The zinc ion in the HNH motif of the endonuclease domain of colicin E7 is not required for DNA binding but is essential for DNA hydrolysis

Ku, Wen Yen; Liu, Yu Wen; Hsu, Ya Chein; Liao, Chen Chung; Liang, Po‐Huang; Yuan, Hanna S.; Chak, Kin‐Fu

doi:10.1093/nar/30.7.1670

Cited by 58 publications

(78 citation statements)

References 46 publications

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“…11-13). HNH proteins include a range of nucleases such as some homing endonucleases (e.g., I-HmuI), colicins (e.g., ColE7, ColE9), and restriction endonucleases (e.g., KpnI, MnlI, and HphI) (14)(15)(16)(17)(18)(19). HNH motifs are less common in eukaryotes.…”

Section: Resultsmentioning

confidence: 99%

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Annealing helicase 2 (AH2), a DNA-rewinding motor with an HNH motif

Yusufzai

Kadonaga

2010

Proc. Natl. Acad. Sci. U.S.A.

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The structure and integrity of DNA is of considerable biological and biomedical importance, and it is therefore critical to identify and to characterize enzymes that alter DNA structure. DNA helicases are ATP-driven motor proteins that unwind DNA. Conversely, HepA-related protein (HARP) protein (also known as SMARCAL1 and DNA-dependent ATPase A) is an annealing helicase that rewinds DNA in an ATP-dependent manner. To date, HARP is the only known annealing helicase. Here we report the identification of a second annealing helicase, which we term AH2, for annealing helicase 2. Like HARP, AH2 catalyzes the ATP-dependent rewinding of replication protein A (RPA)-bound complementary singlestranded DNA, but does not exhibit any detectable helicase activity. Unlike HARP, however, AH2 lacks a conserved RPA-binding domain and does not interact with RPA. In addition, AH2 contains an HNH motif, which is commonly found in bacteria and fungi and is often associated with nuclease activity. AH2 appears to be the only vertebrate protein with an HNH motif. Contrary to expectations, purified AH2 does not exhibit nuclease activity, but it remains possible that AH2 contains a latent nuclease that is activated under specific conditions. These structural and functional differences between AH2 and HARP suggest that different annealing helicases have distinct functions in the cell. T he unwinding of dsDNA occurs frequently during normal cellular processes such as replication, repair, and transcription (for reviews, see refs. 1 and 2). In addition, ssDNA bubbles can arise spontaneously. Because unwound DNA can be stabilized by ssDNA-binding proteins such as replication protein A (RPA), there is considerable potential for the formation of stable RPA-bound ssDNA bubbles.HepA-related protein (HARP, also known as SMARCAL1) is an ATP-driven motor protein that rewinds RPA-bound complementary ssDNA strands (3). Because HARP anneals DNA, it is termed an annealing helicase. HARP binds preferentially to fork DNA relative to ssDNA or dsDNA, and the binding of HARP to fork DNA activates its ATPase activity. In humans, mutations in the HARP gene are responsible for a rare autosomal recessive pleiotropic disorder known as Schimke immunoosseous dysplasia, which typically leads to death in early childhood (4). It thus appears that the annealing helicase activity of HARP is essential for normal growth and development.In addition to its ability to function as an annealing helicase, HARP binds directly to RPA via a conserved N-terminal motif (5-9). This interaction between HARP and RPA is not required for annealing helicase activity, but rather serves to recruit HARP to sites of RPA-bound ssDNA that are formed during DNA damage or replicative stress (5-8). Cells with reduced levels of HARP also exhibit increased levels of RPA-bound ssDNA during S phase relative to that seen with wild-type cells (5, 7). These findings suggest an important role for HARP in DNA structure and dynamics.To date, HARP is the only known annealing helicase. In these studies, we foun...

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Section: Resultsmentioning

confidence: 99%

“…In the experiment shown in Fig. 2A, we tested the binding of AH2 to 30 nt ssDNA [dðCTÞ 15 , which does not form hairpin or duplex structures], 30 bp dsDNA, and 30 nt fork DNA that is identical to the dsDNA except for a 9-nt mismatch at one end. We found that AH2 binds with the highest affinity to fork DNA.…”

Section: Resultsmentioning

confidence: 99%

Annealing helicase 2 (AH2), a DNA-rewinding motor with an HNH motif

Yusufzai

Kadonaga

2010

Proc. Natl. Acad. Sci. U.S.A.

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“…For example, there is only one strictly conserved His residue located at the first ␤-strand in the motif sequence. We used the ␤␤␣-metal motif of the nuclease domain of colicin E7 (PDB ID: 7CEI) 33 as the query motif, which is composed of three fragments: E542-H545 (␤-strand), I561-V564(␤-strand) and P566 -D571 (␣-helix). The query fragments are compared against the nonredundant PDB (nrpdb) chain set composed of nonidentical proteins from NCBI (http://www.ncbi.nlm.nih.gov/Structure/VAST/nrpdb.html).…”

Section: Results and Discussion The ␤␤␣-Metal Motifmentioning

confidence: 99%

“…Previously, a combination of fold alignment methods, together with the PSI-BLAST and the manual procedures, was used to identify the treble clef finger motifs. 24 We used the zinc finger motif of the G protein Rab3A (PDB ID: 1ZDB:B) 33 as the query motif, which is composed of three fragments: S108 -C119, G123-E125, and W135-K148. The top ranking hits are shown in Table III, and the corresponding structures of the zinc fingers are shown in Figure 4.…”

Section: The Treble Clef Finger Motifsmentioning

confidence: 99%

The fragment transformation method to detect the protein structural motifs

Lin

Chen

et al. 2006

Proteins

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To identify functional structural motifs from protein structures of unknown function becomes increasingly important in recent years due to the progress of the structural genomics initiatives. Although certain structural patterns such as the Asp-His-Ser catalytic triad are easy to detect because of their conserved residues and stringently constrained geometry, it is usually more challenging to detect a general structural motifs like, for example, the ␤␤␣-metal binding motif, which has a much more variable conformation and sequence. At present, the identification of these motifs usually relies on manual procedures based on different structure and sequence analysis tools. In this study, we develop a structural alignment algorithm combining both structural and sequence information to identify the local structure motifs. We applied our method to the following examples: the ␤␤␣-metal binding motif and the treble clef motif. The ␤␤␣-metal binding motif plays an important role in nonspecific DNA interactions and cleavage in host defense and apoptosis. The treble clef motif is a zinc-binding motif adaptable to diverse functions such as the binding of nucleic acid and hydrolysis of phosphodiester bonds. Our results are encouraging, indicating that we can effectively identify these structural motifs in an automatic fashion. Our method may provide a useful means for automatic functional annotation through detecting structural motifs associated with particular functions. Proteins 2006;63:636 -643.

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“…Thus, the metal ion within the active site becomes close to the scissile phosphodiester group suggesting that it directly participates in the catalytic process. Indeed, the presence of the metal ions is essential for the catalytic activity [97]. For NColE7 Zn 2+ [96,105] was suggested to be the physiological metal ion, but there is still a debate about the quality of metal ions in colicin nucleases [89,96,[109][110][111][112][113][114][115][116][117] [97,105].…”

Section: Nuclease Colicinsmentioning

confidence: 99%

Rescue of the activity of HNH nuclease mutants - towards controlled enzymes for gene therapy

Gyurcsik

2016

CPPS

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Artificial nucleases are designed for in vivo gene engineering, as the DNA cleavage performed at a specific target site enhances the effectiveness of the cell's DNA repair machinery. The therapeutic potential of the above phenomenon stems from the knowledge that (i) the shifted reading frame can be restored by non-homologous end-joining, or (ii) a DNA of erroneous sequence -causing a genetic disease -can be corrected by homologous recombination in the presence of a suitable DNA template. Besides the advantageous properties of the nowadays applied zinc finger nucleases, TALE nucleases and the CRISPR/Cas9 system, they possess a residual citotoxicity. This is related to offtarget cleavages, which could be prevented by the strict regulation of the enzymes. The studies on enzymes acting naturally in a controlled manner are beneficial to get better insight into their mechanism. Such enzymes or their appropriate domains may be the most promising alternatives to the presently applied ones. As an example, the DNA cleavage of the inactive HNH nuclease mutants is inducible in a multiple way. This property may be used for establishing a control mechanism and thus, in combination with specific DNA-binding domains they are good candidates for the catalytic site of artificial nucleases. Here we collect the results on the properties of the HNH nucleases that allow for their redesign into enzymes with possible therapeutic applications.

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The zinc ion in the HNH motif of the endonuclease domain of colicin E7 is not required for DNA binding but is essential for DNA hydrolysis

Cited by 58 publications

References 46 publications

Annealing helicase 2 (AH2), a DNA-rewinding motor with an HNH motif

Annealing helicase 2 (AH2), a DNA-rewinding motor with an HNH motif

The fragment transformation method to detect the protein structural motifs

Rescue of the activity of HNH nuclease mutants - towards controlled enzymes for gene therapy

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