Structure of DNase I at 2.0 Å resolution suggests a mechanism for binding to and cutting DNA

Suck, Dietrich; Oefner, Christian

doi:10.1038/321620a0

Cited by 341 publications

(238 citation statements)

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“…These results are also in agreement with DNase I footprinting studies of pol III holoenzyme on DNA that showed protection of 27-30 bp at a primer terminus (43). DNase I is a rather large cleavage agent and generally overestimates binding sites by ϳ3-5 nucleotides (44).…”

Section: Discussionsupporting

confidence: 88%

DNA Structure Requirements for the Escherichia coliγ Complex Clamp Loader and DNA Polymerase III Holoenzyme

Yao¹,

Leu²,

Anjelkovic³

et al. 2000

Journal of Biological Chemistry

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The Escherichia coli chromosomal replicase, DNA polymerase III holoenzyme, is highly processive during DNA synthesis. Underlying high processivity is a ringshaped protein, the ␤ clamp, that encircles DNA and slides along it, thereby tethering the enzyme to the template. The ␤ clamp is assembled onto DNA by the multiprotein ␥ complex clamp loader that opens and closes the ␤ ring around DNA in an ATP-dependent manner. This study examines the DNA structure required for clamp loading action. We found that the ␥ complex assembles ␤ onto supercoiled DNA (replicative form I), but only at very low ionic strength, where regions of unwound DNA may exist in the duplex. Consistent with this, the ␥ complex does not assemble ␤ onto relaxed closed circular DNA even at low ionic strength. Hence, a 3-end is not required for clamp loading, but a singlestranded DNA (ssDNA)/double-stranded DNA (dsDNA) junction can be utilized as a substrate, a result confirmed using synthetic oligonucleotides that form forked ssDNA/dsDNA junctions on M13 ssDNA. On a flush primed template, the ␥ complex exhibits polarity; it acts specifically at the 3-ssDNA/dsDNA junction to assemble ␤ onto the DNA. The ␥ complex can assemble ␤ onto a primed site as short as 10 nucleotides, corresponding to the width of the ␤ ring. However, a protein block placed closer than 14 base pairs (bp) upstream from the primer 3 terminus prevents the clamp loading reaction, indicating that the ␥ complex and its associated ␤ clamp interact with ϳ14 -16 bp at a ssDNA/dsDNA junction during the clamp loading operation. A protein block positioned closer than 20 -22 bp from the 3 terminus prevents use of the clamp by the polymerase in chain elongation, indicating that the polymerase has an even greater spatial requirement than the ␥ complex on the duplex portion of the primed site for function with ␤. Interestingly, DNA secondary structure elements placed near the 3 terminus impose similar steric limits on the ␥ complex and polymerase action with ␤. The possible biological significance of these structural constraints is discussed.Escherichia coli DNA polymerase III holoenzyme (pol III 1 holoenzyme) is a highly processive multisubunit replicase (1). Processivity is conferred to the polymerase by the ␤ subunit (2, 3). The ␤ subunit is a ring-shaped protein that completely encircles DNA and slides along the duplex (4, 5). The ␤ ring endows the polymerase with high processivity by binding directly to it, continuously tethering it to the template during synthesis (4). The ␤ ring does not assemble onto DNA by itself; for this, it requires the clamp loading action of the ␥ complex. The ␥ complex is composed of five different proteins (␥, ␦, ␦Ј, , and ) (1). Upon binding ATP, the ␥ complex opens the ␤ ring and positions it around the primed template (7,8). Hydrolysis of two molecules of ATP results in closing the ring around DNA and dissociation of the ␥ complex from the clamp (7, 49).Following the release of the clamp loader from the clamp, the catalytic core polymerase subassembly (pol ...

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Section: Discussionsupporting

confidence: 88%

DNA Structure Requirements for the Escherichia coliγ Complex Clamp Loader and DNA Polymerase III Holoenzyme

Yao¹,

Leu²,

Anjelkovic³

et al. 2000

Journal of Biological Chemistry

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“…was defined by the sequence alignment. Suck and Oefner (1986) proposed another catalytic mechanism of DNase I from the three-dimensional structure of DNase I and the binding of Ca2+-thymidine 3',5'-diphosphate at the active site; that is, Glu 78 accepts a proton from His 134, and His 134 in turn accepts a proton from a water molecule, which then, as a nucleophile, attacks the phosphorus. However, from the structure of the DNA octanucleotide-DNase I complex, they later suggested the alternative mechanism involving the two histidine residues H252 and HI34 (Suck et al, 1988).…”

Section: Conservation Of Functionally Important Residuesmentioning

confidence: 99%

A distant evolutionary relationship between bacterial sphingomyelinase and mammalian DNase I

et al. 1996

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The three-dimensional structure of bacterial sphingomyelinase (SMase) was predicted using a protein fold recognition method; the search of a library of known structures showed that the SMase sequence is highly compatible with the mammalian DNase I structure, which suggested that SMase adopts a structure similar to that of DNase I. The amino acid sequence alignment based on the prediction revealed that, despite the lack of overall sequence similarity (less than 10% identity), those residues of DNase I that are involved in the hydrolysis of the phosphodiester bond, including two histidine residues (His 134 and His 252) of the active center, are conserved in SMase. In addition, a conserved pentapeptide sequence motif was found, which includes two catalytically critical residues, Asp 251 and His 252. A sequence database search showed that the motif is highly specific to mammalian DNase I and bacterial SMase. The functional roles of SMase residues identified by the sequence comparison were consistent with the results from mutant studies. Two Bacillus cereus SMase mutants (H134A and H252A) were constructed by site-directed mutagenesis. They completely abolished their catalytic activity. A model for the SMase-sphingomyelin complex structure was built to investigate how the SMase specifically recognizes its substrate. The model suggested that a set of residues conserved among bacterial SMases, including Trp 28 and Phe 55, might be important in the substrate recognition. The predicted structural similarity and the conservation of the functionally important residues strongly suggest a distant evolutionary relationship between bacterial SMase and mammalian DNase I. These two phosphodiesterases must have acquired the specificity for different substrates in the course of evolution.

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“…It has strong structural specificity for the geometry of the minor groove, and gives staggered cuts on each strand by a 'double hit' mechanism (Suck and Oefner 1986). Because it is a large enzyme of 30 kDa, and may be glycosylated, DNaseI only cleaves the most accessible DNA regions.…”

Section: A Flausmentioning

confidence: 99%

Principles and practice of nucleosome positioningin vitro

Flaus

2011

Frontiers in Life Science

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Structure of DNase I at 2.0 Å resolution suggests a mechanism for binding to and cutting DNA

Cited by 341 publications

References 24 publications

DNA Structure Requirements for the Escherichia coliγ Complex Clamp Loader and DNA Polymerase III Holoenzyme

DNA Structure Requirements for the Escherichia coliγ Complex Clamp Loader and DNA Polymerase III Holoenzyme

A distant evolutionary relationship between bacterial sphingomyelinase and mammalian DNase I

Principles and practice of nucleosome positioningin vitro

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