Title

Evolutionary covariance among DNA replication restart primosome genes

Author

Linda Berg

Date of Award

2012

Degree Name

M.S. in Chemistry

Department

Department of Chemistry

Advisor/Chair

Advisor: Matthew E. Lopper

Abstract

The process of DNA replication can be hindered by DNA damage, and disruptions in replication ultimately lead to cell death if not corrected. Bacteria solve this problem with a mechanism called DNA replication restart," which is catalyzed by primosome proteins. However, not all bacteria encode the full complement of primosome proteins found in the well-studied bacterium, E. coli. This suggests that differences might exist in DNA replication restart pathways among diverse bacteria. N. gonorrhoeae, for example, lacks a DnaT homolog. DnaT is thought to affect interactions between primosome protein PriB and single-stranded DNA. Since N. gonorrhoeae PriB has a weak interaction with single-stranded DNA, as opposed to the strong interaction between PriB and single-stranded DNA seen in E. coli (which encodes a DnaT homolog), we hypothesized that the presence of a DnaT homolog could be used to predict the affinity with which a bacterial PriB binds single-stranded DNA. Binary interactions between PriB and single-stranded DNA of two bacteria, K. pneumoniae (which encodes a DnaT homolog) and Y. enterocolitica (which lacks a DnaT homolog) were analyzed. Both K. pneumoniae and Y. enterocolitica have a high affinity PriB:ssDNA interaction with dissociation constants of 62 ± 14 nM and 84 ± 8 nM, respectively. Thus, the presence of DnaT cannot be used to predict affinities of binary interactions between PriB and single-stranded DNA. However, the experimentally measured binding constants combined with amino acid sequence alignments of the PriB homologs have led to the definition of parameters for high affinity binary interactions between PriB and single-stranded DNA. A recent report describes the identification of a novel PriB protein in K. pneumoniae that is significantly shorter than most sequenced PriB homologs (H.C. Hsieh, and C.Y. Huang, Identification of a novel protein, PriB, in Klebsiella pneumoniae. Biochem Biophys Res Commun 404 (2011) 546-51). The K. pneumoniae PriB protein is proposed to comprise 55 amino acid residues, in contrast to E. coli PriB which comprises 104 amino acid residues and has a length that is typical of most sequenced PriB homologs. Our investigations suggest that the priB gene of K. pneumoniae encodes a 104-amino acid PriB protein, akin to its E. coli counterpart. We have cloned the K. pneumoniae priB gene and purified the 104-amino acid K. pneumoniae PriB protein. Gel filtration experiments reveal that the K. pneumoniae PriB protein is a dimer, and equilibrium DNA binding experiments demonstrate that K. pneumoniae PriB's single-stranded DNA-binding activity is similar to that of E. coli PriB. These results indicate that the PriB homolog of K. pneumoniae is similar in structure and in function to that of E. coli."

Keywords

DNA replication Research, DNA-binding proteins Research, Klebsiella pneumoniae

Rights Statement

Copyright 2012, author

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