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Biomedical subjects

D M Lilley

Publications and source records attributed to D M Lilley.

At least 73 records · Page 4Linked to original sources
PubMed1997

The resolving enzyme CCE1 of yeast opens the structure of the four-way DNA junction.

Junction-resolving enzymes exhibit structure-selective binding to DNA, but may also manipulate the DNA structure. CCE1 is a junction-resolving enzyme found in the yeast mitochondrion. To facilitate the analysis of the CCE1-junction interaction, we have exploited the sequence dependence of the cleavage reaction to devise a junction that is refractory to cleavage by this enzyme, even in the presence of magnesium ions. On binding to four-way DNA junctions, pure recombinant CCE1 opens the global structure into a 4-fold symmetrical configuration of arms with an open, chemically reactive centre. The structure of the CCE1-junction complex is independent of the sequence of the junction, and of the presence or absence of magnesium or other ions. This and other functional properties of CCE1 are strikingly similar to those of RuvC resolving enzyme of Escherichia coli.

Base Sequence↗
PubMed1997

Nucleic acid structure and recognition.

We review the global structures adopted by branched nucleic acids, including three- and four-way helical junctions in DNA and RNA. We find that some general folding principles emerge. First, all the structures exhibit a tendency to undergo pairwise coaxial helical stacking when permitted by the local stereochemistry of strand exchange. Second, metal ions generally play an important role in facilitating folding of branched nucleic acids. These principles can be applied to functionally important branched nucleic acids, such as the Holliday DNA junction of genetic recombination, and the hammerhead ribozyme in RNA.

Journal Article↗
PubMed1997

Characterization of a Holliday junction-resolving enzyme from Schizosaccharomyces pombe.

The rearrangement and repair of DNA by homologous recombination involves the creation of Holliday junctions, which are cleaved by a class of junction-specific endonucleases to generate recombinant duplex DNA products. Only two cellular junction-resolving enzymes have been identified to date: RuvC in eubacteria and CCE1 from Saccharomyces cerevisiae mitochondria. We have identified a protein from Schizosaccharomyces pombe which has 28% sequence identity to CCE1. The YDC2 protein has been cloned and overexpressed in Escherichia coli, and the purified recombinant protein has been shown to be a Holliday junction-resolving enzyme. YDC2 has a high degree of specificity for the structure of the four-way junction, to which it binds as a dimer. The enzyme exhibits a sequence specificity for junction cleavage that differs from both CCE1 and RuvC, and it cleaves fixed junctions at the point of strand exchange. The conservation of the mechanism of Holliday junction cleavage between two organisms as diverse as S. cerevisiae and S. pombe suggests that there may be a common pathway for mitochondrial homologous recombination in fungi, plants, protists, and possibly higher eukaryotes.

Amino Acid Sequence↗
PubMed1996

T4 endonuclease VII. Importance of a histidine-aspartate cluster within the zinc-binding domain.

The DNA junction-resolving enzyme endonuclease VII of bacteriophage T4 contains a zinc-binding region toward the N-terminal end of the primary sequence. In the center of this 39-amino acid section (between residues 38 and 44) lies the sequence HLDHDHE, termed the His-acid cluster. Closely related sequences are found in three other proteins that have similar zinc-binding motifs. We have analyzed the function of these residues by a site-directed mutagenesis approach, modifying single amino acids and studying the properties of the resulting N-terminal protein A fusions. No sequence changes within the His-acid cluster led to a change in zinc content of the protein, indicating that these residues are not involved in the coordination of zinc. We found that the N-terminal aspartate residue (Asp-40) and the two histidine residues (His-41 and His-43) within the cluster are essential for junction-cleavage activity of the proteins. However, all sequence variations within this region generate proteins that retain their ability to bind to four-way DNA junctions (with minor changes in binding affinity in some cases) and to distort their global structure in the same manner as active enzymes. We conclude that the process of cleavage can be uncoupled from those of binding to and distortion of the junction. It is probable that some amino acid side chains of the His-acid cluster participate in the phosphodiester cleavage mechanism of endonuclease VII. The essential aspartate residue might be required for coordination of catalytic metal ions.

Amino Acid Sequence↗
PubMed1996

T4 endonuclease VII selects and alters the structure of the four-way DNA junction; binding of a resolution-defective mutant enzyme.

Bacteriophage T4 endonuclease VII is a nuclease that is selective for four-way DNA junctions and related branched DNA species. Using site-directed mutagenesis we have isolated a mutant protein (E86A) that is inactive in the cleavage of DNA junctions while retaining full selectivity of binding. Using endonuclease VII E86A we have shown: (1) The protein binds as a dimer to DNA junctions, with rapid exchange of subunits in free solution. (2) Binding to junctions is highly selective for the structure of DNA junctions; the complex is not displaced by a 1000-fold excess of duplex competitor DNA. (3) On binding endonuclease VII E86A to junctions, the configuration of the helical arms is significantly altered to a structure that is independent of the presence or absence of metal ions. We suggest a model for the structure of the junction in the protein complex. (4) The protein can bind to the junction in two stereochemically equivalent ways, depending upon the sequence of the junction. T4 endonuclease VII is a junction-selective enzyme that both recognises and manipulates the structure of its substrate.

Amino Acid Sequence↗
PubMed1996

Human MutSalpha recognizes damaged DNA base pairs containing O6-methylguanine, O4-methylthymine, or the cisplatin-d(GpG) adduct.

Bacterial and mammalian mismatch repair systems have been implicated in the cellular response to certain types of DNA damage, and genetic defects in this pathway are known to confer resistance to the cytotoxic effects of DNA-methylating agents. Such observations suggest that in addition to their ability to recognize DNA base-pairing errors, members of the MutS family may also respond to genetic lesions produced by DNA damage. We show that the human mismatch recognition activity MutSalpha recognizes several types of DNA lesion including the 1,2-intrastrand d(GpG) crosslink produced by cis-diamminedichloroplatinum(II), as well as base pairs between O6-methylguanine and thymine or cytosine, or between O4-methylthymine and adenine. However, the protein fails to recognize 1,3-intrastrand adduct produced by trans-diamminedichloroplatinum(II) at a d(GpTpG) sequence. These observations imply direct involvement of the mismatch repair system in the cytotoxic effects of DNA-methylating agents and suggest that recognition of 1,2-intrastrand cis-diamminedichloroplatinum(II) adducts by MutSalpha may be involved in the cytotoxic action of this chemotherapeutic agent.

Adenosine Triphosphatases↗
PubMed1996

The structure-selectivity and sequence-preference of the junction-resolving enzyme CCE1 of Saccharomyces cerevisiae.

CCE1 is a DNA junction-resolving enzyme involved in the resolution of recombining mitochondrial DNA in Saccharomyces cerevisiae. The CCE1 gene was cloned by PCR, and the expressed protein purified to homogeneity. CCE1 was found to bind to four-way DNA junctions, with a strong structural selectivity. The enzyme binds DNA junctions as a dimer, with slow subunit exchange occurring in free solution. While CCE1 binds equally to synthetic four-way DNA junctions of any sequence, it exhibits pronounced sequence-selectivity in cleavage. Both fixed junctions and those capable of branch migration can be cleaved, with a preference for cleavage at the sequence 5'CT/. Cleavage of junctions tethered to adopt specific stacking isomers demonstrated that the target sequences are cleaved fivefold faster when located on a continuous strand compared to an exchanging strand.

Amino Acid Sequence↗
PubMed1996

Effect of flanking sequences on the right- to left-handed transition of a (dA-dT)n tract in supercoiled DNA.

Alternating (dA-dT)n sequences in supercoiled DNA may undergo a transition to a left-handed conformation in the presence of Ni2+ ions and high NaCl concentration (Nejedlý, K., Klysik, J. and Palecek, E., FEBS Lett. 243, 313-317 (1989)). In this work we have found that ionic conditions necessary for the B-to-Z transition are strongly dependent on the sequences flanking the (dA-dT)n tract. In particular, the presence of 5'-homopyrimidine (C3) and 3'-homopurine (G4) blocks adjacent to the tract were found to facilitate the transition to the left-handed form. Within a constant sequence context it was found that the ionic strength required to promote the transition was inversely proportional to the length of the (dA-dT)n sequence.

Base Sequence↗
PubMed1996

The ion-induced folding of the hammerhead ribozyme: core sequence changes that perturb folding into the active conformation.

The hammerhead ribozyme undergoes an ion-dependent folding process into the active conformation. We find that the folding can be blocked at specific stages by changes of sequence or functionality within the core. In the the absence of added metal ions, the global structure of the hammerhead is extended, with a large angle subtended between stems I and II. No core sequence changes appear to alter this geometry, consistent with an unstructured core under these conditions. Upon addition of low concentrations of magnesium ions, the hammerhead folds by an association of stems II and III, to include a large angle between them. This stage is inhibited or altered by mutations within the oligopurine sequence lying between stems II and III, and folding is completely prevented by an A14G mutation. Further increase in magnesium ion concentration brings about a second stage of folding in the natural sequence hammerhead, involving a reorientation of stem I, which rotates around into the same direction of stem II. Because this transition occurs over the same range of magnesium ion concentration over which the hammerhead ribozyme becomes active, it is likely that the final conformation is most closely related to the active form of the structure. Magnesium ion-dependent folding into this conformation is prevented by changes at G5, notably removal of the 2'-hydroxyl group and replacement of the base by cytidine. The ability to dissect the folding process by means of sequence changes suggests that two separate ion-dependent stages are involved in the folding of the hammerhead ribozyme into the active conformation.

Base Sequence↗