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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 and L2 inhibit the DnaA-dependent unwinding of oriC. The localized unwinding of oriC in a supercoiled plasmid was measured as described in ‘Materials and Methods’ section. In the leftmost lane of (A) and (B), which shows the reverse contrast image of ethidium bromide-stained agarose gels, M13oriC2LB5 DNA was linearized with HindIII endonuclease. In (C), densitometric analysis of the ethidium-bromide-stained gels was performed to quantify the relative abundance of the linear DNA. Net intensity (×10−3) is described in Figure 1.
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Figure 6: tL2 and L2 inhibit the DnaA-dependent unwinding of oriC. The localized unwinding of oriC in a supercoiled plasmid was measured as described in ‘Materials and Methods’ section. In the leftmost lane of (A) and (B), which shows the reverse contrast image of ethidium bromide-stained agarose gels, M13oriC2LB5 DNA was linearized with HindIII endonuclease. In (C), densitometric analysis of the ethidium-bromide-stained gels was performed to quantify the relative abundance of the linear DNA. Net intensity (×10−3) is described in Figure 1.

Mentions: We examined tL2 and L2 in assays that measure specific events in the initiation process. One involves the formation of a highly negatively supercoiled DNA named Form 1* (42), which requires the localized unwinding of a region within oriC by DnaA (5,6,34). DnaB helicase from the DnaB–DnaC complex binds to the unwound region, and then unwinds the parental duplex DNA. The inclusion of DNA gyrase to remove the positive superhelicity that would otherwise accumulate in the duplex portion of the oriC plasmid leads to the appearance of Form 1* DNA. Detected by agarose gel electrophoresis (Figure 5), the production of Form 1* DNA was inhibited by tL2 and L2. At the higher levels of tL2 and all but the highest level of L2, the increased amounts of nicked and linear DNA suggest that a nuclease is responsible. However, other experiments failed to detect a nuclease in various preparations of purified tL2 or L2 (Figure 6; data not shown). Instead, we ascribe the increase of these DNAs to DNA gyrase because their appearance was dependent on both of its subunits (data not shown) that introduce breaks in DNA during strand passage that are revealed by treatment with SDS [see (43) and references therein]. The effect of tL2 or L2 on the level of nicked and linear DNA suggests that the ribosomal protein may stabilize DNA gyrase complexed to DNA at the step of strand cleavage, but we have not tested this idea.Figure 5.


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 and L2 inhibit the DnaA-dependent unwinding of oriC. The localized unwinding of oriC in a supercoiled plasmid was measured as described in ‘Materials and Methods’ section. In the leftmost lane of (A) and (B), which shows the reverse contrast image of ethidium bromide-stained agarose gels, M13oriC2LB5 DNA was linearized with HindIII endonuclease. In (C), densitometric analysis of the ethidium-bromide-stained gels was performed to quantify the relative abundance of the linear DNA. Net intensity (×10−3) is described in Figure 1.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105425&req=5

Figure 6: tL2 and L2 inhibit the DnaA-dependent unwinding of oriC. The localized unwinding of oriC in a supercoiled plasmid was measured as described in ‘Materials and Methods’ section. In the leftmost lane of (A) and (B), which shows the reverse contrast image of ethidium bromide-stained agarose gels, M13oriC2LB5 DNA was linearized with HindIII endonuclease. In (C), densitometric analysis of the ethidium-bromide-stained gels was performed to quantify the relative abundance of the linear DNA. Net intensity (×10−3) is described in Figure 1.
Mentions: We examined tL2 and L2 in assays that measure specific events in the initiation process. One involves the formation of a highly negatively supercoiled DNA named Form 1* (42), which requires the localized unwinding of a region within oriC by DnaA (5,6,34). DnaB helicase from the DnaB–DnaC complex binds to the unwound region, and then unwinds the parental duplex DNA. The inclusion of DNA gyrase to remove the positive superhelicity that would otherwise accumulate in the duplex portion of the oriC plasmid leads to the appearance of Form 1* DNA. Detected by agarose gel electrophoresis (Figure 5), the production of Form 1* DNA was inhibited by tL2 and L2. At the higher levels of tL2 and all but the highest level of L2, the increased amounts of nicked and linear DNA suggest that a nuclease is responsible. However, other experiments failed to detect a nuclease in various preparations of purified tL2 or L2 (Figure 6; data not shown). Instead, we ascribe the increase of these DNAs to DNA gyrase because their appearance was dependent on both of its subunits (data not shown) that introduce breaks in DNA during strand passage that are revealed by treatment with SDS [see (43) and references therein]. The effect of tL2 or L2 on the level of nicked and linear DNA suggests that the ribosomal protein may stabilize DNA gyrase complexed to DNA at the step of strand cleavage, but we have not tested this idea.Figure 5.

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