Interaction of terminal transferase with single-stranded DNA

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Terminal transferase is a key enzyme in DNA synthesis.
• It exists in different molecular weight forms, including a 44-kDa dimer and a 58-kDa monomer.
• Understanding the structural and functional differences between these forms is important for elucidating enzyme mechanisms.

Purpose of the Study:

• To characterize the DNA-binding properties of the 58-kDa monomeric terminal transferase.
• To compare the DNA-binding characteristics of the monomer with the previously studied 44-kDa alpha beta dimer.
• To determine if the additional peptide sequences in the monomer influence DNA binding.

Main Methods:

• Isolation of 58-kDa terminal transferase monomer using monoclonal antibody affinity chromatography.
• Enzymatic activity assays.
• Fluorescence-based monitoring of enzyme binding to single-stranded DNA.
• Analysis of binding parameters including site size, cooperativity, and dissociation constants under various conditions (e.g., presence/absence of Mg2+, salt concentration).

Main Results:

• The 58-kDa monomer exhibited comparable enzymatic activity to the 44-kDa dimer.
• Both enzyme forms bound to single-stranded DNA with a similar site size of approximately 11 nucleotides.
• The DNA-binding parameters of the monomer were largely unaffected by buffer composition and mimicked those of the dimer in the presence of Mg2+.
• The cooperativity of the 44-kDa dimer binding to polydeoxyadenosine was significantly influenced by Mg2+ concentration.

Conclusions:

• The additional 14-kDa peptide sequences in the 58-kDa monomer are not part of the DNA-binding site.
• The DNA-binding properties of terminal transferase are conserved between the monomeric and dimeric forms, particularly in the presence of Mg2+.
• These findings suggest that the core DNA-binding domain is similar in both forms of the enzyme.