Raman microspectroscopic study of low-pH-induced changes in DNA structure of polytene chromosomes

The effects of low-pH treatments on DNA structure in polytene chromosomes of Chironomus thummi thummi have been studied by Raman microspectroscopy. Measurements were carried out on chromosomes at low pH and on chromosomes reneutralized after a short exposure to low pH. Protonation of adenine residues and subsequent unpairing of adenine (A) and thymine (T) were found to commence already above pH 3.6 and are completed at pH 2.2. Protonation of the cytosine-guanine base pair also starts above pH 3.6. It leads to an intermediate protonated, most likely Hoogsteen-type, guanine-cytosine base pair. Unpairing of G and C residues starts between pH 2.6 and 2.2 and continues below pH 2.2. Brief treatment of chromosomes at a pH < or = 2.2, i.e., at a pH where both AT and GC base pairs are disrupted, was found to lead to irreversible changes in DNA conformation upon return to neutral pH. These were most pronounced after treatment with 45% acetic acid. More than 10% of the A and T residues was found to have adopted a non-B-DNA conformation. Evidence was found for denaturation of the B-form backbone. The amount of protein extracted from the chromosomes was strongly pH-dependent. Treatment at pH 3.6 did not cause noticeable protein extraction, while treatment with 45% acetic acid extracted more than 50% (by weight) of the chromosomal proteins.