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Two forms of ribosomal protein L2 of Escherichia coli that inhibit DnaA in DNA replication.

Chodavarapu S, Felczak MM, Kaguni JM - Nucleic Acids Res. (2011)

Bottom Line: We purified an inhibitor of oriC plasmid replication and determined that it is a truncated form of ribosomal protein L2 evidently lacking 59 amino acid residues from the C-terminal region encoded by rplB.We show that this truncated form of L2 or mature L2 physically interacts with the N-terminal region of DnaA to inhibit initiation from oriC by apparently interfering with DnaA oligomer formation, and the subsequent assembly of the prepriming complex on an oriC plasmid.Both forms of L2 also inhibit the unwinding of oriC by DnaA.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824-1319, USA.

ABSTRACT
We purified an inhibitor of oriC plasmid replication and determined that it is a truncated form of ribosomal protein L2 evidently lacking 59 amino acid residues from the C-terminal region encoded by rplB. We show that this truncated form of L2 or mature L2 physically interacts with the N-terminal region of DnaA to inhibit initiation from oriC by apparently interfering with DnaA oligomer formation, and the subsequent assembly of the prepriming complex on an oriC plasmid. Both forms of L2 also inhibit the unwinding of oriC by DnaA. These in vitro results raise the possibility that one or both forms of L2 modulate DnaA function in vivo to regulate the frequency of initiation.

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tL2 specifically inhibits DNA replication of an oriC-containing plasmid. In (A), assays to measure DNA replication of an oriC plasmid (M13oriC2LB5 DNA; 46 fmol or 200 ng) were performed with DnaA (1 pmol; 50 ng), and the indicated amounts of tL2 or L2 as described in ‘Materials and Methods’ section. In (B), reactions were assembled as in (A) but lacked DnaA and instead were supplemented with RNA polymerase (0.56 µg), which supports DNA synthesis that is independent of DnaA and the oriC sequence. Reactions in (C) were assembled as in (A), but contained a single-stranded DNA carrying a DnaA box in a hairpin (M13 A-site DNA; 17 fmol or 80 ng) instead of the oriC-containing plasmid and lacked HU and DNA gyrase. Reactions were incubated at 30°C for 20 (A and B) or 10 min (C) to measure DNA synthesis.
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Figure 3: tL2 specifically inhibits DNA replication of an oriC-containing plasmid. In (A), assays to measure DNA replication of an oriC plasmid (M13oriC2LB5 DNA; 46 fmol or 200 ng) were performed with DnaA (1 pmol; 50 ng), and the indicated amounts of tL2 or L2 as described in ‘Materials and Methods’ section. In (B), reactions were assembled as in (A) but lacked DnaA and instead were supplemented with RNA polymerase (0.56 µg), which supports DNA synthesis that is independent of DnaA and the oriC sequence. Reactions in (C) were assembled as in (A), but contained a single-stranded DNA carrying a DnaA box in a hairpin (M13 A-site DNA; 17 fmol or 80 ng) instead of the oriC-containing plasmid and lacked HU and DNA gyrase. Reactions were incubated at 30°C for 20 (A and B) or 10 min (C) to measure DNA synthesis.

Mentions: The inhibitory effect of tL2 on DNA replication of the oriC-containing plasmid suggests that L2 is also inhibitory, which we confirmed (Figure 3A). We also compared both proteins in other DNA replication assays. In a replication system that includes RNA polymerase and any plasmid DNA, but does not require DnaA (36,37), both forms of L2 were modestly stimulatory (Figure 3B). In contrast, only L2 inhibited DNA replication of a single-stranded DNA carrying a DnaA box in a hairpin structure (Figure 3C). With this single-stranded DNA for which only a subset of DnaA functions is necessary (38), DnaA bound to the hairpin loads the DnaB–DnaC complex (38). After DnaC dissociates from DnaB, primase interacts with DnaB to form primers that are extended by DNA polymerase III holoenzyme to convert the single-stranded DNA to a duplex molecule. These results indicate that tL2 specifically inhibits oriC plasmid replication, whereas L2 additionally inhibits DNA replication of the single-stranded DNA. The following experiments investigate their mechanism of inhibition.Figure 3.


Two forms of ribosomal protein L2 of Escherichia coli that inhibit DnaA in DNA replication.

Chodavarapu S, Felczak MM, Kaguni JM - Nucleic Acids Res. (2011)

tL2 specifically inhibits DNA replication of an oriC-containing plasmid. In (A), assays to measure DNA replication of an oriC plasmid (M13oriC2LB5 DNA; 46 fmol or 200 ng) were performed with DnaA (1 pmol; 50 ng), and the indicated amounts of tL2 or L2 as described in ‘Materials and Methods’ section. In (B), reactions were assembled as in (A) but lacked DnaA and instead were supplemented with RNA polymerase (0.56 µg), which supports DNA synthesis that is independent of DnaA and the oriC sequence. Reactions in (C) were assembled as in (A), but contained a single-stranded DNA carrying a DnaA box in a hairpin (M13 A-site DNA; 17 fmol or 80 ng) instead of the oriC-containing plasmid and lacked HU and DNA gyrase. Reactions were incubated at 30°C for 20 (A and B) or 10 min (C) to measure DNA synthesis.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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Figure 3: tL2 specifically inhibits DNA replication of an oriC-containing plasmid. In (A), assays to measure DNA replication of an oriC plasmid (M13oriC2LB5 DNA; 46 fmol or 200 ng) were performed with DnaA (1 pmol; 50 ng), and the indicated amounts of tL2 or L2 as described in ‘Materials and Methods’ section. In (B), reactions were assembled as in (A) but lacked DnaA and instead were supplemented with RNA polymerase (0.56 µg), which supports DNA synthesis that is independent of DnaA and the oriC sequence. Reactions in (C) were assembled as in (A), but contained a single-stranded DNA carrying a DnaA box in a hairpin (M13 A-site DNA; 17 fmol or 80 ng) instead of the oriC-containing plasmid and lacked HU and DNA gyrase. Reactions were incubated at 30°C for 20 (A and B) or 10 min (C) to measure DNA synthesis.
Mentions: The inhibitory effect of tL2 on DNA replication of the oriC-containing plasmid suggests that L2 is also inhibitory, which we confirmed (Figure 3A). We also compared both proteins in other DNA replication assays. In a replication system that includes RNA polymerase and any plasmid DNA, but does not require DnaA (36,37), both forms of L2 were modestly stimulatory (Figure 3B). In contrast, only L2 inhibited DNA replication of a single-stranded DNA carrying a DnaA box in a hairpin structure (Figure 3C). With this single-stranded DNA for which only a subset of DnaA functions is necessary (38), DnaA bound to the hairpin loads the DnaB–DnaC complex (38). After DnaC dissociates from DnaB, primase interacts with DnaB to form primers that are extended by DNA polymerase III holoenzyme to convert the single-stranded DNA to a duplex molecule. These results indicate that tL2 specifically inhibits oriC plasmid replication, whereas L2 additionally inhibits DNA replication of the single-stranded DNA. The following experiments investigate their mechanism of inhibition.Figure 3.

Bottom Line: We purified an inhibitor of oriC plasmid replication and determined that it is a truncated form of ribosomal protein L2 evidently lacking 59 amino acid residues from the C-terminal region encoded by rplB.We show that this truncated form of L2 or mature L2 physically interacts with the N-terminal region of DnaA to inhibit initiation from oriC by apparently interfering with DnaA oligomer formation, and the subsequent assembly of the prepriming complex on an oriC plasmid.Both forms of L2 also inhibit the unwinding of oriC by DnaA.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824-1319, USA.

ABSTRACT
We purified an inhibitor of oriC plasmid replication and determined that it is a truncated form of ribosomal protein L2 evidently lacking 59 amino acid residues from the C-terminal region encoded by rplB. We show that this truncated form of L2 or mature L2 physically interacts with the N-terminal region of DnaA to inhibit initiation from oriC by apparently interfering with DnaA oligomer formation, and the subsequent assembly of the prepriming complex on an oriC plasmid. Both forms of L2 also inhibit the unwinding of oriC by DnaA. These in vitro results raise the possibility that one or both forms of L2 modulate DnaA function in vivo to regulate the frequency of initiation.

Show MeSH
Related in: MedlinePlus