Mechanism and Stoichiometry of Interaction of DnaG Primase with DnaB Helicase of Escherichia coli in RNA Primer Synthesis

Mitkova, A.; Khopde, Sujata M.; Biswas, Subhasis B.

doi:10.1074/jbc.m308956200

Cited by 66 publications

(82 citation statements)

References 60 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The data suggested that the single DnaB replication fork helicase loaded on the lagging strand could recruit DnaG primase molecules to reprime not only on the lagging strand but also on the leading strand ( Figure 2B). The recent finding that multiple primase monomers can bind a DnaB hexamer [12] supports the idea that a single helicase can mediate the recruitment of more than one DnaG molecule, thus possibly mediating both lagging and leading strand priming events.…”

mentioning

confidence: 56%

Gaps and forks in DNA replication: Rediscovering old models

2006

View full text Add to dashboard Cite

show abstract

mentioning

confidence: 56%

Gaps and forks in DNA replication: Rediscovering old models

2006

View full text Add to dashboard Cite

show abstract

“…Protein titration led to a concentration-dependent quenching of F-ID1, where the total fluorescence of the system is equal to the denominator of Equation 2. Thus, the raw anisotropy data were corrected for quenching using Equation 3, as described previously (63).…”

Section: Determination Of Fementioning

confidence: 99%

High Affinity Heme Binding to a Heme Regulatory Motif on the Nuclear Receptor Rev-erbβ Leads to Its Degradation and Indirectly Regulates Its Interaction with Nuclear Receptor Corepressor

Carter

Gupta

Ragsdale

2016

Journal of Biological Chemistry

View full text Add to dashboard Cite

Rev-erb␣ and Rev-erb␤ are heme-binding nuclear receptors (NR) that repress the transcription of genes involved in regulating metabolism, inflammation, and the circadian clock. Previous gene expression and co-immunoprecipitation studies led to a model in which heme binding to Rev-erb␣ recruits nuclear receptor corepressor 1 (NCoR1) into an active repressor complex. However, in contradiction, biochemical and crystallographic studies have shown that heme decreases the affinity of the ligand-binding domain of Rev-erb NRs for NCoR1 peptides. One explanation for this discrepancy is that the ligand-binding domain and NCoR1 peptides used for in vitro studies cannot replicate the key features of the full-length proteins used in cellular studies. However, the combined in vitro and cellular results described here demonstrate that heme does not directly promote interactions between full-length Rev-erb␤ (FLRev-erb␤) and an NCoR1 construct encompassing all three NR interaction domains. NCoR1 tightly binds both apo-and heme-replete FLRev-erb␤⅐DNA complexes; furthermore, heme, at high concentrations, destabilizes the FLRev-erb␤⅐NCoR1 complex. The interaction between FLRev-erb␤ and NCoR1 as well as Reverb␤ repression at the Bmal1 promoter appear to be modulated by another cellular factor(s), at least one of which is related to the ubiquitin-proteasome pathway. Our studies suggest that heme is involved in regulating the degradation of Rev-erb␤ in a manner consistent with its role in circadian rhythm maintenance. Finally, the very slow rate constant (10 ؊6 s ؊1 ) of heme dissociation from Rev-erb␤ rules out a prior proposal that Reverb␤ acts as an intracellular heme sensor.Nuclear receptors (NRs) 2 are eukaryotic transcription factors that activate or repress gene expression in response to binding small signaling molecules such as steroid hormones, lipophilic vitamins, and fatty acids (1). Genes regulated by NRs are involved in a myriad of cellular processes ranging from metabolic homeostasis to growth and development. The subjects of this article, Rev-erb␣ and Rev-erb␤, are NRs that have overlapping functions and tissue expression patterns (2-9) and are under circadian control (10 -12). Rev-erb NRs repress the transcription of key genes, e.g. Bmal1, CLOCK, and Rev-erb␣ itself, involved in regulating the molecular clock (13)(14)(15). This is accomplished in part through the formation of a heterodimeric Bmal1-CLOCK complex that activates the transcription of clock-controlled genes including Rev-erb␣/␤, completing a critical feedback loop required for circadian rhythm maintenance (6, 7). Rev-erb␣ and Rev-erb␤ also regulate the expression of genes involved in gluconeogenesis, lipid homeostasis, and immune responses, ultimately synchronizing these processes to the diurnal cycle (16 -21).The multiple functions of NRs (DNA, ligand, and coregulator binding) are accomplished through their modular structure (22, 23). The N-terminal A/B domain is hypervariable, is involved in ligand-independent transcriptional regulation, and is subject to ...

show abstract

“…In vitro experiments with the E. coli DnaB and DnaG proteins showed that DnaB modulates the activity of DnaG in several ways. One role of DnaB is to transport the bound DnaG to its recognition sites (5,7,12). In addition, DnaB also modulates primer synthesis in a manner independent of its translocation ability.…”

mentioning

confidence: 99%

In the Bacillus stearothermophilus DnaB-DnaG Complex, the Activities of the Two Proteins Are Modulated by Distinct but Overlapping Networks of Residues

Thirlway

Soultanas

2006

J Bacteriol

View full text Add to dashboard Cite

We demonstrate the primase activity of Bacillus stearothermophilus DnaG and show that it initiates at 3-ATC-5 and 3-ATT-5 sites synthesizing primers that are 22 or 23 nucleotides long. In the presence of the helicase DnaB the size distribution of primers is different, and a range of additional smaller primers are also synthesized. Nine residues from the N-and C-terminal domains of DnaB, as well as its linker region, have been reported previously to affect this interaction. In Bacillus stearothermophilus only three residues from the linker region (I119 and I125) and the N-terminal domain (Y88) of DnaB have been shown previously to have direct structural importance, and I119 and I125 mediate DnaG-induced effects on DnaB activity. The functions of the other residues (L138, T191, E192, R195, and M196) are still a mystery. Here we show that the E15A, Y88A, and E15A Y88A mutants bind DnaG but are not able to modulate primer size, whereas the R195A M196A mutant inhibited the primase activity. Therefore, four of these residues, E15 and Y88 (N-terminal domain) and R195 and M196 (C-terminal domain), mediate DnaB-induced effects on DnaG activity. Overall, the data suggest that the effects of DnaB on DnaG activity and vice versa are mediated by distinct but overlapping networks of residues.

show abstract

Mechanism and Stoichiometry of Interaction of DnaG Primase with DnaB Helicase of Escherichia coli in RNA Primer Synthesis

Cited by 66 publications

References 60 publications

Gaps and forks in DNA replication: Rediscovering old models

Gaps and forks in DNA replication: Rediscovering old models

High Affinity Heme Binding to a Heme Regulatory Motif on the Nuclear Receptor Rev-erbβ Leads to Its Degradation and Indirectly Regulates Its Interaction with Nuclear Receptor Corepressor

In the Bacillus stearothermophilus DnaB-DnaG Complex, the Activities of the Two Proteins Are Modulated by Distinct but Overlapping Networks of Residues

Contact Info

Product

Resources

About