Replication initiation at a distance: determination of the cis- and trans-acting elements of replication origin alpha of plasmid R6K.

Plasmid R6K, which contains 3 replication origins called alpha, gamma, and beta, is a favorable system to investigate the molecular mechanism(s) of action at a distance, i.e. replication initiation at a considerable distance from the primary initiator protein binding sites (iterons). The centrally located gamma origin contains 7 iterons that bind to the plasmid-encoded initiator protein, pi. Ori alpha, located at a distance of approximately 4 kb from gamma, contains a single iteron that does not directly bind to pi but is believed to access the protein by pi-mediated alpha-gamma iteron-iteron interaction that loops out the intervening approximately 3.7 kb of DNA. Although the cis-acting components and the trans-acting proteins required for ori gamma function have been analyzed in detail, such information was lacking for ori alpha. Here, we have identified both the sequence elements located at alpha and those at gamma, that together promoted alpha activity. The data support the conclusion that besides the single iteron, a neighboring DNA primase recognition element called G site is essential for alpha-directed plasmid maintenance. Sequences preceding the iteron and immediately following the G site, although not absolutely necessary, appear to play a role in efficient plasmid maintenance. In addition, while both dnaA1 and dnaA2 boxes that bind to DnaA protein and are located at gamma were essential for alpha activity, only dnaA2 was required for initiation at gamma. Mutations in the AT-rich region of gamma also abolished alpha function. These results are consistent with the interpretation that a protein-DNA complex consisting of pi and DnaA forms at gamma and activates alpha at a distance by DNA looping.

Investigations of pi initiator protein-mediated interaction between replication origins alpha and gamma of the plasmid R6K.

A typical plasmid replicon of Escherichia coli, such as ori gamma of R6K, contains tandem iterons (iterated initiator protein binding sites), an AT-rich region that melts upon initiator-iteron interaction, two binding sites for the bacterial initiator protein DnaA, and a binding site for the DNA-bending protein IHF. R6K also contains two structurally atypical origins called alpha and beta that are located on either side of gamma and contain a single and a half-iteron, respectively. Individually, these sites do not bind to initiator protein pi but access it by DNA looping-mediated interaction with the seven pi-bound gamma iterons. The pi protein exists in 2 interconvertible forms: inert dimers and active monomers. Initiator dimers generally function as negative regulators of replication by promoting iteron pairing ("handcuffing") between pairs of replicons that turn off both origins. Contrary to this existing paradigm, here we show that both the dimeric and the monomeric pi are necessary for ori alpha-driven plasmid maintenance. Furthermore, efficient looping interaction between alpha and gamma or between 2 gamma iterons in vitro also required both forms of pi. Why does alpha-gamma iteron pairing promote alpha activation rather than repression? We show that a weak, transitory alpha-gamma interaction at the iteron pairs was essential for alpha-driven plasmid maintenance. Swapping the alpha iteron with one of gamma without changing the original sequence context that caused enhanced looping in vitro caused a significant inhibition of alpha-mediated plasmid maintenance. Therefore, the affinity of alpha iteron for pi-bound gamma and not the sequence context determined whether the origin was activated or repressed.

Replication termination mechanism as revealed by Tus-mediated polar arrest of a sliding helicase.

The replication terminator protein Tus of Escherichia coli promotes polar fork arrest at sequence-specific replication termini (Ter) by antagonizing DNA unwinding by the replicative helicase DnaB. Here, we report that Tus is also a polar antitranslocase. We have used this activity as a tool to uncouple helicase arrest at a Tus-Ter complex from DNA unwinding and have shown that helicase arrest occurred without the generation of a DNA fork or a bubble of unpaired bases at the Tus-Ter complex. A mutant form of Tus, which reduces DnaB-Tus interaction but not the binding affinity of Tus for Ter DNA, was also defective in arresting a sliding DnaB. A model of polar fork arrest that proposes melting of the Tus-Ter complex and flipping of a conserved C residue of Ter at the blocking but not the nonblocking face has been reported. The model suggests that enhanced stability of Tus-Ter interaction caused by DNA melting and capture of a flipped base by Tus generates polarity strictly by enhanced protein-DNA interaction. In contrast, the observations presented here show that polarity of helicase and fork arrest in vitro is generated by a mechanism that not only involves interaction between the terminator protein and the arrested enzyme but also of Tus with Ter DNA, without any melting and base flipping in the termination complex.

Characterization and functional validation of glyoxalase II from rice.

Glyoxalase II, one of the enzymes of the glyoxalase pathway, cDNA cloned from rice (OsglyII) consists of 1623 nucleotides with an open reading frame of 1010 bp encoding a polypeptide of 336 amino acids and an estimated isoelectric point of 8.08. The recombinant protein purified from Escherichia coli using Ni-NTA affinity chromatography showed molecular mass of approximately 37 kDa. Catalytic parameters of the protein were determined using S-D-lactoylglutathione as a thioester substrate. The K(m) (61 microM) and K(cat) (301 s(-1)) values were lower than those reported for Arabidopsis, human and yeast and showed pH optima at 7.2. The E. coli overexpressing OsglyII were able to grow on higher concentration of methylglyoxal. Transcript analysis in rice showed that OsglyII gene expression is stimulated within 15 min in response to various abiotic stresses as well as treatment with abscisic acid or salicylic acid. This multistress response of OsglyII gene documents its future utility in developing tolerance to various stresses in crop plants.

Cloning and characterization of a mitochondrial glyoxalase II from Brassica juncea that is upregulated by NaCl, Zn, and ABA.

A cDNA (1061 bp) Bj glyII was cloned from a mannitol induced library of Brassica juncea. It encoded a protein of 335 amino acids with a molecular weight of 36.52 kDa. The deduced amino acid sequence of the clone showed 92% and 56% identity with Pennisetum and rice glyoxalase II, respectively, and 30% identity was observed with the human glyoxalase II. Search for the identical residues revealed the presence of highly conserved THHHXDH domain which is involved in zinc binding. p-NN and pSORT analysis of this sequence revealed a N-terminal mitochondrial target peptide. The cDNA was cloned in pMAL and a fusion protein with MBP (78 kDa) was expressed in Escherichia coli. The recombinant protein was purified approximately sixfold by affinity purification on amylose column and showed its pH optima at 7.0. The K(m) was determined to be 120 microM using S-d-lactoylglutathione as substrate. The expression of Bj glyII under various abiotic stress conditions showed that it is upregulated by salinity, heavy metal stress, and ABA.