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The Protein Journal

, Volume 32, Issue 6, pp 477–483 | Cite as

Yeast Two-Hybrid Analysis of PriB-Interacting Proteins in Replication Restart Primosome: A Proposed PriB–SSB Interaction Model

  • Yen-Hua Huang
  • Min-Jon Lin
  • Cheng-Yang HuangEmail author
Article

Abstract

PriB is one of the components of the replication restart primosome. The activity mediated by the primosomal proteins, including PriB, is required for reinitiating chromosomal DNA replication in bacteria after DNA damage. As such, the study of the interactions between PriB and members in the primosome is essential to better understand their mechanism of action. In this study, we investigated PriB-interacting proteins in the primosome through the yeast two-hybrid system. Yeast two-hybrid analysis using two strains, Y187 and AH109, revealed that PriB interacts with PriA, DnaT, SSB, and itself and does not interact with DnaA, DnaB, DnaC, or DnaG. In addition, mutational analysis showed that PriB may bind to SSB via K82, K84, and K89 in the L45 loop. Based on these preliminary data, we proposed a PriB–SSB interaction model. Further work can focus on determination of how PriB binds to the PriA–SSB complex in replication restart.

Keywords

PriB PriA Primosome ssDNA SSB 

Abbreviations

ssDNA

Single-stranded DNA

SSB

ssDNA-binding protein

OB

Oligonucleotide/oligosaccharide binding

NEM

N-ethylmaleimide

PDB

Protein data bank

–LT

–Trp/–Leu

–HULT

–Trp/–Leu/–His/–urea

3KA

K82A/K84A/K89A mutant

aa

Amino acid residues

Notes

Acknowledgments

This research was supported by a grant from the National Research Program for Genome Medicine, Taiwan (NSC 100-3112-B-040-001 to C. Y. Huang).

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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Yen-Hua Huang
    • 1
  • Min-Jon Lin
    • 1
    • 2
  • Cheng-Yang Huang
    • 1
    • 2
    Email author
  1. 1.School of Biomedical SciencesChung Shan Medical UniversityTaichung CityTaiwan
  2. 2.Department of Medical ResearchChung Shan Medical University HospitalTaichung CityTaiwan

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