Crystal structure and DNA ‐binding mode of K lebsiella pneumoniae primosomal P ri B protein

• Published in: Genes to cells : devoted to molecular & cellular mechanisms 2012-10, Vol.17 (10), p.837-849
• Main Authors: Huang, Yen‐Hua, Lo, Yu‐Hua, Huang, Wenya, Huang, Cheng‐Yang
• Format: Article
• Language: English
• ISSN: 1356-9597; 1365-2443
• DOI: 10.1111/gtc.12001

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 P ri B in K lebsiella pneumoniae ( K p P ri B ) 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 C T value of 22.4. The crystal structure of Kp PriB ( P rotein D ata B ank entry: 4 APV ) determined at a resolution of 2.1 Å was similar to that of E scherichia coli P ri B ( E c P ri B ). K p P ri B formed a single complex with single‐stranded DNA (ss DNA ) of different lengths, suggesting a highly cooperative process. Structure‐based mutational analysis revealed that substitution at K 18, F 42, R 44, W 47, K 82, K 84, or K 89 but not R 34 in K p P ri B had a significant effect on both ss DNA and double‐stranded DNA (ds DNA ) binding. Based on these findings, the known ss DNA interaction sites of P ri B were expanded to include R 44 and F 42, thus allowing nucleic acids to wrap around the whole P ri B protein.