Hairpin-duplex equilibrium reflected in the A-->B transition in an undecamer quasi-palindrome present in the locus control region of the human beta-globin gene cluster.
Our recent work on an A-->G single nucleotide polymorphism (SNP) at the quasi-palindromic sequence d(TGGGG[A/G]CCCCA) of HS4 of the human beta-globin locus control region in an Indian population showed a significant association between the G allele and the occurrence of beta-thalassemia. Using UV-thermal denaturation, gel assay, circular dichroism (CD) and nuclease digestion experiments we have demonstrated that the undecamer quasi- palindromic sequence d(TGGGGACCCCA) (HPA11) and its reported polymorphic (SNP) version d(TGG GGGCCCCA) (HPG11) exist in hairpin-duplex equilibria. The biphasic nature of the melting profiles for both the oligonucleotides persisted at low as well as high salt concentrations. The HPG11 hairpin showed a higher T(m) than HPA11. The presence of unimolecular and bimolecular species was also shown by non-denaturating gel electrophoresis experiments. The CD spectra of both oligonucleotides showed features of the A- as well as B-type conformations and, moreover, exhibited a concentration dependence. The disappearance of the 265 nm positive CD signal in an oligomer concentration-dependent manner is indicative of an A-->B transition. The results give unprecedented insight into the in vitro structure of the quasi-palindromic sequence and provide the first report in which a hairpin-duplex equilibrium has been correlated with an A-->B interconversion of DNA. The nuclease-dependent degradation suggests that HPG11 is more resistant to nuclease than HPA11. Multiple sequence alignment of the HS4 region of the beta-globin gene cluster from different organisms revealed that this quasi-palindromic stretch is unique to Homo sapiens. We propose that quasi-palindromic sequences may form stable mini- hairpins or cruciforms in the HS4 region and might play a role in regulating beta-globin gene expression by affecting the binding of transcription factors.
Kaushik
Mahima
M
Department of Chemistry, University of Delhi (North Campus), Delhi 110007, India.
Kukreti
Ritushree
R
Grover
Deepak
D
Brahmachari
Samir K
SK
Kukreti
Shrikant
S
D001483:Base Sequence; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D030541:Databases, Genetic; D003851:Deoxyribonucleases / Q000378:metabolism; D005786:Gene Expression Regulation; D005914:Globins / Q000235:genetics*; D006358:Hot Temperature; D006801:Humans; D019901:Locus Control Region / Q000235:genetics*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000235:genetics; D009690:Nucleic Acid Conformation / Q000187:drug effects; D009691:Nucleic Acid Denaturation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011110:Polymorphism, Genetic / Q000235:genetics; D012492:Salts / Q000494:pharmacology; D016415:Sequence Alignment; D013816:Thermodynamics; D014466:Ultraviolet Rays
D009838:Oligodeoxyribonucleotides; D012492:Salts; D005914:Globins; D004247:DNA; D003851:Deoxyribonucleases