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Biomedical subjects

G Kneale

Publications and source records attributed to G Kneale.

16 recordsLinked to original sources
PubMed2000

RNA-dependent cytoplasmic anchoring of a transcription factor subunit during Xenopus development.

The CCAAT box transcription factor (CBTF) is a multimeric transcription factor that activates expression of the haematopoietic regulatory factor, GATA-2. The 122 kDa subunit of this complex, CBTF(122), is cytoplasmic in fertilized Xenopus eggs and subsequently translocates to the nucleus prior to activation of zygotic GATA-2 transcription at gastrulation. Here we present data suggesting both a role for CBTF(122) prior to its nuclear translocation and the mechanism that retains it in the cytoplasm before the midblastula transition (MBT). CBTF(122) and its variant CBTF(98) are associated with translationally quiescent mRNP complexes. We show that CBTF(122) RNA binding activity is both necessary and sufficient for its cytoplasmic retention during early development. The introduction of an additional nuclear localization signal to CBTF(122) is insufficient to overcome this retention, suggesting that RNA binding acts as a cytoplasmic anchor for CBTF(122). Destruction of endogenous RNA by microinjection of RNase promotes premature nuclear translocation of CBTF(122). Thus, the nuclear translocation of CBTF(122) at the MBT is likely to be coupled to the degradation of maternal mRNA that occurs at that stage.

5' Untranslated Regions↗
PubMed1993

Substrate recognition and selectivity in the type IC DNA modification methylase M.EcoR124I.

The type I DNA modification methylase M.EcoR124I binds sequence specifically to DNA and protects a 25bp fragment containing its cognate recognition sequence from digestion by exonuclease III. Using modified synthetic oligonucleotide duplexes we have investigated the catalytic properties of the methylase, and have established that a specific adenine on each strand of DNA is the site of methylation. We show that the rate of methylation of each adenine is increased at least 100 fold by prior methylation at the other site. However, this is accompanied by a significant decrease in the affinity of the methylase for these substrates according to competitive gel retardation assays. In contrast, methylation of an adenine in the recognition site which is not a target for the enzyme results in only a small decrease in both DNA binding affinity and rate of methylation by the enzyme.

Base Sequence↗
PubMed1992

High-level expression of the cloned genes encoding the subunits of and intact DNA methyltransferase, M.EcoR124.

We have cloned the genes coding for the two subunits (HsdM and HsdS) of the type-I DNA methyltransferase (MTase), M.EcoR124, into the specially constructed expression vector, pJ119. These subunits have been synthesized together as an intact MTase. We have also cloned the individual subunit-encoding genes under the control of the T7 gene 10 promoter or the lacUV5 promoter. High levels of expression have been obtained in all cases. While HsdM was found to be soluble, HsdS was insoluble. However, in the presence of the co-produced HsdM subunit, HsdS was found in the soluble fraction as part of an active MTase. We have partially purified the cloned multi-subunit enzyme and shown that it is capable of DNA methylation both in vivo and in vitro.

Amino Acid Sequence↗
PubMed1992

Purification and biochemical characterisation of the EcoR124 type I modification methylase.

Large scale purification of the type I modification methylase EcoR124 has been achieved from an over-expressing strain by a two step procedure using ion-exchange and heparin chromatography. Pure methylase is obtained at a yield of 30 mg per gm of cell paste. Measurements of the molecular weight and subunit stoichiometry show that the enzyme is a trimeric complex of 162 kDa consisting of two subunits of HsdM (58 kDa) and one subunit of HsdS (46 kDa). The purified enzyme can methylate a DNA fragment bearing its cognate recognition sequence. Binding of the methylase to synthetic DNA fragments containing either the EcoR124 recognition sequence GAAN6RTCG, or the recognition sequence GAAN7RTCG of the related enzyme EcoR124/3, was followed by fluorescence competition assays and by gel retardation analysis. The results show that the methylase binds to its correct sequence with an affinity of the order 10(8) M-1 forming a 1:1 complex with the DNA. The affinity for the incorrect sequence, differing by an additional base pair in the non-specific spacer, is almost two orders of magnitude lower.

Anilino Naphthalenesulfonates↗
PubMed1990

Influence of pH on the conformation and stability of mismatch base-pairs in DNA.

A series of self-complementary dodecanucleotide duplexes containing two symmetrically disposed mismatches have been studied by pH-dependent, ultraviolet light melting techniques. The results indicate that A.C, and C.C mismatches are strongly stabilized by protonation and that the degree of stabilization of the A.C mismatch depends greatly on the flanking bases. In one case, a duplex containing two A.C mismatches is more stable than the native sequence below pH 5.5. The G.A mismatch displays conformational flexibility, with a protonated G(syn).A(anti) base-pair occurring in certain base stacking environments but not in others. The A.A and T.C mismatches are not stabilized at low pH. These solution studies correlate well with predictions based on X-ray crystallographic data.

Base Composition↗
PubMed1987

The structure of guanosine-thymidine mismatches in B-DNA at 2.5-A resolution.

The structure of the deoxyoligomer d(C-G-C-G-A-A-T-T-T-G-C-G) was determined at 2.5-A resolution by single crystal x-ray diffraction techniques. The final R factor is 18% with the location of 71 water molecules. The oligomer crystallizes in a B-DNA-type conformation, with two strands interacting to form a dodecamer duplex. The double helix consists of four A X T and six G X C Watson-Crick base pairs and two G X T mismatches. The G X T pairs adopt a "wobble" structure with the thymine projecting into the major groove and the guanine into the minor groove. The mispairs are accommodated in the normal double helix by small adjustments in the conformation of the sugar phosphate backbone. A comparison with the isomorphous parent compound containing only Watson-Crick base pairs shows that any changes in the structure induced by the presence of G X T mispairs are highly localized. The global conformation of the duplex is conserved. The G X T mismatch has already been studied by x-ray techniques in A and Z helices where similar results were found. The geometry of the mispair is essentially identical in all structures so far examined, irrespective of the DNA conformation. The hydration is also similar with solvent molecules bridging the functional groups of the bases via hydrogen bonds. Hydration may be an important factor in stabilizing G X T mismatches. A characteristic of Watson-Crick paired A X T and G X C bases is the pseudo 2-fold symmetry axis in the plane of the base pairs. The G X T wobble base pair is pronouncedly asymmetric. This asymmetry, coupled with the disposition of functional groups in the major and minor grooves, provides a number of features which may contribute to the recognition of the mismatch by repair enzymes.

Base Sequence↗
PubMed1986

Refined crystal structure of an octanucleotide duplex with G . T mismatched base-pairs.

Single crystal X-ray diffraction techniques have been used to determine the structure of the DNA octamer d(G-G-G-G-C-T-C-C) at a resolution of 2.25 A. The asymmetric unit consists of two strands coiled about each other to produce an A-type DNA helix. The double helix contains six G . C Watson-Crick base-pairs and two G . T mismatched base-pairs. The mismatches adopt a "wobble" type structure in which both bases retain their major tautomer forms. The double helix is able to accommodate this G . T pairing with little distortion of the overall helical conformation. Crystals of this octamer melt at a substantially lower temperature than do those of a related octamer also containing two G . T base-pairs. We attribute this destabilization to disruption of the hydration network around the mismatch site combined with changes in intermolecular packing. Full details are given of conformational parameters, base stacking, intermolecular contacts and hydration involving 52 solvent molecules.

Base Composition↗
PubMed1986

Structural characterisation of the bromouracil.guanine base pair mismatch in a Z-DNA fragment.

The deoxyoligonucleotide d(BrU-G-C-G-C-G) was crystallised at pH 8.2 and its structure analysed by X-ray diffraction. The unit cell, of dimensions a = 17.94, b = 30.85, c = 49.94A contains four DNA duplexes in space group P2(1)2(1)2(1). The duplexes are in the Z conformation, with four Watson-Crick G.C base pairs and two BrU.G base pairs. The structure was refined to an R factor of 0.16 at a resolution of 2.2A with 64 solvent molecules located. The BrU.G base pair mismatch is of the wobble type, with both bases in the major tautomer form and hydrogen bonds linking 0-2 of BrU with N-1 of G and N3 of BrU with 0-6 of G. There is no indication of the presence of ionised base pairs, in spite of the high pH of crystallisation. The results are discussed in terms of the mutagenic properties of 5- bromouracil.

Bromodeoxyuridine↗
PubMed1986

Molecular structure of the G.A base pair in DNA and its implications for the mechanism of transversion mutations.

The synthetic deoxydodecamer d(C-G-C-G-A-A-T-T-A-G-C-G) was analyzed by x-ray diffraction methods, and the structure was refined to a residual error of R = 0.17 at 2.5-A resolution (2 sigma data) with 83 water molecules located. The sequence crystallizes as a full turn of a B-DNA helix and contains 2 purine X purine (G.A) base pairs and 10 Watson-Crick base pairs. The analysis shows conclusively that adenine is in the syn orientation with respect to the sugar moiety whereas guanine adopts the usual trans orientation. Nitrogen atoms of both bases are involved in hydrogen bonding with the N-1 of guanine 2.84 A from the N-7 of adenine and the N-6 of adenine within 2.74 A of the O-6 of guanine. The C-1'...C-1' separation is 10.7 A close to that for standard Watson-Crick base pairs. The incorporation of the purine.purine base pairs at two steps in the dodecamer causes little perturbation of either the local or the global conformation of the double helix. Comparison of the structural features with those of the G.T wobble pair and the standard G.C pair suggests a rationale for the differential enzymatic repair of the two types of base-pair mismatches.

Chemical Phenomena↗
PubMed1985

G . T base-pairs in a DNA helix: the crystal structure of d(G-G-G-G-T-C-C-C).

The synthetic deoxyoctanucleotide d(G-G-G-G-T-C-C-C) crystallizes as an A-type DNA double helix containing two adjacent G . T base-pair mismatches. The structure has been refined to an R-factor of 14% at 2.1 A resolution with 104 solvent molecules located. The two G . T mismatches adopt the "wobble" form of base-pairing. The mismatched bases are linked by a network of water molecules interacting with the exposed functional groups in both the major and minor grooves. The presence of two mispaired bases in the octamer has surprisingly little effect on the global structure of the helix or the backbone and glycosidic torsional angles. Base stacking around the mismatch is perturbed, but the central G-T step shows particularly good base overlap, which may contribute to the relatively high stability of this oligomer.

Base Composition↗
PubMedDate not supplied

High-resolution structure of a DNA helix containing mismatched base pairs.

The concept of complementary base pairing, integral to the double-helical structure of DNA, provides an effective and elegant mechanism for the faithful transmission of genetic information. Implicit in this model, however, is the potential for incorporating non-complementary base pairs (mismatches) during replication or subsequently, for example, during genetic recombination. As such errors are usually damaging to the organism, they are generally detected and repaired. Occasionally, however, the propagation of erroneous copies of the genome confers a selective advantage, leading to genetic variation and evolutionary change. An understanding of the nature of base-pair mismatches at a molecular level, and the effect of incorporation of such errors on the secondary structure of DNA is thus of fundamental importance. We now report the first single-crystal X-ray analysis of a DNA fragment, d(GGGGCTCC), which contains two non-complementary G X T base pairs, and discuss the implications of the results for the in vivo recognition of base-pair mismatches.

Base Composition↗