Crystal structure of bacteriophage T4 5' nuclease in complex with a branched DNA reveals how flap endonuclease-1 family nucleases bind their substrates.
Bacteriophage T4 RNase H, a flap endonuclease-1 family nuclease, removes RNA primers from lagging strand fragments. It has both 5' nuclease and flap endonuclease activities. Our previous structure of native T4 RNase H (PDB code 1TFR) revealed an active site composed of highly conserved Asp residues and two bound hydrated magnesium ions. Here, we report the crystal structure of T4 RNase H in complex with a fork DNA substrate bound in its active site. This is the first structure of a flap endonuclease-1 family protein with its complete branched substrate. The fork duplex interacts with an extended loop of the helix-hairpin-helix motif class 2. The 5' arm crosses over the active site, extending below the bridge (helical arch) region. Cleavage assays of this DNA substrate identify a primary cut site 7-bases in from the 5' arm. The scissile phosphate, the first bond in the duplex DNA adjacent to the 5' arm, lies above a magnesium binding site. The less ordered 3' arm reaches toward the C and N termini of the enzyme, which are binding sites for T4 32 protein and T4 45 clamp, respectively. In the crystal structure, the scissile bond is located within the double-stranded DNA, between the first two duplex nucleotides next to the 5' arm, and lies above a magnesium binding site. This complex provides important insight into substrate recognition and specificity of the flap endonuclease-1 enzymes.
Devos
Juliette M
JM
Department of Chemistry, The University of Toledo, Toledo, Ohio 43606, USA.
Tomanicek
Stephen J
SJ
Jones
Charles E
CE
Nossal
Nancy G
NG
Mueser
Timothy C
TC
D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000201:enzymology*; D001483:Base Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004279:DNA, Viral / Q000737:chemistry* / Q000378:metabolism*; D045585:Flap Endonucleases / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012367:RNA, Viral / Q000378:metabolism; D016914:Ribonuclease H / Q000737:chemistry / Q000378:metabolism; D013059:Spectrum Analysis, Raman; D013379:Substrate Specificity
D004279:DNA, Viral; D012367:RNA, Viral; D045585:Flap Endonucleases; D016914:Ribonuclease H