrane complex; the membrane obtained from the seqA mutant failed to bind the hemimethylated oriC DNA in vitro (2, 24). Although these results show the predominant role of the SeqA protein in oriC sequestration, the auxiliary role played by the membrane in the process should not be disregarded. Shakibai et al. (23) observed that the hemimethylated oriC binding activity of SeqA is stimulated by addition of a membrane protein preparation designated SeqB. It has also been found that the membrane obtained from a seqA mutant stimulates the protective activity of a His-tagged SeqA protein against DNase I attack of hemimethylated oriC DNA (7).In order to isolate auxiliary factors of SeqA, we adopted a strategy of searching for factors among the dnaA46 suppressor gene products. The rationale behind this approach was based on the observation that the seqA mutation partially suppresses the temperature sensitivity of dnaA46 (17). Likewise, mutation of the auxiliary factor gene should also result in suppression of dnaA46. Previously, this type of work has been undertaken by Katayama's group, who used a system involving random insertion of Tn10-Tet r into the chromosome. In this way, they found one new suppressor mutation for dnaA46, hslU (12).Isolation of dnaA46 suppressors by mini-Tn10 insertion. Briefly, random insertion of mini-Tn10 into the chromosome containing dnaA46 (KA413⌬H) ( Table 1) was accomplished by infection of the mutant with a lambda phage (1098) carrying mini-Tn10 (27), followed by plat...

Deletion of a Novel F-Box Protein, MUS-10, in Neurospora crassa Leads to Altered Mitochondrial Morphology, Instability of mtDNA and Senescence

“…These findings are concurrent with the hypothesis that SCF complexes can regulate mitochondrial function (Cohen et al 2008;Deng et al 2008). A YccV-like domain, which has been shown to bind hemimethylated DNA (D'Alencon et al 2003), was also observed in MUS-10, suggesting that it may be capable of interacting with DNA ( Figure 5). …”

Analysis of Global Gene Expression and Double-Strand-Break Formation in DNA Adenine Methyltransferase- and Mismatch Repair-DeficientEscherichia coli

“…example, dnaA expression is reduced in both dam and dam mutS strains (signal log ratio ϭ Ϫ0.368, P ϭ 0.035, and Ϫ0.412, P ϭ 0.025, respectively), as shown previously in dam mutant strains (8,25). Methylation of d(GATC) sites in the dnaAp2 promoter, two of which are in the Ϫ35 and Ϫ10 sequences, may affect the affinity of DNA binding proteins that regulate the expression of dnaA (8,12). In addition, we find expression of the phase variation flu gene (agn43) to be slightly downregulated (signal log ratio ϭ Ϫ0.383, P ϭ 0.016) in dam mutant E. coli.…”

DNA polymerase δ-interacting protein 2 is a processivity factor for DNA polymerase λ during 8-oxo-7,8-dihydroguanine bypass

“…When PolDIP2 was tested with Pol ι in the presence of its preferred metal ion, Mn 2+ (Fig. S2C), only very slight stimulation was observed for both dATP and dCTP incorporation, either opposite a normal G (lanes 2-10) or opposite 8-oxo-G (lanes [12][13][14][15][16][17][18][19][20]. Thus, PolDIP2 did not influence the fidelity of TLS by Pol ι across the lesion (9).…”

“…The N-terminal half (amino acids 74-200) of PolDIP2 shares 34% similarity with the YccV Escherichia coli protein (1), which was shown to bind DNA (13). However, electrophoretic mobility shift assays (EMSAs) with the 5′-labeled 39/100-mer p/t, whether undamaged (Fig.…”

“…S4B), being able to synthesize long products even in the presence of the competitor DNA (lanes 5-7). However, addition of PolDIP2 increased the time-dependent accumulation of long products (lanes [11][12][13]. Plotting the size distribution and relative amounts of the synthesized products over time for Pols λ and η, respectively, as detected in the presence of the competitor DNA ( Fig.…”