Characterization of the Chimeric PriB-SSBc Protein.

En-Shyh Lin,Cheng-Yang Huang,Yen-Hua Huang

doi:10.3390/ijms221910854

Abstract

PriB is a primosomal protein required for the replication fork restart in bacteria. Although PriB shares structural similarity with SSB, they bind ssDNA differently. SSB consists of an N-terminal ssDNA-binding/oligomerization domain (SSBn) and a flexible C-terminal protein–protein interaction domain (SSBc). Apparently, the largest difference in structure between PriB and SSB is the lack of SSBc in PriB. In this study, we produced the chimeric PriB-SSBc protein in which Klebsiella pneumoniae PriB (KpPriB) was fused with SSBc of K. pneumoniae SSB (KpSSB) to characterize the possible SSBc effects on PriB function. The crystal structure of KpSSB was solved at a resolution of 2.3 Å (PDB entry 7F2N) and revealed a novel 114-GGRQ-117 motif in SSBc that pre-occupies and interacts with the ssDNA-binding sites (Asn14, Lys74, and Gln77) in SSBn. As compared with the ssDNA-binding properties of KpPriB, KpSSB, and PriB-SSBc, we observed that SSBc could significantly enhance the ssDNA-binding affinity of PriB, change the binding behavior, and further stimulate the PriA activity (an initiator protein in the pre-primosomal step of DNA replication), but not the oligomerization state, of PriB. Based on these experimental results, we discuss reasons why the properties of PriB can be retrofitted when fusing with SSBc.

Highlights

Single-stranded DNA-binding proteins (SSBs) play crucial roles in DNA replication, repair, recombination, and replication fork restart in both prokaryotes [1]and eukaryotes [2,3,4]
The crystal structure of K. pneumoniae SSB (KpSSB) was solved (PDB entry 7F2N) and sites, namely, Asn14, Lys74, and Gln77, in Single-stranded DNA (ssDNA)-binding/oligomerization domain (SSBn). Based on these results from biochemical revealed a novel GGRQ motif in SSBc that pre-occupies and interacts with the ssDNAanalysis of PriB-SSBc, we found that SSBc could significantly enhance the ssDNA-binding binding sites, namely, Asn14, Lys74, and Gln77, in SSBn
Based on these results from bioaffinity, change the binding behavior, and further stimulate the PriA activity, but not the chemical analysis of PriB-SSBc, we found that SSBc could significantly enhance the oligomerization state, of PriB

Summary

Introduction

Single-stranded DNA (ssDNA)-binding proteins (SSBs) play crucial roles in DNA replication, repair, recombination, and replication fork restart in both prokaryotes [1]and eukaryotes [2,3,4]. Single-stranded DNA (ssDNA)-binding proteins (SSBs) play crucial roles in DNA replication, repair, recombination, and replication fork restart in both prokaryotes [1]. SSBs typically recognize ssDNA [6,7,8,9] via a highly conserved oligonucleotide/oligosaccharidebinding (OB) fold formed from a five-stranded β-barrel capped by an α-helix [10,11]. The functions of SSB have been studied extensively in Escherichia coli (EcSSB) [12,13]. EcSSB consists of an N-terminal ssDNA-binding/oligomerization domain (SSBn) and a flexible. SSBc can be further subdivided into two sub-domains, namely, the intrinsically disordered linker (IDL) and the highly conserved acidic tail DDDIPF (SSB-Ct) at the C-terminus. IDL, but not just only SSB-Ct, had been found to be involved in binding to at least

Methods

Results

Conclusion