Crystal structure and DNA-binding mode of Klebsiella pneumoniae primosomal PriB protein.

Genes Cells. 2012;17(10):837-49.

Huang YH, Lo YH, Huang W, Huang CY

Department of Biomedical Sciences, Chung Shan Medical University, Taiwan

 

Abstract

PriB is a primosomal DNA replication protein required for the re-initiation of replication in bacteria. In this study, we investigated the gene expression of PriB in Klebsiella pneumoniae (KpPriB) and characterized the gene product through crystal structural and functional analyses. Quantitative polymerase chain reaction analysis (Q-PCR) indicated that the 104-aa priB was expressed in K. pneumoniae with a CT value of 22.4. The crystal structure of KpPriB (Protein Data Bank entry: 4APV) determined at a resolution of 2.1 Å was similar to that of Escherichia coli PriB (EcPriB). KpPriB formed a single complex with single-stranded DNA (ssDNA) of different lengths, suggesting a highly cooperative process. Structure-based mutational analysis revealed that substitution at K18, F42, R44, W47, K82, K84, or K89 but not R34 in KpPriB had a significant effect on both ssDNA and double-stranded DNA (dsDNA) binding. Based on these findings, the known ssDNA interaction sites of PriB were expanded to include R44 and F42, thus allowing nucleic acids to wrap around the whole PriB protein.

PMID:22938024

 

Supplement

The initiation and re-initiation of chromosomal DNA replication in bacteria for genome duplication is a complex process that relies on divergent multi-protein assembly for entry of the replicative DNA helicase. In Escherichia coli, replication starts with the loading of DnaB helicase at the unique oriC site—a process accomplished by DnaA (an oriC-specific recognition protein) and DnaC (a loader protein). However, the replication fork initiated at oriC can be arrested anywhere along the DNA, leading to failure of replication. Thus, a reloading DnaB helicase for oriC-independent DNA replication is required for bacterial survival. The replication restart primosome is a multi-protein complex that reactivates stalled DNA replication at the forks after DNA damage. Figure 1 shows a hand-off mechanism for PriA-directed primosome assembly (Figure 1), where (i) PriA recognizes and binds to a replication fork; (ii) PriB joins to PriA to form a PriA-PriB-DNA ternary complex; and (iii) DnaT participates in this nucleocomplex to form a triprotein complex, in which, the recruitment of DnaT results in the release of ssDNA by PriB, and then loads the DnaB/C complex.

Cheng-Yang Huang-1

In this study, we have solved the crystal structure of KpPriB, and found that KpPriB has a classical OB-fold ssDNA-binding surface. Unlike SSB (single-stranded DNA binding protein), KpPriB binds ssDNA with the highly electrostatic positive L45 loop surface. Interestingly, sequence comparisons and operon organization analyses have shown that PriB evolved from SSB via gene duplication with subsequent rapid sequence diversification. We then asked the question as to why PriB has become a kind of SSB using different binding strategy to ssDNA, and how PriB participates in DNA replication differently from its ancestor. Because PriB is highly positively charged, this feature leads us to assess whether PriB can bind to ssDNA and dsDNA. Based on the mutational analysis and the electrophoretic mobility shift analysis, we identified and characterized the ssDNA- and dsDNA-binding properties of PriB, and proposed several binding modes. Figure 2 shows that according to crystal structures of some dimeric proteins complexed with dsDNA found in Protein Data Bank (PDB), as well as results from our mutational analysis, we speculate that PriB binds dsDNA in two possible ways. First, PriB may bind to dsDNA via the HU binding mode. According to the structure of HU-DNA complex, we manually superimpose the location of dsDNA with our PriB structure and find that this complex structure seems to match the residues of PriB important for dsDNA binding. Secondly, PriB may bind dsDNA in a manner similar to bind ssDNA. The structure-based mutational analysis indicated that residues in PriB crucial for ssDNA binding were also crucial for dsDNA binding. Since these residues responsible for ssDNA and dsDNA binding were almost overlapped, PriB may use a similar way to bind to the phosphate backbone of ssDNA and dsDNA via several positively charged residues.

Cheng-Yang Huang-2 

The importance of this study

We have found that not only ssDNA, PriB can also bind to dsDNA. Thus, the hand-off mechanism for primosome assembly may need to be further studied.

 

Acknowledgements: 

This research was supported by a grant from the National Science Council, Taiwan (NSC 102-2320-B-040-019 to C.Y. Huang).

 

Cheng-Yang Huang-3Contact:

Department of Biomedical Sciences

ChungShanMedicalUniversity

No.110, Sec.1, Chien-Kuo N. Rd.

Taichung City, Taiwan

cyhuang@csmu.edu.tw

http://w3.csmu.edu.tw/~cyhuang/.

 

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