The role of specific amino acid residues in the active site of Escherichia coli DNA polymerase I on translesion DNA synthesis across from and past an N-2-aminofluorene adduct.
Understanding how carcinogenic DNA adducts compromise accurate DNA replication is an important goal in cancer research. A central part of these studies is to determine the molecular mechanism that allows a DNA polymerase to incorporate a nucleotide across from and past a bulky adduct in a DNA template. To address the importance of polymerase architecture on replication across from this type of bulky DNA adduct, three active-site mutants of Escherichia coli DNA polymerase I (Klenow fragment) were used to study DNA synthesis on DNA modified with the carcinogen N-2-aminofluorene (AF). Running-start synthesis studies showed that full-length synthesis past the AF adduct was inhibited for all of the mutants, but that this inhibition was substantially less for the F762A mutant. Single nucleotide extension and steady-state kinetic experiments showed that the Y766S mutant displayed higher rates of insertion of each incorrect nucleotide relative to WT across from the dG-AF adduct. This effect was not observed for F762A or E710A mutants. Similar experiments that measured synthesis one nucleotide past the dG-AF adduct revealed an enhanced preference by the F762A mutant for dG opposite the T at this position. Finally, synthesis at the +1 and +2 positions was inhibited to a greater extent for the Y766S and E710A mutants compared with both the WT and F762A mutants. Taken together, this work is consistent with the model that polymerase geometry plays a crucial role in both the insertion and extension steps during replication across from bulky DNA lesions.
Lone
Samer
S
Department of Chemistry, Wayne State University, Detroit, Michigan 48202, USA.
Romano
Louis J
LJ
D015073:2-Acetylaminofluorene / Q000737:chemistry*; D001665:Binding Sites; D004247:DNA / Q000096:biosynthesis*; D018736:DNA Adducts / Q000378:metabolism; D004256:DNA Polymerase I / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*
D018736:DNA Adducts; D004247:DNA; D015073:2-Acetylaminofluorene; D004256:DNA Polymerase I