The Recombinant Product of theChryptomonasφ Plastid GenehlpAis an Architectural Hu‐Like Protein that Promotes the Assembly of Complex Nucleoprotein Structures

Abstract

The HlpA protein which is encoded by the hlpA gene in the plastid genome of the cryptomonad alga Chryptomonas phi is structurally related to the non-sequence-specific DNA-binding and DNA-bending HU family of chromatin-associated proteins. The expression of the HlpA protein complements the mutant phenotype of Bacillus subtilis cells impaired in the Hbsu protein (B. subtilis HU), as measured by the resistance of the cells to methylmethane sulphonate. To analyse the interactions of HlpA with DNA, we expressed the protein in Escherichia coli and purified it to homogeneity. HlpA interacts preferentially with four-way junction DNA or DNA minicircles, when compared with linear DNA, recognising DNA structure. HlpA and E. coli HU display comparable affinities for all types of DNA tested; however, HlpA exhibits a stronger tendency to self-associate in the presence of DNA. Accordingly, HlpA oligomerises more readily than HU in protein crosslinking experiments. In the presence of topoisomerase I, HlpA constrains negative superhelical turns in closed circular plasmid DNA. The HlpA protein mediates the joining of distant recombination sites into a complex nucleoprotein structure, as judged by beta-mediated site-specific recombination. The results presented provide evidence that HlpA is a functional plastid equivalent of nuclear and mitochondrial HMG1-like proteins and bacterial HU proteins.

The Essential HupB and HupN Proteins of Pseudomonas putida Provide Redundant and Nonspecific DNA-bending Functions

“…These results indicated that both HupB and HupB can complement in full the loss of Hbsu in B. subtilis and strengthened the notion that each protein is capable by itself of meeting in vivo any physiological demand of generic DNA bending. In this respect, it is worth noticing that the HU-like hlpA gene of the plastid genome of the cryptomonad alga Chryptomonas ⌽ was not as efficient as the P. putida hup genes in the same complementation assay (54).…”

“…They grew at the very same rate and yield that the wild type cells in a variety of rich and minimal media tested and were as resistant to UV radiation as the wild type (not shown). As a more specific test, we examined the effect of a lack of HupB or HupN on the activity of the 54 promoter Ps of the TOL plasmid pWW0 of P. putida mt-2 (55). This promoter and its cognate activator, XylR, form part of an intricate regulatory system that controls the expression of a plasmid-encoded pathway for the biodegradation of toluene, m-xylene, and p-xylene (26).…”

“…This promoter and its cognate activator, XylR, form part of an intricate regulatory system that controls the expression of a plasmid-encoded pathway for the biodegradation of toluene, m-xylene, and p-xylene (26). XylR belongs to the family of the prokaryotic enhancerbinding proteins that act in concert with the 54 -containing form of RNAP. The activation mechanism of Ps requires the looping out of the distant XylR⅐UAS complex to contact the 54 -RNAP bound downstream (56).…”

New Insights into Host Factor Requirements for Prokaryotic %-Recombinase-mediated Reactions in Mammalian Cells

“…The host factor is also provided in eukaryotic environments, but its identity remains unknown. In vitro reactions with HMG1 proteins of both plant and animal origins have proven to work as efficient accessory factors for ␤-mediated recombination (18,23). HMG1 and other members of the same family are thus good candidates for host factors in ␤-mediated recombination reactions in eukaryotic cells.…”

“…More precisely, histone-like proteins Hbsu and HU can efficiently contribute to the recombination process. The identity of this host factor in eukaryotic environments is still unknown, although different HMG1-like proteins of mammalian and plant origin have the ability to facilitate the formation of nucleoprotein structures to different extents, because they can efficiently replace a bacterial chromatin-associated protein required for the site-specific ␤-mediated recombination (15,18,23,24). HMG1/2, which are abundant components of the eukaryotic nucleus, bind any linear DNA with moderate affinity and no sequence specificity (25) but bind with high affinity to DNA that is already sharply bent (26 -28); thus they are considered structural chromatin proteins expressed in all eukaryotic cell types.…”