Spectroscopic studies of the structural domains of mammalian DNA beta-polymerase.
The 8- and 31-kDa fragments of beta-polymerase, prepared by controlled proteolysis as described (Kumar, A., Widen, S. G., Williams, K. R., Kedar, P., Karpel, R. L., and Wilson, S. H. (1990) J. Biol. Chem. 265, 2124-2131), constitute domains that are structurally and functionally dissimilar. There is little disruption of secondary structure upon proteolysis of the intact enzyme, as suggested from CD spectra of the fragments. beta-Polymerase is capable of binding both single- and double-stranded nucleic acids: the 8-kDa fragment binds specifically to single-stranded lattices, whereas the 31-kDa domain displays affinity exclusively for double-stranded polynucleotides. These domains are connected by a highly flexible protease-hypersensitive segment that may allow the coordinate functioning of the two binding activities in the intact protein. beta-Polymerase binds to poly(ethenoadenylic acid) with higher affinity, similar cooperativity, but lesser salt dependence than the 8-kDa fragment. Under physiological conditions, the intact enzyme displays greater binding free energy for single-stranded polynucleotides than the 8-kDa fragment, suggesting that the latter may carry a truncated binding site. Binding of double-stranded calf thymus DNA brings about a moderate quenching of the Tyr and Trp fluorescence emission of both the 31-kDa fragment and beta-polymerase and induces a 6-nm blue shift in the Trp emission maximum of the intact enzyme, but not in the fragment. This latter result is likely due to a change in the relative orientation of the 8- and 31-kDa domains in the intact protein upon interaction with double-stranded DNA; alternatively, the binding mode of intact protein may differ from that of the fragment. Simultaneous interaction of both domains with polynucleotides most likely does not occur since double-stranded DNA binding to the 31-kDa domain of intact beta-polymerase induces the displacement of single-stranded polynucleotides from the 8-kDa domain. These results are evaluated in light of the role of beta-polymerase in DNA repair.
Department of Chemistry and Biochemistry, University of Maryland Baltimore County 21228.
J R
JR
Casas-Finet
A
A
Kumar
G
G
Morris
S H
SH
Wilson
R L
RL
Karpel
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D005454:Fluorescence Polarization; D011487:Protein Conformation; D051381:Rats; D012984:Software; D014357:Trypsin / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
D014443:Tyrosine; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D014357:Trypsin