Nucleic acid recognition by tandem helical repeats

Protein domains constructed from tandem α-helical repeats have until recently been primarily associated with protein scaffolds or RNA recognition. Recent crystal structures of human mitochondrial termination factor MTERF1 and Bacillus cereus alkylpurine DNA glycosylase AlkD bound to DNA revealed two new superhelical tandem repeat architectures capable of wrapping around the double helix in unique ways. Unlike DNA sequence recognition motifs that rely mainly on major groove read-out, MTERF and ALK motifs locate target sequences and aberrant nucleotides within DNA by resculpting the double-helix through extensive backbone contacts. Comparisons between MTERF and ALK repeats, together with recent advances in ssRNA recognition by Pumilio/FBF (PUF) domains, provide new insights into the fundamental principles of protein-nucleic acid recognition.     

63 EMSA detection of p53 sequence non-specific binding in PCR produced human DNA analogues

p53 is a tumor suppressor that induces cell cycle arrest and apoptosis in response to DNA damage and cancerogenesis. Its ability to bind DNA, and thus play its biological role, is possible in two manners: sequence-specific binding to its consensus sequence (p53CON) and sequence non-specific binding, which occurs preferably in higher DNA structures. Recently, it has been proven that DNA quadruplexes occur in regulation areas of most cancer genes. In our study, we have tested human DNA cloned into plasmid vectors. The DNAs were obtained by chromatin immunoprecipitation of regions which were bound by p53 with high affinity, although they do not contain p53CON. The sequence studied in this work is located in a noncoding region of human chromosome 7. We suggest that structure-specific binding is responsible for higher affinity of p53 binding in these areas. It has been previously found that some single-stranded regions appeared in these areas, suggesting the presence of higher DNA structures by S1 nuclease digestion (unpublished results). Because we were unable to detect the exact location of p53 binding with sufficient resolution by standard methods, we have amplified different parts of immunoprecipitated DNAs by PCR and found, using EMSA, to what part of the insert p53 binds with the highest affinity. This area is represented by cca 150 nucleotides. The strongest preference of p53 was found for the region which contained repeated short tracts of 3–4 Ts and a short polyPu.polyPy sequence. It is known that dAn:dTn blocks can cause DNA curvature, and the polyPu.polyPy sequence is able to form an intramolecular triplex.