Energetics and specificity of Rat DNA polymerase beta interactions with template-primer and gapped DNA substrates.
Interactions between rat polymerase beta (pol beta) and the template-primer, as well as gapped DNAs, were studied using the quantitative fluorescence titration technique. Stoichiometries of rat pol beta complexes with DNA substrates are much higher than stoichiometries predicted by the structures of co-crystals. The data can be understood in the context of the two single-stranded (ss)DNA-binding modes of the enzyme, the (pol beta)(16) and (pol beta)(5) binding modes, which differ by the number of nucleotides occluded by the protein. The 8-kDa domain of the enzyme engages the double-stranded (ds)DNA downstream from the primer, while the 31-kDa domain has similar affinity for the ss-ds DNA junction and the dsDNA. The affinity of rat pol beta for the gapped DNA is not affected by the size of the gap. The results indicate a plausible model for recognition of the gapped DNA by rat pol beta. The enzyme binds the ss-ds DNA junction of the gap using the 31-kDa domain. This binding induces an allosteric transition, resulting in the association of the 8-kDa domain with the dsDNA, leading to an amplification of the affinity for the gap. The 5' terminal phosphate, downstream from the primer, has little effect on the affinity, but affects the ssDNA conformation of the gap.
Jezewska
M J
MJ
Department of Human Biological Chemistry and Genetics, The University of Texas Medical Branch, Galveston, Texas 77555-1053.
Rajendran
S
S
Bujalowski
W
W
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D019951:DNA Polymerase beta / Q000737:chemistry* / Q000378:metabolism*; D017931:DNA Primers; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D051381:Rats; D013379:Substrate Specificity; D013698:Templates, Genetic
D017931:DNA Primers; D004277:DNA, Single-Stranded; D004247:DNA; D019951:DNA Polymerase beta