Search PubMed⌕ Search

Biomedical subjects

Michelle M Spiering

Publications and source records attributed to Michelle M Spiering.

5 recordsLinked to original sources
PubMed2005

Interaction between the T4 helicase-loading protein (gp59) and the DNA polymerase (gp43): a locking mechanism to delay replication during replisome assembly.

The T4 helicase-loading protein (gp59) has been proposed to coordinate leading- and lagging-strand DNA synthesis by blocking leading-strand synthesis during the primosome assembly. In this work, we unambiguously demonstrate through a series of biochemical and biophysical experiments, including single-molecule fluorescence microscopy, that the inhibition of leading-strand holoenzyme progression by gp59 is the result of a complex formed between gp59 and leading-strand polymerase (gp43) on DNA that is instrumental in preventing premature replication during the assembly of the T4 replisome. We find that both the polymerization and 3' --> 5' exonuclease activities of gp43 are totally inhibited within this complex. Chemical cross-linking of the complex followed by tryptic digestion and peptide identification through matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry identified Cys169 of gp43 and Cys215 of gp59 as residues in a region of a protein-protein contact. With the available crystal structures for both gp43 and gp59, a model of the complex was constructed based on shape complementarity, revealing that parts of the C-terminal domain from gp59 insert into the interface created by the thumb and exonuclease domains of gp43. This insertion effectively locks the polymerase into a conformation where switching between the pol and editing modes is prevented. Thus, continued assembly of the replisome through addition of the primosome components and elements of the lagging-strand holoenzyme can occur without leading-strand DNA replication.

Bacteriophage T4↗
PubMed2005

Assembly of the bacteriophage T4 primosome: single-molecule and ensemble studies.

Within replisomes for DNA replication, the primosome is responsible for unwinding double-stranded DNA and synthesizing RNA primers. Assembly of the bacteriophage T4 primosome on individual molecules of ssDNA or forked DNA (fDNA) has been studied by using FRET microscopy. On either DNA substrate, an ordered process of assembly begins with tight 1:1 binding of ssDNA-binding protein (gp32) and helicase-loading protein (gp59) to the DNA. Magnesium adenosine 5'-O-(3-thiotriphosphate) (MgATPgammaS) mediates the weak binding of helicase (gp41) to DNA coated with gp32 and gp59, whereas MgATP induces gp32 and gp59 to dissociate, leaving gp41 bound to the DNA. Finally, primase (gp61) binds to the gp41.DNA complex. Ensemble studies were used to determine protein stoichiometries and binding constants. These single-molecule studies provide an unambiguous description of the pathway for assembly of the primosome on the lagging strand of DNA at a replication fork.

Adenosine Triphosphate↗
PubMed2003

Metal stoichiometry and functional studies of the diphtheria toxin repressor.

Diphtheria toxin repressor (DtxR) is a transition metal ion-activated repressor in Corynebacterium diphtheriae. DtxR is an iron sensor; metal-bound DtxR represses transcription of genes downstream of the tox operator. Wild-type DtxR [DtxR(wt)] and several mutant forms were overexpressed and purified from Escherichia coli. DtxR was isolated without bound metal. Metal reconstitution gave a binding stoichiometry of 2 per monomer for DtxR(wt) and 1 per monomer for DtxR(H79A) and DtxR(M10A). DNA binding of DtxR(H79A) and DtxR(M10A) indicates that metal site 2 is essential for activity. Metal binding lowers the dimerization K(d) of DtxR from low micromolar to 33 nM. Gel electrophoretic mobility-shift assays show that Fe(2+) and not Fe(3+) activates DtxR for DNA binding. This finding suggests that gene regulation by DtxR may be sensitive not only to iron levels but also to redox state of the iron. Mutations in the tox operator sequence indicate that DtxR dimers binding to DNA may be highly cooperative.

Bacterial Proteins↗
PubMed2003

Trace elements and nitric oxide function.

Nitric oxide (NO) has emerged over the last 15 y as a mammalian metabolic intermediate that is involved in the regulation of critical physiological functions such as blood vessel homeostasis, neuronal transmission and host response to infection. NO is synthesized by the enzyme nitric oxide synthase, which converts the amino acid L-arginine to citrulline and NO. NO functions in biological systems in two very important ways. First, it has been found to be a messenger by which cells communicate with one another (signal transduction), and second, it plays a critical role in the host response to infection. In this second function, it appears that the toxic properties of NO have been harnessed by the immune system to kill or at least slow the growth of invading organisms. The nonspecific chemical reactivity with key cellular targets is responsible for this action. In signaling, NO directly activates the enzyme soluble guanylate cyclase (sGC). Once activated, sGC converts GTP to cGMP and pyrophosphate. The cGMP formed is responsible for the well-documented actions of NO such as blood vessel dilation. With the initial discovery of NO signaling, several important questions emerged that centered largely on the issue of how a signaling system functions when the signaling agent is chemically reactive (short lived), highly diffusible and toxic. Critical, especially in signaling, are the control of NO biosynthesis and interaction with the biological receptors at a concentration that will not harm the host. Why did Nature choose NO for the roles it has? That question engenders only speculation. How does NO work (i.e., what does NO do, and how does it do it without harm yet with specificity)? Answers to these questions can now be offered as the molecular level details emerge to form an interesting picture.

Animals↗