ABSTRACT
Synthesis of oligoribonucleotide primers for lagging-strand DNA synthesis in the DNA replication system of bacteriophage T7 is catalyzed by the primase domain of the gene 4 helicase-primase. The primase consists of a zinc-binding domain (ZBD) and an RNA polymerase (RPD) domain. The ZBD is responsible for recognition of a specific sequence in the ssDNA template whereas catalytic activity resides in the RPD. The ZBD contains a zinc ion coordinated with four cysteine residues. We have examined the ligation state of the zinc ion by X-ray absorption spectroscopy and biochemical analysis of genetically altered primases. The ZBD of primase engaged in catalysis exhibits considerable asymmetry in coordination to zinc, as evidenced by a gradual increase in electron density of the zinc together with elongation of the zinc-sulfur bonds. Both wild-type primase and primase reconstituted from purified ZBD and RPD have a similar electronic change in the level of the zinc ion as well as the configuration of the ZBD. Single amino acid replacements in the ZBD (H33A and C36S) result in the loss of both zinc binding and its structural integrity. Thus the zinc in the ZBD may act as a charge modulation indicator for the surrounding sulfur atoms necessary for recognition of specific DNA sequences.
Subject(s)
Bacteriophage T7/enzymology , DNA Primase/chemistry , DNA Primase/metabolism , DNA, Viral/metabolism , Zinc/metabolism , Amino Acid Substitution/drug effects , Amino Acid Substitution/genetics , Bacteriophage T7/drug effects , Biocatalysis/drug effects , Chlorides/pharmacology , Crystallography, X-Ray , Cysteine/metabolism , Enzyme Stability/drug effects , Histidine/metabolism , Ligands , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Oligoribonucleotides/biosynthesis , Protein Structure, Secondary , Protein Structure, Tertiary , Structure-Activity Relationship , Templates, Genetic , X-Ray Absorption Spectroscopy , Zinc Compounds/pharmacologyABSTRACT
The NSLS X28C white-light beamline has been upgraded with a focusing mirror in order to provide increased x-ray density and a wide selection of beam shapes at the sample position. The cylindrical single crystal silicon mirror uses an Indalloy 51 liquid support bath as both a mechanism for heat transfer and a buoyant support to counter the effects of gravity and correct for minor parabolic slope errors. Calorimetric measurements were performed to verify that the calculated more than 200-fold increase in flux density is delivered by the mirror at the smallest beam spot. The properties of the focused beam relevant to radiolytic footprinting, namely, the physical dimensions of the beam, the effective hydroxyl radical dose delivered to the sample, and sample heating upon irradiation, have been studied at several mirror angles.
