DNA base flipping by both members of the PspGI restriction-modification system

Carpenter, Michael A.; Bhagwat, Ashok S.

doi:10.1093/nar/gkn528

Cited by 6 publications

(6 citation statements)

References 32 publications

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“…With regard to HP0593 MTase, metal ions are not essential for enzyme activity but stimulate the reaction. Addition of Ca 2+ or Mg 2+ , but not Mn 2+ , Co 2+ or Cd 2+ to the reaction buffer stimulated the activity of the PspGI MTase [56]. Recently, Chan et al, [57] demonstrated that in case of HpyAV restriction endonuclease from H. pylori , activity was stimulated by Mn 2+ and Co 2+ .…”

Section: Resultsmentioning

confidence: 99%

Functional Analysis of an Acid Adaptive DNA Adenine Methyltransferase from Helicobacter pylori 26695

Banerjee

Rao

2011

PLoS ONE

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HP0593 DNA-(N6-adenine)-methyltransferase (HP0593 MTase) is a member of a Type III restriction-modification system in Helicobacter pylori strain 26695. HP0593 MTase has been cloned, overexpressed and purified heterologously in Escherichia coli. The recognition sequence of the purified MTase was determined as 5′-GCAG-3′and the site of methylation was found to be adenine. The activity of HP0593 MTase was found to be optimal at pH 5.5. This is a unique property in context of natural adaptation of H. pylori in its acidic niche. Dot-blot assay using antibodies that react specifically with DNA containing m6A modification confirmed that HP0593 MTase is an adenine-specific MTase. HP0593 MTase occurred as both monomer and dimer in solution as determined by gel-filtration chromatography and chemical-crosslinking studies. The nonlinear dependence of methylation activity on enzyme concentration indicated that more than one molecule of enzyme was required for its activity. Analysis of initial velocity with AdoMet as a substrate showed that two molecules of AdoMet bind to HP0593 MTase, which is the first example in case of Type III MTases. Interestingly, metal ion cofactors such as Co2+, Mn2+, and also Mg2+ stimulated the HP0593 MTase activity. Preincubation and isotope partitioning analyses clearly indicated that HP0593 MTase-DNA complex is catalytically competent, and suggested that DNA binds to the MTase first followed by AdoMet. HP0593 MTase shows a distributive mechanism of methylation on DNA having more than one recognition site. Considering the occurrence of GCAG sequence in the potential promoter regions of physiologically important genes in H. pylori, our results provide impetus for exploring the role of this DNA MTase in the cellular processes of H. pylori.

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Section: Resultsmentioning

confidence: 99%

Functional Analysis of an Acid Adaptive DNA Adenine Methyltransferase from Helicobacter pylori 26695

Banerjee

Rao

2011

PLoS ONE

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“…The complete rotation of a nucleotide from the DNA duplex, around the phosphate backbone, and into the catalytic pocket of an enzyme is commonly known as nucleotide flipping. It was first observed for the methyltransferase enzyme HhaI ( 1 ) and has since been observed in many other DNA methyltransferase systems ( 2–6 ) and several DNA-repair enzyme complexes ( 7 , 8 ). Recently, it was demonstrated that the Ecl18kI restriction enzyme uses nucleotide flipping as part of its DNA recognition mechanism ( 9 ).…”

Section: Introductionmentioning

confidence: 95%

“…This report was the first example of an enzyme using nucleotide flipping in a situation where the flipped base is not the target of some chemical modification by the enzyme. This seemed a surprising and energetically extravagant method for recognizing a short-DNA sequence, but was soon found to apply to the evolutionarily related enzymes, PspGI ( 6 , 10–14 ) and EcoRII-C ( 11 , 15 ) the catalytic subunit of EcoRII ( Table 1 ).…”

Section: Introductionmentioning

confidence: 99%

Time-resolved fluorescence studies of nucleotide flipping by restriction enzymes

Neely

Тамулайтис

Chen

et al. 2009

Nucleic Acids Research

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Restriction enzymes Ecl18kI, PspGI and EcoRII-C, specific for interrupted 5-bp target sequences, flip the central base pair of these sequences into their protein pockets to facilitate sequence recognition and adjust the DNA cleavage pattern. We have used time-resolved fluorescence spectroscopy of 2-aminopurine-labelled DNA in complex with each of these enzymes in solution to explore the nucleotide flipping mechanism and to obtain a detailed picture of the molecular environment of the extrahelical bases. We also report the first study of the 7-bp cutter, PfoI, whose recognition sequence (T/CCNGGA) overlaps with that of the Ecl18kI-type enzymes, and for which the crystal structure is unknown. The time-resolved fluorescence experiments reveal that PfoI also uses base flipping as part of its DNA recognition mechanism and that the extrahelical bases are captured by PfoI in binding pockets whose structures are quite different to those of the structurally characterized enzymes Ecl18kI, PspGI and EcoRII-C. The fluorescence decay parameters of all the enzyme-DNA complexes are interpreted to provide insight into the mechanisms used by these four restriction enzymes to flip and recognize bases and the relationship between nucleotide flipping and DNA cleavage.

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“…Significant sequence similarity between Ecl18kI and PspGI/EcoRII suggests that these enzymes should also flip the nucleotide at the center of the CCXGG sequence, when X is adenine or thymine, and might or might not flip it when X is guanine or cytosine. Direct biochemical evidence for adenine or thymine flipping by PspGI/EcoRII is difficult to provide, but fluorescence experiments with 2-aminopurine (2AP) demonstrate that this adenine analog (which shares some hydrogen bonding requirements with guanine) can be flipped by PspGI/EcoRII ( 12 , 13 ). Evidence for flipping of a natural base in the CCNGG context by PspGI has been obtained for cytosine: this base in the central position of the CCNGG sequence was found to deaminate faster in the presence of the enzyme ( 14 ).…”

Section: Introductionmentioning

confidence: 99%

Central base pair flipping and discrimination by PspGI

Szczepanowski

Carpenter

Czapinska

et al. 2008

Nucleic Acids Research

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PspGI is a representative of a group of restriction endonucleases that recognize a pentameric sequence related to CCNGG. Unlike the previously investigated Ecl18kI, which does not have any specificity for the central base pair, PspGI prefers A/T over G/C in its target site. Here, we present a structure of PspGI with target DNA at 1.7 Å resolution. In this structure, the bases at the center of the recognition sequence are extruded from the DNA and flipped into pockets of PspGI. The flipped thymine is in the usual anti conformation, but the flipped adenine takes the normally unfavorable syn conformation. The results of this and the accompanying manuscript attribute the preference for A/T pairs over G/C pairs in the flipping position to the intrinsically lower penalty for flipping A/T pairs and to selection of the PspGI pockets against guanine and cytosine. Our data show that flipping can contribute to the discrimination between normal bases. This adds a new role to base flipping in addition to its well-known function in base modification and DNA damage repair.

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DNA base flipping by both members of the PspGI restriction-modification system

Cited by 6 publications

References 32 publications

Functional Analysis of an Acid Adaptive DNA Adenine Methyltransferase from Helicobacter pylori 26695

Functional Analysis of an Acid Adaptive DNA Adenine Methyltransferase from Helicobacter pylori 26695

Time-resolved fluorescence studies of nucleotide flipping by restriction enzymes

Central base pair flipping and discrimination by PspGI

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