Functional significance of intermediate cleavages in the 3'ETS of the pre-rRNA from Schizosaccharomyces pombe

Pathways for the maturation of ribosomal RNAs are complex with numerous intermediate cleavage sites that are not always conserved closely in the course of evolution. Both in eukaryotes and bacteria genetic analyses and in vitro studies have strongly implicated RNase III-like enzymes in the processing of rRNA precursors. In Schizosacharomyces pombe, for example, the RNase III-like Pac1 nuclease has been shown to cleave the free 3′ETS at two known intermediate sites but, in the presence of RAC protein, the same RNA also is cleaved at the 3′-end of the 25 S rRNA sequence. In this study normal and mutant 3′ETS sequences were digested with the Pac1 enzyme to further evaluate its role in rRNA processing. Accurate cleavage at the known intermediate processing sites was dependent on the integrity of the helical structure at these sites as well as a more distal upper stem region in the conserved extended hairpin structure of the 3′ETS. The cleavage of mutant 3′ETS sequences also generally correlated with the known effects of these mutations on rRNA production, in vivo. One mutant, however, was efficiently processed in vivo but was not a substrate for the Pac1 nuclease, in vitro. In contrast, in the presence of RAC protein, the same RNA remained susceptible to Pac1 nuclease cleavage at the 3′-end of the 25 rRNA sequence, indicating that the removal of the 3′ETS does not require cleavage at the intermediate sites. These results suggest that basic maturation pathways may be less complex than previously reported raising similar questions about other intermediate processing sites, which have been identified by analyses of termini, and/or processing, in vitro.     

Restriction endonuclease BpuJI specific for the 5'-CCCGT sequence is related to the archaeal Holliday junction resolvase family

Type IIS restriction endonucleases (REases) recognize asymmetric DNA sequences and cleave both DNA strands at fixed positions downstream of the recognition site. REase BpuJI recognizes the asymmetric sequence 5′-CCCGT, however it cuts at multiple sites in the vicinity of the target sequence. We show that BpuJI is a dimer, which has two DNA binding surfaces and displays optimal catalytic activity when bound to two recognition sites. BpuJI is cleaved by chymotrypsin into an N-terminal domain (NTD), which lacks catalytic activity but binds specifically to the recognition sequence as a monomer, and a C-terminal domain (CTD), which forms a dimer with non-specific nuclease activity. Fold recognition approach reveals that the CTD of BpuJI is structurally related to archaeal Holliday junction resolvases (AHJR). We demonstrate that the isolated catalytic CTD of BpuJI possesses end-directed nuclease activity and preferentially cuts 3nt from the 3′-terminus of blunt-ended DNA. The nuclease activity of the CTD is repressed in the apo-enzyme and becomes activated upon specific DNA binding by the NTDs. This leads to a complicated pattern of specific DNA cleavage in the vicinity of the target site. Bioinformatics analysis identifies the AHJR-like domain in the putative Type III enzymes and functionally uncharacterized proteins.     

The nature of the 5'-terminus is a major determinant for DNA processing by Schizosaccharomyces pombe Rad2p, a FEN-1 family nuclease

The nuclease activity of FEN-1 is essential for both DNA replication and repair. Intermediate DNA products formed during these processes possess a variety of structures and termini. We have previously demonstrated that the 5′→3′ exonuclease activity of the Schizosaccharomyces pombe FEN-1 protein Rad2p requires a 5′-phosphoryl moiety to efficiently degrade a nick-containing substrate in a reconstituted alternative excision repair system. Here we report the effect of different 5′-terminal moieties of a variety of DNA substrates on Rad2p activity. We also show that Rad2p possesses a 5′→3′ single-stranded exonuclease activity, similar to Saccharomyces cerevisiae Rad27p and phage T5 5′→3′ exonuclease (also a FEN-1 homolog). FEN-1 nucleases have been associated with the base excision repair pathway, specifically processing cleaved abasic sites. Because several enzymes cleave abasic sites through different mechanisms resulting in different 5′-termini, we investigated the ability of Rad2p to process several different types of cleaved abasic sites. With varying efficiency, Rad2p degrades the products of an abasic site cleaved by Escherichia coli endonuclease III and endonuclease IV (prototype AP endonucleases) and S.pombe Uve1p. These results provide important insights into the roles of Rad2p in DNA repair processes in S.pombe.     

Patterns of sequence conservation at termini of long terminal repeat (LTR) retrotransposons and DNA transposons in the human genome: lessons from phage Mu

Long terminal repeat (LTR) retrotransposons and DNA transposons are transposable elements (TEs) that perform cleavage and transfer at precise DNA positions. Here, we present statistical analyses of sequences found at the termini of precise TEs in the human genome. The results show that the terminal di- and trinucleotides of these TEs are highly conserved. 5′TG…CA3′ occurs most frequently at the termini of LTR retrotransposons, while 5′CAG…CTG3′ occurs most frequently in DNA transposons. Interestingly, these sequences are the most flexible base pair steps in DNA. Both the sequence preference and the degree of conservation of each position within the human LTR dinucleotide termini are remarkably similar to those experimentally demonstrated in transposable phage Mu. We discuss the significance of these observations and their implication for the function of terminal residues in the transposition of precise TEs.     

Crystal structure of the Escherichia coli dcm very-short-patch DNA repair endonuclease bound to its reaction product-site in a DNA superhelix

Very-short-patch repair (Vsr) enzymes occur in a variety of bacteria, where they initiate nucleotide excision repair of G:T mismatches arising by deamination of 5-methyl-cytosines in specific regulatory sequences. We have now determined the structure of the archetypal dcm-Vsr endonuclease from Escherichia coli bound to the cleaved authentic hemi-deaminated/hemi-methylated dcm sequence 5′-C-OH-3′ 5′-p-T-p-A-p-G-p-G-3′/3′-G-p-G-p-T-p^Me5C-p-C formed by self-assembly of a 12mer oligonucleotide into a continuous nicked DNA superhelix. The structure reveals the presence of a Hoogsteen base pair within the deaminated recognition sequence and the substantial distortions of the DNA that accompany Vsr binding to product sites.     

Unidirectional translocation from recognition site and a necessary interaction with DNA end for cleavage by Type III restriction enzyme

Type III restriction enzymes have been demonstrated to require two unmethylated asymmetric recognition sites oriented head-to-head to elicit double-strand break 25–27 bp downstream of one of the two sites. The proposed DNA cleavage mechanism involves ATP-dependent DNA translocation. The sequence context of the recognition site was suggested to influence the site of DNA cleavage by the enzyme. In this investigation, we demonstrate that the cleavage site of the R.EcoP15I restriction enzyme does not depend on the sequence context of the recognition site. Strikingly, this study demonstrates that the enzyme can cleave linear DNA having either recognition sites in the same orientation or a single recognition site. Cleavage occurs predominantly at a site proximal to the DNA end in the case of multiple site substrates. Such cleavage can be abolished by the binding of Lac repressor downstream (3′ side) but not upstream (5′ side) of the recognition site. Binding of HU protein has also been observed to interfere with R.EcoP15I cleavage activity. In accordance with a mechanism requiring two enzyme molecules cooperating to elicit double-strand break on DNA, our results convincingly demonstrate that the enzyme translocates on DNA in a 5′ to 3′ direction from its recognition site and indicate a switch in the direction of enzyme motion at the DNA ends. This study demonstrates a new facet in the mode of action of these restriction enzymes.     

Specific terminal labeling of DNA molecules

We report a simple method to directly label or modify a specific terminus of linear DNA molecules. The method is based upon our finding that a presumably triple-stranded structure by RecA-mediated formation at the terminus formed with deoxyoligonucleotides, whose sequence is complementary to the 5' terminus of one of the strands of a double-stranded DNA molecule, is quite stable and can serve as a template for DNA polymerase reaction, with the nucleotides being incorporated by an exchange reaction. This novel type of nucleotide incorporation has made it possible to label a specific terminus of target double-stranded DNA molecules by a direct means (without amplification) regardless of its molecular size, a procedure previously unavailable. As an application, we show that large DNA molecules can be fixed to a solid support in a specific orientation, thus being utilized for various analytical purposes of DNA molecules.     

Synthesis,characterization and solution structure of tethered oligonucleotides containing an internal 3'-phosphoglycolate, 5'-phosphate gapped lesion

Bleomycins (BLMs) are antitumor antibiotics that in the presence of iron and oxygen mediate DNA damage by 4′-hydrogen atom abstraction of pyrimidines 3′ to guanines. The resulting 4′-deoxyribose radicals can be trapped by O₂ and ultimately result in the formation of base-propenal and gapped DNA with 3′-phosphoglycolate (3′-PG) and 5′-phosphate (5′-P) ends. The role of this lesion in triggering double-strand cleavage of duplex DNA by a single BLM molecule and the mechanism by which this lesion is repaired in vivo remain unsolved problems. The structure of these lesions is an essential step in addressing both of these problems. Duplex DNAs (13mers containing tethered hexaethylene glycol linkers) with GTAC and GGCC cleavage sites have been synthesized in which gaps containing 3′-PG and 5′-P ends at the sites of BLM cleavage have been inserted. The former sequence represents a hot spot for double-strand cleavage, while the latter is a hot spot for single-strand cleavage. Analytical methods to characterize the lesioned products have been developed. These oligonucleotides have been examined using 2D NMR methods and molecular modeling. The studies reveal that the lesioned DNAs are B-form and the 3′-PG and 5′-P are extrahelical. The base opposite the gap and the base pairs adjacent to the gap remain well stacked in the DNA duplex. Titrations of the lesioned GGCC oligomer with HOO-CoBLM leads to a mixture of complexes, in contrast to results of a similar titration with the lesioned GTAC oligomer.     

Synthesis and polymerase incorporation of 5'-amino-2',5'-dideoxy-5'-N-triphosphate nucleotides

Owing to the markedly increased reactivity of amino functional groups versus hydroxyls, the 5′-amino-5′-deoxy nucleoside and nucleotide analogs have proven widely useful in biological, pharmaceutical and genomic applications. However, synthetic procedures leading to these analogs have not been fully explored, which may possibly have limited the scope of their utility. Here we describe the synthesis of the 5′-amino-2′,5′-dideoxy analogs of adenosine, cytidine, guanosine, inosine and uridine from their respective naturally occurring nucleosides via the reduction of 5′-azido-2′,5′-dideoxy intermediates using the Staudinger reaction, and the high yield conversion of these modified nucleosides and 5′-amino-5′-deoxythymidine to the corresponding 5′-N-triphosphates through reaction with trisodium trimetaphosphate in the presence of tris(hydroxymethyl)aminomethane (Tris). We also show that each of these nucleotide analogs can be efficiently incorporated into DNA by the Klenow fragment of Escherichia coli DNA polymerase I when individually substituted for its naturally occurring counterpart. Mild acid treatment of the resulting DNA generates polynucleotide fragments that arise from specific cleavage at each modified nucleotide, providing a sequence ladder for each base. Because the ladders are generated after the extension, the corresponding products may be manipulated by enzymatic and/or purification processes. The potential utility of this extension–cleavage procedure in genomic sequence analysis is discussed.     

Efficient processing of TFO-directed psoralen DNA interstrand crosslinks by the UvrABC nuclease

Photoreactive psoralens can form interstrand crosslinks (ICLs) in double-stranded DNA. In eubacteria, the endonuclease UvrABC plays a key role in processing psoralen ICLs. Psoralen-modified triplex-forming oligonucleotides (TFOs) can be used to direct ICLs to specific genomic sites. Previous studies of pyrimidine-rich methoxypsoralen–modified TFOs indicated that the TFO inhibits cleavage by UvrABC. Because different chemistries may alter the processing of TFO-directed ICLs, we investigated the effect of another type of triplex formed by purine-rich TFOs on the processing of 4′-(hydroxymethyl)-4,5′,8-trimethylpsoralen (HMT) ICLs by the UvrABC nuclease. Using an HMT-modified TFO to direct ICLs to a specific site, we found that UvrABC made incisions on the purine-rich strand of the duplex ~3 bases from the 3′-side and ~9 bases from the 5′-side of the ICL, within the TFO-binding region. In contrast to previous reports, the UvrABC nuclease cleaved the TFO-directed psoralen ICL with a greater efficiency than that of the psoralen ICL alone. Furthermore, the TFO was dissociated from its duplex binding site by UvrA and UvrB. As mutagenesis by TFO-directed ICLs requires nucleotide excision repair, the efficient processing of these lesions supports the use of triplex technology to direct DNA damage for genome modification.     

Cleavage of Circular, Superhelical Simian Virus 40 DNA to a Linear Duplex by S1 Nuclease

S(1) nuclease, the single-strand specific nuclease from Aspergillus oryzae can cleave both strands of circular covalently closed, superhelical simian virus 40 (SV40) DNA to generate unit length linear duplex molecules with intact single strands. But circular, covalently closed, nonsuperhelical DNA, as well as linear duplex molecules, are relatively resistant to attack by the enzyme. These findings indicate that unpaired or weakly hydrogen-bonded regions, sensitive to the single strand-specific nuclease, occur or can be induced in superhelical DNA. Nicked, circular SV40 DNA can be cleaved on the opposite strand at or near the nick to yield linear molecules. S(1) nuclease may be a useful reagent for cleaving DNAs at regions containing single-strand nicks. Unlike the restriction endonucleases, S(1) nuclease probably does not cleave SV40 DNA at a specific nucleotide sequence. Rather, the sites of cleavage occur within regions that are readily denaturable in a topologically constrained superhelical molecule. At moderate salt concentrations (75 mM) SV40 DNA is cleaved once, most often within either one of the two following regions: the segments defined as 0.15 to 0.25 and 0.45 to 0.55 SV40 fractional length, clockwise, from the EcoR(I) restriction endonuclease cleavage site (defined as the zero position on the SV40 DNA map). In higher salt (250 mM) cleavage occurs preferentially within the 0.45 to 0.55 segment of the map.     

Cation-dependent cleavage of the duplex form of the subtype-B HIV-1 RNA dimerization initiation site

The crystal structure of subtype-B HIV-1 genomic RNA Dimerization Initiation Site duplex revealed chain cleavage at a specific position resulting in 3′-phosphate and 5′-hydroxyl termini. A crystallographic analysis showed that Ba²⁺, Mn²⁺, Co²⁺ and Zn²⁺ bind specifically on a guanine base close to the cleaved position. The crystal structures also point to a necessary conformational change to induce an ‘in-line’ geometry at the cleavage site. In solution, divalent cations increased the rate of cleavage with pH/pKa compensation, indicating that a cation-bound hydroxide anion is responsible for the cleavage. We propose a ‘Trojan horse’ mechanism, possibly of general interest, wherein a doubly charged cation hosted near the cleavage site as a ‘harmless’ species is further transformed in situ into an ‘aggressive’ species carrying a hydroxide anion.     

Structure specific DNA recognition by the SLX1–SLX4 endonuclease complex

The SLX1–SLX4 structure-specific endonuclease complex is involved in processing diverse DNA damage intermediates, including resolution of Holliday junctions, collapse of stalled replication forks and removal of DNA flaps. The nuclease subunit SLX1 is inactive on its own, but become activated upon binding to SLX4 via its conserved C-terminal domain (CCD). Yet, how the SLX1–SLX4 complex recognizes specific DNA structure and chooses cleavage sites remains unknown. Here we show, through a combination of structural, biochemical and computational analyses, that the SAP domain of SLX4 is critical for efficient and accurate processing of 5′-flap DNA. It binds the minor groove of DNA about one turn away from the flap junction, and the 5′-flap is implicated in binding the core domain of SLX1. This binding mode accounts for specific recognition of 5′-flap DNA and specification of cleavage site by the SLX1–SLX4 complex.     

Type II restriction endonuclease R.KpnI is a member of the HNH nuclease superfamily

The restriction endonuclease (REase) R.KpnI is an orthodox Type IIP enzyme, which binds to DNA in the absence of metal ions and cleaves the DNA sequence 5′-GGTAC^C-3′ in the presence of Mg²⁺ as shown generating 3′ four base overhangs. Bioinformatics analysis reveals that R.KpnI contains a ββα-Me-finger fold, which is characteristic of many HNH-superfamily endonucleases, including homing endonuclease I-HmuI, structure-specific T4 endonuclease VII, colicin E9, sequence non-specific Serratia nuclease and sequence-specific homing endonuclease I-PpoI. According to our homology model of R.KpnI, D148, H149 and Q175 correspond to the critical D, H and N or H residues of the HNH nucleases. Substitutions of these three conserved residues lead to the loss of the DNA cleavage activity by R.KpnI, confirming their importance. The mutant Q175E fails to bind DNA at the standard conditions, although the DNA binding and cleavage can be rescued at pH 6.0, indicating a role for Q175 in DNA binding and cleavage. Our study provides the first experimental evidence for a Type IIP REase that does not belong to the PD…D/EXK superfamily of nucleases, instead is a member of the HNH superfamily.     

Flap Endonuclease 1 Mechanism Analysis Indicates Flap Base Binding Prior to Threading

FEN1 cleaves 5′ flaps at their base to create a nicked product for ligation. FEN1 has been reported to enter the flap from the 5′-end and track to the base. Current binding analyses support a very different mechanism of interaction with the flap substrate. Measurements of FEN1 binding to a flap substrate show that the nuclease binds with similar high affinity to the base of a long flap even when the 5′-end is blocked with biotin/streptavidin. However, FEN1 bound to a blocked flap is more sensitive to sequestration by a competing substrate. These results are consistent with a substrate interaction mechanism in which FEN1 first binds the flap base and then threads the flap through an opening in the protein from the 5′-end to the base for cleavage. Significantly, when the unblocked flap length is reduced from five to two nucleotides, FEN1 can be sequestered from the substrate to a similar extent as a blocked, long flap substrate. Apparently, interactions related to threading occur only when the flap is greater than two to four nucleotides long, implying that short flaps are cleaved without a threading requirement.     

Specific cleavage of kinetoplast minicircle DNA from Leishmania tarentolae by mung bean nuclease and identification of several additional minicircle sequence classes

Multiple sequence classes of kinetoplast minicircle DNA from Leishmania tarentolae were cleaved by mung bean nuclease in the presence of formamide, yielding unit length linear molecules which retained the anomalous electrophoretic mobility in acrylamide characteristic of minicircle DNA. No specific cleavage site sequence common to all minicircle sequence classes was apparent, although the main region of nuclease cleavage was localized approximately 350 bp from the unique SmaI restriction site of the conserved region found in all minicircle sequence classes. Covalent closure of the minicircle substrate was not a requirement for cleavage, as linearized network-derived or cloned minicircles were also cleaved by mung bean nuclease at similar locations. The partial sequences of several new minicircle sequence classes released from the network by mung bean nuclease are also reported.     

Mechanisms of DNA cleavage by copper complexes of 3-clip-phen and of its conjugate with a distamycin analogue

Mechanisms of DNA oxidation by copper complexes of 3-Clip-Phen and its conjugate with a distamycin analogue, in the presence of a reductant and air, were studied. Characterisation of the production of 5-methylenefuranone (5-MF) and furfural, associated with the release of nucleobases, indicated that these copper complexes oxidised the C1′ and C5′ positions of 2-deoxyribose, respectively, which are accessible from the DNA minor groove. Oxidation at C1′ was the major degradation route. Digestion of DNA oxidation products by P1 nuclease and bacterial alkaline phosphatase allowed characterisation of glycolic acid residues, indicating that these copper complexes also induced C4′ oxidation. However, this pathway was not associated with base propenal release. The ability of the copper complex of the 3-Clip-Phen conjugate with the distamycin analogue to produce sequence-selective DNA cleavage allowed confirmation of these mechanisms of DNA oxidation by PAGE. Comparison of DNA cleavage activity showed that conjugation of 3-Clip-Phen with a DNA minor groove binder, like the distamycin analogue, decreased both its ability to perform C1′ oxidation as well as the initial rate of the reaction, but this conjugate is still active after 5 h at 37°C, making it an efficient DNA cleaver.     

Mung bean nuclease: mode of action and specificity vs synthetic esters of 3'-nucleotides

Mung bean nuclease hydrolyzes synthetic esters of 3′-nucleotides to nucleosides and phosphate esters; esters of 2′-nucleotides, and 2′→ 5′ internucleotide linkages, are resistant. Esters of ribonucleotides are cleaved at 100-fold the rate for deoxyribonucleotides, the increased rate being due to presence of the 2′-hydroxyl and not to differences in conformation. Introduction of a 5′-substituent leads to a 3-fold increase in rate. The rates of hydrolysis vary up to 10-fold with the nature of the base, in the order adenine > hypoxanthine > uracil; and up to 6-fold with the nature of the ester radical. This form of cleavage of esters of 3′-nucleotides is also characteristic for nuclease-3′-nucleotidase activities from potato tubers and wheat, suggesting that one type of enzyme is responsible for all these activities.     

High sequence specificity of micrococcal nuclease.

The substrate specificity of micrococcal nuclease (EC 3.1.4.7.) has been studied. The enzyme recognises features of nucleotide composition, nucleotide sequence and tertiary structure of DNA. Kinetic analysis indicates that the rate of cleavage is 30 times greater at the 5' side of A or T than at G or C. Digestion of end-labelled linear DNA molecules of known sequence revealed that only a limited number of sites are cut, generating a highly specific pattern of fragments. The frequency of cleavage at each site has been determined and it may reflect the poor base overlap in the 5' T-A 3' stack as well as the length of contiguous A and T residues. The same sequence preferences are found when DNA is assembled into nucleosomes. Deoxyribonuclease 1 (EC 3.1.4.5.) recognises many of the same sequence features. Micrococcal nuclease also mimics nuclease S1 selectively cleaving an inverted repeat in supercoiled pBR322. The value of micrococcal nuclease as a "non-specific" enzymatic probe for studying nucleosome phasing is questioned.