Binding of DNA to alpha/beta-type small, acid-soluble proteins from spores of Bacillus or Clostridium species prevents formation of cytosine dimers, cytosine-thymine dimers, and bipyrimidine photoadducts after UV irradiation

Abstract

Small, acid-soluble proteins (SASP) of the alpha/beta-type from spores of Bacillus and Clostridium species bind to DNA; this binding prevents formation of cyclobutane-type thymine dimers upon UV irradiation, but promotes formation of the spore photoproduct, an adduct between adjacent thymine residues. alpha/beta-Type SASP also bound to poly(dG).poly(dC) and poly(dA-dG).poly(dC-dT). While UV irradiation of poly(dG).poly(dC) produced cyclobutane-type cytosine dimers as well as fluorescent bipyrimidine adducts, the yields of both types of photoproduct were greatly reduced upon irradiation of alpha/beta-type SASP-poly(dG).poly(dC) complexes. UV irradiation of poly(dA-dG).poly(dC-dT) produced a significant amount of a cyclobutane dimer between cytosine and thymine, as well as a 6-4 bipyrimidine adduct. Again, binding of alpha/beta-type SASP to poly(dA-dG).poly(dC-dT) greatly reduced formation of these two photoproducts, although formation of the cytosine-thymine analog of the spore photoproduct was not observed. These data provide further evidence for the dramatic change in DNA structure and photoreactivity which takes place on binding of alpha/beta-type SASP and suggest that binding of these proteins to DNA in vivo prevents formation of most deleterious photoproducts upon UV irradiation.

Synthesis and characterization of a 29‐amino acid residue DNA‐binding peptide derived from α/β‐type small, acid‐soluble spore proteins (SASP) of bacteria

“…Consequently, we tested the effect of SASP-peptide on the UV photochemistry of poly(dG), poly{[~H]dC). As found previously [17], cyclobutane-type dimers b~tween adjacent cytosine residues (CC dimers) were the major photoproduct in UVirradiated i)oly(dG).po!y([3H]dC), and CC dimer formation was suppressed upon ~/fl-type SASP binding (Table It). Binding of the SASP-peptide also reduced CC dimer formation in poly(dG), poly(dC) over 7-fold, a reduction similar to that obtained with several intact c~/fl-typ¢ SASP (Table II) [17].…”

“…As found previously [17], cyclobutane-type dimers b~tween adjacent cytosine residues (CC dimers) were the major photoproduct in UVirradiated i)oly(dG).po!y([3H]dC), and CC dimer formation was suppressed upon ~/fl-type SASP binding (Table It). Binding of the SASP-peptide also reduced CC dimer formation in poly(dG), poly(dC) over 7-fold, a reduction similar to that obtained with several intact c~/fl-typ¢ SASP (Table II) [17]. Note, however, that SspC "~", a mutant version of SspC which has lost all DNA-binding activity, has virtually no effect on poly(dG).poly(dC) photochemistry (Table II).…”

“…1). Since Clostridial ~'-type SASP behave comparable to those of Bacilhts species in experiments measuring SASP interaction with DNA [2,17,18], it seems fair to assume that these proteins share the same overall conserved tertiary structure (at least when complexed with DNA). For this to be true the 'gap' residues in Clostridial proteins must cons,,:.tute a protruding loop bridging two more essem:ial strttctural elements, defined by the sets of highly conserve:l residues centered around positions 19-20 and around position 41 (Fig.…”

“…First, it has a net charge of 2+, compared with 2-for the amino-terminal portion of the molecule (calculated for the consensus sequence at pH 7.0). Second, it conrains the site of the most functionally disruptive sitedirected mutation yet produced: SspC ")~ has agly ~ ala substitution at position 403 and lacks all of the signature properties of c~/,6-type SASP -conferring UV resistance on spores [15], promotion of plasmid negative supercoiling [15], protection of DNA against DNase I digestion [2,15], and alteration of DNA photochemistry in vitro [17,18]. For such a chemically minor change to have such profound functional consequences it seems most probable that the change directly disrupts the protein/DNA interface.…”

“…Analyses of the photoproducts formed by UV irradiation of poly(dG)'poly(~H-dC) with or without the SASP.peptide or intact ~/fl-type SASP were performed as described previously [17][18][19]. Complexes between polynucleotide and proteins or SASP-peptide were incubated for 2 h at 37*C before exposure to UV light mostly ~t 254 nm (fluent: 10 k J/m=).…”