Biomedical subjects

K J Woodford

Publications and source records attributed to K J Woodford.

6 recordsLinked to original sources

PubMed1998

The mouse Ms6-hm hypervariable microsatellite forms a hairpin and two unusual tetraplexes.

The mouse Ms6-hm microsatellite consists of a tandem array of the pentamer d(CAGGG)n. This microsatellite is extremely hypervariable, showing a germ line mutation rate of 2.5%/gamete. The mechanism responsible for this instability is not known. The ability to form intrastrand structures is a conserved feature of many hypervariable sequences, and it has been suggested that the formation of such structures might account for instability by affecting DNA replication, repair, or recombination. Here we show that this microsatellite is able to form intrastrand structures as well. Under physiological conditions, the Ms6-hm microsatellite forms a hairpin as well as two different unusual intrastrand tetraplexes. The hairpin forms in the absence of monovalent cation and contains G.A, G.C, and G.G base pairs in a 1:1:1 ratio. In the presence of K+, a tetraplex is formed in which the adenines are unpaired and extrahelical, and the cytosines are involved in C.C pairs. In Na+, a tetraplex forms that contains C.C+ pairs, with the adenines being intrahelical and hydrogen-bonded to guanines. Tetraplex formation in the presence of Na+ requires both cytosines and adenines and might reflect the altered internal dimensions of this tetraplex, perhaps resulting from the ability of the C.C+ pairs to become intercalated in this sequence context. Our demonstration of the stabilization of tetraplexes by hydrogen bonding between adenines and guanines expands the hydrogen-bonding possibilities for tetraplexes and suggests that the category of sequences with tetraplex-forming potential may be larger than previously appreciated.

Animals↗

PubMed1997

DNA secondary structures and the evolution of hypervariable tandem arrays.

Tandem repeats are ubiquitous in nature and constitute a major source of genetic variability in populations. This variability is associated with a number of genetic disorders in humans including triplet expansion diseases such as Fragile X syndrome and Huntington's disease. The mechanism responsible for the variability/instability of these tandem arrays remains contentious. We show here that formation of secondary structures, in particular intrastrand tetraplexes, is an intrinsic property of some of the more unstable arrays. Tetraplexes block DNA polymerase progression and may promote instability of tandem arrays by increasing the likelihood of reiterative strand slippage. In the course of doing this work we have shown that some of these tetraplexes involve unusual base interactions. These interactions not only generate tetraplexes with novel properties but also lead us to conclude that the number of sequences that can form stable tetraplexes might be much larger than previously thought.

Animals↗

PubMed1996

The development and use of a DNA polymerase arrest assay for the evaluation of parameters affecting intrastrand tetraplex formation.

We show here that a K+-dependent block to DNA synthesis is a sensitive and specific indicator of intrastrand tetraplex formation that can be used, both to identify sequences with tetraplex-forming potential and to examine parameters that affect tetraplex formation. We show that tetraplex formation is determined by a complex combination of factors including the size and base composition of its constituent loops and stems. In the process of carrying out this study we have found that the number of sequences with the ability to form tetraplexes is larger than previously thought, and that such sequences are ubiquitous in eukaryote genomes.

Base Composition↗

PubMed1996

The chicken beta-globin gene promoter forms a novel "cinched" tetrahelical structure.

We have previously shown that the G-rich sequence G16CG(GGT)2GG in the promoter region of the chicken beta-globin gene poses a formidable barrier to DNA synthesis in vitro (Woodford et al., 1994, J. Biol. Chem. 269, 27029-27035). The K+ requirement, template-strand specificity, template concentration independence, and involvement of Hoogsteen bonding suggested that the underlying basis of this new type of DNA synthesis arrest site might be an intrastrand tetrahelical structure. However, the arrest site lacks the four G-rich repeats that are a hallmark of previously described intramolecular tetraplexes and contains a number of noncanonical bases that would be expected to greatly destabilize such a structure. Here we report evidence for an unusual K+-dependent intrastrand "cinched" tetraplex. This structure has several unique features including the incorporation of bases other than guanine into the stem of the tetraplex, interaction between loop bases and bases in the flanking region, and base pairing between bases 3 and 5 of the tetrahelix-forming region to form a molecular "cinch." This finding extends the range of sequences capable of tetraplex formation as well as our appreciation of the conformational complexity of the chicken beta-globin promoter.

Animals↗

PubMed1995

CGG repeats associated with DNA instability and chromosome fragility form structures that block DNA synthesis in vitro.

A large increase in the length of a CGG tandem array is associated with a number of triplet expansion diseases, including fragile X syndrome, the most common cause of heritable mental retardation in humans. Expansion results in the appearance of a fragile site on the X chromosome in the region of the CGG array. We show here that CGG repeats readily form a series of barriers to DNA synthesis in vitro. There barriers form only when the (CGG)n strand is used as the template, are K(+)-dependent, template concentration-independent, and involve hydrogen bonding between guanines. Chemical modification experiments suggest these blocks to DNA synthesis result from the formation of a series of intrastrand tetraplexes. A number of lines of evidence suggest that both triplet expansion and chromosome fragility are the result of replication defects. Our data are discussed in the light of such evidence.

Acetaldehyde↗

PubMed1994

A novel K(+)-dependent DNA synthesis arrest site in a commonly occurring sequence motif in eukaryotes.

We have found that a strong DNA synthesis arrest site forms in the chicken beta-globin promoter in vitro under physiological conditions. The arrest site is located in a G+C-rich region in which the guanines are located predominately on the top strand and the pyrimidines on the bottom strand. This region is non-palindromic and has no mirror symmetry. Arrest of DNA synthesis is only observed when the G-rich strand of the promoter is used as the template, and shows an absolute requirement for K+. The sequence G16CG(GGT)3 is necessary and sufficient to arrest DNA synthesis. This arrest is template concentration independent and is eliminated by blocking the N7 positions of the last 4 guanine residues in the arrest site. These observations suggest that the basis of the block to chain extension is the formation of an unusual tetraplex-like structure by the template strand. Sequences able to form intrastrand tetraplexes are ubiquitous in eukaryotes. We show that known intrastrand tetraplex-forming sequences arrest DNA synthesis in vitro, suggesting that this may be a general property of DNA tetraplexes. We suggest that the arrest of DNA synthesis by some of these structures may account for some of the high frequency of recombination associated with these loci, perhaps by promoting strand slippage or providing an opportunity for strand exchange.

Animals↗