Vaccinia topoisomerase mutants illuminate roles for Phe59, Gly73, Gln69 and Phe215.
Vaccinia topoisomerase provides a model system for structure-function analysis of the type IB topoisomerase family. Here we performed an alanine scan of eight positions in the beta4 and beta5 strands of the N-terminal domain (Leu57, Ile58, Phe59, Val60, Gly61, Ser62, Gln69 and Gly73) and eight positions in the alpha8-alpha9 loop of the C-terminal catalytic domain (Ser241, Ile242, Ser243, Pro244, Leu245, Pro246, Ser247, and Pro248). Mutants F59A, G73A, and Q69A displayed rate defects in relaxing supercoiled DNA that were attributed to effects on DNA binding rather than transesterification chemistry. Replacing Gln69 conservatively with Asn, Glu or Lys failed to restore relaxation activity. Gln69 is located along a concave DNA-binding surface of the N-terminal domain and it makes direct contact with the +2A base of the 5'-CCCTT/3-GGGAA target site for DNA cleavage. Gly73 is located at the junction between the N-terminal domain and catalytic domain and it is likely to act as a swivel for the large domain movements that coordinate DNA ingress and closure of the topoisomerase clamp around the duplex. Previous alanine scanning had identified Phe215 in helix alpha7 of the catalytic domain as contributing to DNA relaxation activity. Here we find that F215L resembles F215A in its diminished relaxation activity and its sensitivity to inhibition by salt. The Phe215 side chain makes van der Waals contacts to Ile98, Met121 and Phe101, which we propose stabilize a three helix bundle and promote clamp closure.
Tian
Ligeng
L
Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10021, USA.
Shuman
Stewart
S
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D020134:Catalytic Domain; D053837:DNA Cleavage; D026942:DNA Topoisomerases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D008274:Magnesium; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation / Q000235:genetics*; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D012965:Sodium Chloride; D014616:Vaccinia virus / Q000201:enzymology* / Q000235:genetics
D004279:DNA, Viral; D012965:Sodium Chloride; D026942:DNA Topoisomerases; D008274:Magnesium