Identification of the Active Oligomeric State of an Essential Adenine DNA Methyltransferase from Caulobacter crescentus

Shier, Vincent K.; Hancey, Carey J.; Benkovic, Stephen J.

doi:10.1074/jbc.m010688200

Cited by 31 publications

(25 citation statements)

References 48 publications

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“…In experiments involving crosslinking of the BamHI MTase subunits, it was shown that the enzyme in a free state exists as a dimer (Malygin et al, 2001). A similar behavior was observed for adenine M.CcrM from C. crescentus dimerized at physiological concentrations, and the dimerization does not affect DNA methylation (Shier et al, 2001).…”

Section: Introductionsupporting

confidence: 51%

Structure of the Q237W mutant of HhaI DNA methyltransferase: an insight into protein-protein interactions

Dong

Zhou

Zhang

et al. 2004

Biological Chemistry

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We have determined the structure of a mutant (Q237W) of HhaI DNA methyltransferase, complexed with the methyl-donor product AdoHcy. The Q237W mutant proteins were crystallized in the monoclinic space group C2 with two molecules in the crystallographic asymmetric unit. Protein-protein interface calculations in the crystal lattices suggest that the dimer interface has the specific characteristics for homodimer protein-protein interactions, while the two active sites are spatially independent on the outer surface of the dimer. The solution behavior suggests the formation of HhaI dimers as well. The same HhaI dimer interface is also observed in the previously characterized binary (M.HhaI-AdoMet) and ternary (M.HhaI-DNA-AdoHcy) complex structures, crystallized in different space groups. The dimer is characterized either by a non-crystallographic two-fold symmetry or a crystallographic symmetry. The dimer interface involves three segments: the amino-terminal residues 2-8, the carboxy-terminal residues 313-327, and the linker (amino acids 179-184) between the two functional domainsthe catalytic methylation domain and the DNA target recognition domain. Both the amino-and carboxy-terminal segments are part of the methylation domain. We also examined protein-protein interactions of other structurally characterized DNA MTases, which are often found as a 2-fold related 'dimer' with the largest dimer interface area for the group-b MTases. A possible evolutionary link between the Type I and Type II restriction-modification systems is discussed.

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Section: Introductionsupporting

confidence: 51%

Structure of the Q237W mutant of HhaI DNA methyltransferase: an insight into protein-protein interactions

Dong

Zhou

Zhang

et al. 2004

Biological Chemistry

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“…The product was digested with BfaI and SapI and ligated into a custom IMPACT (New England Biolabs) vector placed under the control of a T7 promoter (14) digested with NdeI and SapI. This vector places a self-cleaving intein and chitin binding domain at the C terminus of gp59 and allows affinity purification on a chitin column followed by cleavage of the intein with dithiothreitol (DTT) to yield the wild-type gp59.…”

Section: Methodsmentioning

confidence: 99%

Identification and Mapping of Protein-Protein Interactions between gp32 and gp59 by Cross-linking

Ishmael

Alley

Benkovic

2001

Journal of Biological Chemistry

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The bacteriophage T4 59 protein (gp59) plays a vital role in recombination and replication by promoting the assembly of the gene 41 helicase (gp41) onto DNA, thus enabling replication as well as strand exchange in recombination. Loading of the helicase onto gp32 (the T4 single strand binding protein)-coated single-stranded DNA requires gp59 to remove gp32 and replace it with gp41. Cross-linking studies between gp32 and gp59 reveal an interaction between Cys-166 of gp32 and Cys-42 of gp59. Since Cys-166 lies in the DNA binding core domain of gp32, this interaction may affect the association of gp32 with DNA. In the presence of gp32 or DNA, gp59 is capable of forming a multimer consisting of at least five gp59 subunits. Kinetics studies suggest that gp59 and gp41 exist in a one-to-one ratio, predicting that gp59 is capable of forming a hexamer (Raney, K. D., Carver, T. E., and Benkovic, S. J. (1996) J. Biol. Chem. 271, 14074 -14081). The C-terminal A-domain of gp32 is needed for gp59 oligomer formation. Cross-linking has established that gp59 can interact with gp32-A (a truncated form of gp32 lacking the A-domain) but cannot form higher species. The results support a model in which gp59 binds to gp32 on a replication fork, destabilizing the gp32-singlestranded DNA interaction concomitant with the oligomerization of gp59 that results in a switching of gp41 for gp32 at the replication fork.Assembly of the gp41 replicative helicase at the bacteriophage T4 DNA replication fork requires the displacement of single-strand DNA-binding proteins (gp32) 1 coating the lagging strand. The helicase assembly protein, gp59, is required to effectively load the helicase under these conditions, thus assuming a critical role in bacteriophage T4 DNA replication as well as recombination (1-3). Mutations in gene 59 result in arrested DNA synthesis and reduced phage burst size (1), indicating that the gene product is essential for recombinationdependent DNA replication (which occurs in the late stages of Escherichia coli T4 infection). Since gp32 is also involved in early, origin-initiated DNA replication, it is likely that gp59 also facilitates replication in the early stages of T4 infection. Furthermore, gp59 mutants exhibit recombination deficiency, UV sensitivity, and sensitivity to chemical mutagens, suggesting a role of gp59 in recombination and recombination-mediated DNA repair (2, 3).gp59 is a basic (pI ϭ 10.18), 26-kDa protein (4, 5). Hydrodynamic studies indicate that it exists as a monomer in solution (4, 5). It has a high affinity for DNA and has been shown to bind duplex DNA, single-stranded DNA, and forked DNA substrates (4, 6). Moreover, Mueser et al. (7) demonstrate that gp59 binds with a higher affinity to a DNA fork than ssDNA. The crystal structure of gp59 reveals that it is composed predominantly of ␣-helices and contains two domains, a C-and an N-terminal domain. Models generated from the crystal structure of gp59 and its structural similarity to DNA-binding proteins of the high mobility group family propose that dup...

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“…CcrM binds to and methylates adenosine in the sequence 5Ј-GANTC-3Ј, where "N" is any nucleotide (167,300). Like EcoDam, CcrM is a functional monomer and acts processively (20), although evidence suggests that it is a dimer at physiologic concentration (234). However, unlike EcoDam, CcrM has a distinct preference for hemimethylated DNA as a substrate, based on the observation that the turnover rate for hemimethylated DNA containing a GANTC target site(s) was significantly higher than that for DNA containing nonmethylated sites (20).…”

Section: Orphan Dna Mtasesmentioning

confidence: 99%

Epigenetic Gene Regulation in the Bacterial World

Casadesús

Low

2006

Microbiol Mol Biol Rev

568

562

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SUMMARY Like many eukaryotes, bacteria make widespread use of postreplicative DNA methylation for the epigenetic control of DNA-protein interactions. Unlike eukaryotes, however, bacteria use DNA adenine methylation (rather than DNA cytosine methylation) as an epigenetic signal. DNA adenine methylation plays roles in the virulence of diverse pathogens of humans and livestock animals, including pathogenic Escherichia coli, Salmonella, Vibrio, Yersinia, Haemophilus, and Brucella. In Alphaproteobacteria, methylation of adenine at GANTC sites by the CcrM methylase regulates the cell cycle and couples gene transcription to DNA replication. In Gammaproteobacteria, adenine methylation at GATC sites by the Dam methylase provides signals for DNA replication, chromosome segregation, mismatch repair, packaging of bacteriophage genomes, transposase activity, and regulation of gene expression. Transcriptional repression by Dam methylation appears to be more common than transcriptional activation. Certain promoters are active only during the hemimethylation interval that follows DNA replication; repression is restored when the newly synthesized DNA strand is methylated. In the E. coli genome, however, methylation of specific GATC sites can be blocked by cognate DNA binding proteins. Blockage of GATC methylation beyond cell division permits transmission of DNA methylation patterns to daughter cells and can give rise to distinct epigenetic states, each propagated by a positive feedback loop. Switching between alternative DNA methylation patterns can split clonal bacterial populations into epigenetic lineages in a manner reminiscent of eukaryotic cell differentiation. Inheritance of self-propagating DNA methylation patterns governs phase variation in the E. coli pap operon, the agn43 gene, and other loci encoding virulence-related cell surface functions.

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Identification of the Active Oligomeric State of an Essential Adenine DNA Methyltransferase from Caulobacter crescentus

Cited by 31 publications

References 48 publications

Structure of the Q237W mutant of HhaI DNA methyltransferase: an insight into protein-protein interactions

Structure of the Q237W mutant of HhaI DNA methyltransferase: an insight into protein-protein interactions

Identification and Mapping of Protein-Protein Interactions between gp32 and gp59 by Cross-linking

Epigenetic Gene Regulation in the Bacterial World

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