Magnesium Acetate Induces a Conformational Change in Escherichia coli Primase

Iterative profile searches and structural modeling show that bacterial DnaG-type primases, small primase-like proteins from bacteria and archaea, type IA and type II topoisomerases, bacterial and archaeal nucleases of the OLD family and bacterial DNA repair proteins of the RecR/M family contain a common domain, designated Toprim (topoisomerase-primase) domain. The domain consists of ∼100 amino acids and has two conserved motifs, one of which centers at a conserved glutamate and the other one at two conserved aspartates (DxD). Examination of the structure of Topo IA and Topo II and modeling of the Toprim domains of the primases reveal a compact β/α fold, with the conserved negatively charged residues juxtaposed, and inserts seen in Topo IA and Topo II. The conserved glutamate may act as a general base in nucleotide polymerization by primases and in strand rejoining by topoisomerases and as a general acid in strand cleavage by topoisomerases and nucleases. The role of this glutamate in catalysis is supported by sitedirected mutagenesis data on primases and Topo IA. The DxD motif may coordinate Mg 2+ that is required for the activity of all Toprim-containing enzymes. The common ancestor of all life forms could encode a prototype Toprim enzyme that might have had both nucleotidyl transferase and polynucleotide cleaving activity.

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“…This is consistent with the detection of conformational changes in the primases in the presence of Mg 2+ (50).…”

Section: Structure Of the Toprim Domain And Its Function In Catalysissupporting

confidence: 91%

Toprim--a conserved catalytic domain in type IA and II topoisomerases, DnaG-type primases, OLD family nucleases and RecR proteins

Aravind

Leipe

Koonin

1998

Nucleic Acids Research

358

327

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“…The observed NTP-binding site in Sa DnaG is formed by multiple highly-conserved residues, many of which have been demonstrated previously to be critical for catalysis (Fig S1/S2a, Table 2). For example, the invariant acidic amino acids that ligand the metal ions involved in nucleotide coordination have been shown to be critical for metal binding and priming activity in both E. coli DnaG and in the related phage protein, T7 gp4 (Godson et al, 2000,; Lee and Richardson, 2005; Rodina and Godson, 2006; Urlacher and Griep, 1995). The positively-charged amino acids that emerge from the basic ridge to contact the nucleotidyl triphosphate moiety likewise have been examined, and found to be important for primase function (Keck et al, 2000; Rodina and Godson, 2006).…”

Section: Resultsmentioning

confidence: 99%

Binding Mechanism of Metal⋅NTP Substrates and Stringent-Response Alarmones to Bacterial DnaG-Type Primases

Rymer

Solorio²,

Tehranchi

et al. 2012

Structure

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“…The Mg 2ϩ -dependent specificity of primase binding may reflect the requirement of Mg 2ϩ for forming the specific conformation of primase when interacting with DNA template. It has been reported that Mg(OAc) 2 could induce a conformational change in primase that resulted in an altered pattern of protease cleavage (23). For E. coli RNA polymerase, which has similar properties as E. coli primase, it has also been found that Mg 2ϩ plays roles in transience of formation of RNA polymerase and P R promoter complexes (20), and the location of the Mg 2ϩ binding site was reported recently (28).…”

Section: Vol 178 1996 Interaction Of Primase With G4ori C -Ssb Compmentioning

confidence: 93%

Interaction of Escherichia coli primase with a phage G4ori(c)-E. coli SSB complex

Sun

Godson

1996

J Bacteriol

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We earlier reported that Escherichia coli single-stranded DNA-binding protein (SSB) bound in a fixed position to the stem-loop structure of the origin of complementary DNA strand synthesis in phage G4 (G4ori c ), leaving stem-loop I and the adjacent 5 CTG 3, the primer RNA initiation site, as an SSB-free region (W. Sun and G. N. Godson Primase is required to initiate DNA replication in prokaryotes and eucaryotes. It functions to synthesize short primer RNAs (pRNA) on the separated strands of the replication fork, which provide primed templates with free 3Ј-OH terminus for DNA polymerase to elongate and synthesize the complementary DNA strand (4,13,14,25). Several prokaryotic priming systems have been established to study Escherichia coli primase (referred to as primase in this paper) function in vitro, such as cloned E. coli chromosomal origin of DNA replication (oriC) (9, 24), the origin of complementary DNA strand synthesis (ori c ) of single-stranded (ss) E. coli phages (e.g., X174[1]; G4 [3,25]; and st-1, ␣3, and K [2, 17]), double-stranded phage (e.g., [26]), and E. coli plasmid origins of DNA replication (e.g., R1 [11]). The simplest system is ss phage ori c exemplified by phage G4. In this system, primase in association with E. coli ss DNA-binding protein (SSB) synthesizes 29 nucleotides (nt) of pRNA from a unique 5Ј CTG 3Ј sequence in a region of the viral DNA strand called G4ori c (10). This is in contrast to other priming systems in which primase acts in concert with several other initiation proteins and synthesizes pRNA at many sites on the template DNA, although almost always initiating at 5Ј CTG 3Ј sequence (27). The G4ori c has been extensively studied as a model origin of DNA replication. Deletion studies have shown that the G4ori c region is a 140-nt, noncoding sequence containing potential secondary structure: stem-loops I, II, and III (5). Primase synthesizes pRNA at a 5Ј CTG 3Ј site, close to the 3Ј side of stem-loop I (7,8,18). Initiation of DNA replication in G4-related ss phages ␣3, st-1, and K (17) is similar to that of G4ori c .Several early studies provided information on the interaction of primase with the ori c DNA and SSB. A nuclease footprinting study on the interaction of primase with SSB-coated Kori c demonstrated that regions of the stem-loop structure were involved in primase binding (16). Then a stoichiometric study using gel filtration reported that two primase molecules bound on one G4ori c that was cloned into phage M13 ss-DNA (19). In a more recent study (21) to dissect the interactions of SSB and primase with G4ori c , we have demonstrated that two SSB tetramers bound on G4ori c in a fixed position. This resulted in the formation of a unique structure with two SSB tetramers (i.e., an SSB octamer) bound to the stem-loop region and other SSB tetramers bound on the 5Ј and 3Ј sides, leaving approximately 30 nt of free uncoated ss-DNA between the central SSB octamer and the adjacent SSB tetramers (or octamers). The 5Ј CTG 3Ј pRNA initiation site was located in the SSB-free linke...

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Magnesium Acetate Induces a Conformational Change in Escherichia coli Primase

Cited by 12 publications

References 31 publications

Toprim--a conserved catalytic domain in type IA and II topoisomerases, DnaG-type primases, OLD family nucleases and RecR proteins

Toprim--a conserved catalytic domain in type IA and II topoisomerases, DnaG-type primases, OLD family nucleases and RecR proteins

Binding Mechanism of Metal⋅NTP Substrates and Stringent-Response Alarmones to Bacterial DnaG-Type Primases

Interaction of Escherichia coli primase with a phage G4ori(c)-E. coli SSB complex

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