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Interaction between Escherichia coli DNA polymerase IV and single-stranded DNA-binding protein is required for DNA synthesis on SSB-coated DNA.

Furukohri A, Nishikawa Y, Akiyama MT, Maki H - Nucleic Acids Res. (2012)

Bottom Line: We found that purified SSB binds directly to Pol IV in a pull-down assay, whereas SSBΔC8, a mutant protein lacking the C-terminal tail, failed to interact with Pol IV.These results show that the interaction between Pol IV and SSB is mediated by the C-terminal tail of SSB.These results demonstrate that Pol IV requires the interaction with the C-terminal tail of SSB to replicate DNA efficiently when the template ssDNA is covered with SSB.

View Article: PubMed Central - PubMed

Affiliation: The Department of Molecular Biology, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan. furukori@bs.naist.jp

ABSTRACT
DNA polymerase IV (Pol IV) is one of three translesion polymerases in Escherichia coli. A mass spectrometry study revealed that single-stranded DNA-binding protein (SSB) in lysates prepared from exponentially-growing cells has a strong affinity for column-immobilized Pol IV. We found that purified SSB binds directly to Pol IV in a pull-down assay, whereas SSBΔC8, a mutant protein lacking the C-terminal tail, failed to interact with Pol IV. These results show that the interaction between Pol IV and SSB is mediated by the C-terminal tail of SSB. When polymerase activity was tested on an SSBΔC8-coated template, we observed a strong inhibition of Pol IV activity. Competition experiments using a synthetic peptide containing the amino acid sequence of SSB tail revealed that the chain-elongating capacity of Pol IV was greatly impaired when the interaction between Pol IV and SSB tail was inhibited. These results demonstrate that Pol IV requires the interaction with the C-terminal tail of SSB to replicate DNA efficiently when the template ssDNA is covered with SSB. We speculate that at the primer/template junction, Pol IV interacts with the tail of the nearest SSB tetramer on the template, and that this interaction allows the polymerase to travel along the template while disassembling SSB.

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Related in: MedlinePlus

Proteins bound to Pol IV and lysozyme affinity column. Fractions eluted by heating in NuPAGE-loading buffer from the lysozyme control column (L) and the Pol IV column (IV) were separated on NuPAGE Bis–Tris gel and visualized by silver staining. Asterisks indicate protein bands specific to the Pol IV column that were analyzed by mass spectrometry.
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gks264-F1: Proteins bound to Pol IV and lysozyme affinity column. Fractions eluted by heating in NuPAGE-loading buffer from the lysozyme control column (L) and the Pol IV column (IV) were separated on NuPAGE Bis–Tris gel and visualized by silver staining. Asterisks indicate protein bands specific to the Pol IV column that were analyzed by mass spectrometry.

Mentions: When we loaded the cell lysate onto the Pol IV column and tried to elute binding proteins with a low-pH buffer, >90% of the protein remained bound in the column (data not shown). We then retrieved the resin from the column and heated it with SDS–PAGE sample buffer to recover all proteins in the column. Pol IV column-interacting proteins were then compared by SDS–PAGE with proteins recovered similarly from the lysozyme-immobilized control column (Figure 1, compare lane 1 with lane 2). In the Pol IV column eluate, specific bands of 45, 42 and 22 kDa were detected and were identified by mass spectrometry as the β clamp, Pol IV and SSB, respectively. The Pol IV must come from the column, because there can be no endogenous Pol IV in the lysate of the ΔdinB strain used. Other proteins observed here should be Pol IV column-interacting proteins, but they were different from those observed in Godoy et al.’s (25) report. In that study, GroEL, RecA and UmuD′ were detected as the major Pol IV-interacting proteins, whereas the β clamp and SSB were not recovered. It is possible that the former proteins were washed out by the low-pH buffer in our experiments, although we could not detect any of them in low-pH fractions by mass spectrometry (data not shown). Their interactions with Pol IV may be weak, and/or the proteins may bind to Pol IV only when the SOS is induced.Figure 1.


Interaction between Escherichia coli DNA polymerase IV and single-stranded DNA-binding protein is required for DNA synthesis on SSB-coated DNA.

Furukohri A, Nishikawa Y, Akiyama MT, Maki H - Nucleic Acids Res. (2012)

Proteins bound to Pol IV and lysozyme affinity column. Fractions eluted by heating in NuPAGE-loading buffer from the lysozyme control column (L) and the Pol IV column (IV) were separated on NuPAGE Bis–Tris gel and visualized by silver staining. Asterisks indicate protein bands specific to the Pol IV column that were analyzed by mass spectrometry.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3401449&req=5

gks264-F1: Proteins bound to Pol IV and lysozyme affinity column. Fractions eluted by heating in NuPAGE-loading buffer from the lysozyme control column (L) and the Pol IV column (IV) were separated on NuPAGE Bis–Tris gel and visualized by silver staining. Asterisks indicate protein bands specific to the Pol IV column that were analyzed by mass spectrometry.
Mentions: When we loaded the cell lysate onto the Pol IV column and tried to elute binding proteins with a low-pH buffer, >90% of the protein remained bound in the column (data not shown). We then retrieved the resin from the column and heated it with SDS–PAGE sample buffer to recover all proteins in the column. Pol IV column-interacting proteins were then compared by SDS–PAGE with proteins recovered similarly from the lysozyme-immobilized control column (Figure 1, compare lane 1 with lane 2). In the Pol IV column eluate, specific bands of 45, 42 and 22 kDa were detected and were identified by mass spectrometry as the β clamp, Pol IV and SSB, respectively. The Pol IV must come from the column, because there can be no endogenous Pol IV in the lysate of the ΔdinB strain used. Other proteins observed here should be Pol IV column-interacting proteins, but they were different from those observed in Godoy et al.’s (25) report. In that study, GroEL, RecA and UmuD′ were detected as the major Pol IV-interacting proteins, whereas the β clamp and SSB were not recovered. It is possible that the former proteins were washed out by the low-pH buffer in our experiments, although we could not detect any of them in low-pH fractions by mass spectrometry (data not shown). Their interactions with Pol IV may be weak, and/or the proteins may bind to Pol IV only when the SOS is induced.Figure 1.

Bottom Line: We found that purified SSB binds directly to Pol IV in a pull-down assay, whereas SSBΔC8, a mutant protein lacking the C-terminal tail, failed to interact with Pol IV.These results show that the interaction between Pol IV and SSB is mediated by the C-terminal tail of SSB.These results demonstrate that Pol IV requires the interaction with the C-terminal tail of SSB to replicate DNA efficiently when the template ssDNA is covered with SSB.

View Article: PubMed Central - PubMed

Affiliation: The Department of Molecular Biology, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan. furukori@bs.naist.jp

ABSTRACT
DNA polymerase IV (Pol IV) is one of three translesion polymerases in Escherichia coli. A mass spectrometry study revealed that single-stranded DNA-binding protein (SSB) in lysates prepared from exponentially-growing cells has a strong affinity for column-immobilized Pol IV. We found that purified SSB binds directly to Pol IV in a pull-down assay, whereas SSBΔC8, a mutant protein lacking the C-terminal tail, failed to interact with Pol IV. These results show that the interaction between Pol IV and SSB is mediated by the C-terminal tail of SSB. When polymerase activity was tested on an SSBΔC8-coated template, we observed a strong inhibition of Pol IV activity. Competition experiments using a synthetic peptide containing the amino acid sequence of SSB tail revealed that the chain-elongating capacity of Pol IV was greatly impaired when the interaction between Pol IV and SSB tail was inhibited. These results demonstrate that Pol IV requires the interaction with the C-terminal tail of SSB to replicate DNA efficiently when the template ssDNA is covered with SSB. We speculate that at the primer/template junction, Pol IV interacts with the tail of the nearest SSB tetramer on the template, and that this interaction allows the polymerase to travel along the template while disassembling SSB.

Show MeSH
Related in: MedlinePlus