Book of Abstracts: Albany 2005

category image Volume 22
No. 6
June 2005

Biophysical Features Of Slipped CTG/CAG Structures And Their Biological Recognition

CTG/CAG instability is the cause of >14 diseases including myotonic dystrophy (DM1) and Huntingtons disease (HD). The expansion of CTG/CAG repeats is likely to involve slipped-strand DNAs as mutagenic intermediates. There are two types of slipped-DNAs: S-DNAs are slipped homoduplexes formed between two strands having the same number of repeats; and SI-DNAs which are slipped intermediate heteroduplexes formed between two strands having different numbers of repeats. S-DNAs may be mutagenic intermediates during error-prone DNA metabolism, such as repair or recombination. SI-DNAs may occur at replication forks, forming between the nascent and template strands or at strand breaks in non-replicating DNAs. The biophysical features of slipped-DNAs, including the slip-outs and slip-out junctions may critically determine whether and how they are recognized and processed by DNA metabolizing proteins.

The two forms of heteroduplex SI-DNAs were dramatically different: slip-outs of CAG repeats assumed a random-coil single-stranded conformation, while slip-outs of CTG assumed an intrastrand hairpin conformation. These biophysical differences may account for the preferential binding of single-stranded binding protein (SSB) and hMSH2 to the CAG slip-out. Furthermore, slip-out junctions of excess CAG or excess CTG repeats were cleaved differentially by DNA junction resolving enzymes. Slipped DNAs were bound by antibodies specific for Z-DNA and DNA junctions, revealing shared characteristics between the antigenic DNA structures and slipped-DNAs.

Circular heteroduplexes containing different numbers of repeats were prepared for use in an in vitro repair assay and these were found to assume similar biophysical characteristics to SI-DNAs. The distinct features of slipped-DNAs with an excess of CTG repeats compared to those with an excess of CAG repeats may permit them to be recognized and processed by distinct mechanisms, leading to subsequent repeat expansions, contractions or no change. The processing of slipped DNAs will provide insight into the role of slipped strand DNAs in disease-associated repeat instability.

?Respect the personality of DNA?
Commandment VIII from Arthur Kornberg?s Ten Commandments of DNA

Mariana Kekis1,2
Rachel Lau1
S. Erin Montgomery1,2
Kerrie Nichol Edamura2
Yuh-Hwa Wang3
Christopher E. Pearson1,2,*

1Genetics & Genomic Biology
The Hospital for Sick Children
555 University Avenue
11-135 Elm Wing
Toronto, Canada
2Department of Molecular & Medical Genetics
University of Toronto
3Department of Biochemistry
University of Medicine and Dentistry of New Jersey

*Phone: 416-81-8256
Fax: 416-813-4931
Email: cepearson@genet.sickkids.on.ca