Herpes simplex virus-1 helicase-primase: roles of each subunit in DNA binding and phosphodiester bond formation.
The helicase-primase complex from herpes simplex virus-1 contains three subunits, UL5, UL52, and UL8. We generated each of the potential two-subunit complexes, UL5-UL52, UL5-UL8, and UL52-UL8, and used a series of kinetic and photo-cross-linking studies to provide further insights into the roles of each subunit in DNA binding and primer synthesis. UL8 increases the rate of primer synthesis by UL5-UL52 by increasing the rate of primer initiation (two NTPs --> pppNpN), the rate-limiting step in primer synthesis. The UL5-UL8 complex lacked any detectable catalytic activity (DNA-dependent ATPase, primase, or RNA polymerase using a RNA primer-template and NTPs as substrates) but could still bind DNA, indicating that UL52 must provide some key amino acids needed for helicase function. The UL52-UL8 complex lacked detectable DNA-dependent ATPase activity and could not synthesize primers on single-stranded DNA. However, it exhibited robust RNA polymerase activity using a RNA primer-template and NTPs as substrates. Thus, UL52 must contain the entire primase active site needed for phosphodiester bond formation, while UL5 minimally contributes amino acids needed for the initiation of primer synthesis. Photo-cross-linking experiments using single-stranded templates containing 5-iodouracil either before, in, or after the canonical 3'-GPyPy (Py is T or C) initiation site for primer synthesis showed that only UL5 cross-linked to the DNA. This occurred for the UL5-UL52, UL5-UL52-UL8, and UL5-UL8 complexes and whether the reaction mixtures contained NTPs. Photo-cross-linking of a RNA primer-template, the product of primer synthesis, containing 5-iodouracil in the template generated the same apparent cross-linked species.
Cavanaugh
Nisha A
NA
Department of Chemistry and Biochemistry, University of Colorado, UCB-215, Boulder, Colorado 80309, USA.
Ramirez-Aguilar
Kathryn A
KA
Urban
Milan
M
Kuchta
Robert D
RD
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D020134:Catalytic Domain; D002460:Cell Line; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism*; D019915:DNA Primase / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D010755:Organophosphates / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D018411:Spodoptera; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*
D017931:DNA Primers; D004277:DNA, Single-Stranded; D010755:Organophosphates; D021122:Protein Subunits; D014764:Viral Proteins; D004247:DNA; D019915:DNA Primase; C062021:helicase-primase, Human herpesvirus 1; D004265:DNA Helicases