scholarly journals Interaction of the Initiator Protein of an IncB Plasmid with Its Origin of DNA Replication

2003 ◽  
Vol 185 (7) ◽  
pp. 2210-2218 ◽  
Author(s):  
T. Betteridge ◽  
J. Yang ◽  
A. J. Pittard ◽  
J. Praszkier
Keyword(s):  
Binding Site ◽  
Sequence Motif ◽  
Initiator Protein ◽  
Origin Of Dna Replication ◽  
Replication Initiator Protein ◽  
Replication Initiator ◽  
Dnaa Binding Site ◽  
Dnaa Box

ABSTRACT The replication initiator protein RepA of the IncB plasmid pMU720 was purified and used in DNase I protection assays in vitro. RepA protected a 68-bp region of the origin of replication of pMU720. This region, which lies immediately downstream of the DnaA box, contains four copies of the sequence motif 5′AANCNGCAA3′. Mutational analyses identified this sequence as the binding site specifically recognized by RepA (the RepA box). Binding of RepA to the RepA boxes was ordered and sequential, with the box closest to the DnaA binding site (box 1) occupied first and the most distant boxes (boxes 3 and 4) occupied last. However, only boxes 1, 2, and 4 were essential for origin activity, with box 3 playing a lesser role. Changing the spacing between box 1 and the other three boxes affected binding of RepA in vitro and origin activity in vivo, indicating that the RepA molecules bound to ori B interact with one another.

scholarly journals Role of RepA and DnaA Proteins in the Opening of the Origin of DNA Replication of an IncB Plasmid

2004 ◽  
Vol 186 (12) ◽  
pp. 3785-3793 ◽  
Author(s):  
T. Betteridge ◽  
J. Yang ◽  
A. J. Pittard ◽  
J. Praszkier
Keyword(s):  
Escherichia Coli ◽  
Binding Sites ◽  
Origin Of Replication ◽  
Initiator Protein ◽  
Origin Of Dna Replication ◽  
Replication Initiator Protein ◽  
Replication Initiator

ABSTRACT The replication initiator protein RepA of the IncB plasmid pMU720 was shown to induce localized unwinding of its cognate origin of replication in vitro. DnaA, the initiator protein of Escherichia coli, was unable to induce localized unwinding of this origin of replication on its own but enhanced the opening generated by RepA. The opened region lies immediately downstream of the last of the three binding sites for RepA (RepA boxes) and covers one turn of DNA helix. A 6-mer sequence, 5′-TCTTAA-3′, which lies within the opened region, was essential for the localized unwinding of the origin in vitro and origin activity in vivo. In addition, efficient unwinding of the origin of replication of pMU720 in vitro required the native positioning of the binding sites for the initiator proteins. Interestingly, binding of RepA to RepA box 1, which is essential for origin activity, was not required for the localized opening of the origin in vitro.

scholarly journals Effect of CIS on Activity intrans of the Replication Initiator Protein of an IncB Plasmid

2000 ◽  
Vol 182 (14) ◽  
pp. 3972-3980 ◽  
Author(s):  
J. Praszkier ◽  
S. Murthy ◽  
A. J. Pittard
Keyword(s):  
Amino Acids ◽  
Multicopy Plasmid ◽  
Origin Of Replication ◽  
Coding Region ◽  
Initiator Protein ◽  
Initiation Of Replication ◽  
Replication Initiator Protein ◽  
Position And Orientation ◽  
Replication Initiator

ABSTRACT RepA, the replication initiator protein of the IncB plasmid pMU720, acts preferentially in cis. The cis activity of RepA is thought to be mediated by CIS, a 166-bp region of DNA separating the coding region of repA from the origin of replication (ori) of pMU720. To investigate thetrans activity of RepA, the repA gene, without its cognate ori, was cloned on a multicopy plasmid, pSU39. The ori on which RepA acts was cloned on pAM34, a plasmid whose replicon is inactive without induction by isopropyl-β-d-thiogalactopyranoside (IPTG). Thus, in the absence of IPTG, replication of the pAM34 derivatives was dependent on activation of the cloned ori by RepA produced intrans from the pSU39 derivatives. The effect ofCIS, when present either on the RepA-producing or theori plasmid or both, on the efficiency of replication of the ori plasmid in vivo, was determined. The presence ofCIS, in its native position and orientation, on the RepA-producing plasmid reduced the efficiency of replication of theori plasmid. This inhibitory activity of CISwas sequence specific and involved interaction with the C-terminal 20 to 37 amino acids of RepA. By contrast, CIS had no effect when present on the ori plasmid. Initiation of replication from the ori in trans was independent of transcription into CIS.

scholarly journals Putative Cooperative ATP–DnaA Binding to Double-Stranded DnaA Box and Single-Stranded DnaA-Trio Motif upon Helicobacter pylori Replication Initiation Complex Assembly

2021 ◽  
Vol 22 (12) ◽  
pp. 6643
Author(s):  
Pawel Jaworski ◽  
Dorota Zyla-Uklejewicz ◽  
Malgorzata Nowaczyk-Cieszewska ◽  
Rafal Donczew ◽  
Thorsten Mielke ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Replication Initiation ◽  
Dna Unwinding ◽  
Rich Region ◽  
Initiator Protein ◽  
New Class ◽  
H Pylori ◽  
General Architecture ◽  
Dnaa Box

oriC is a region of the bacterial chromosome at which the initiator protein DnaA interacts with specific sequences, leading to DNA unwinding and the initiation of chromosome replication. The general architecture of oriCs is universal; however, the structure of oriC and the mode of orisome assembly differ in distantly related bacteria. In this work, we characterized oriC of Helicobacter pylori, which consists of two DnaA box clusters and a DNA unwinding element (DUE); the latter can be subdivided into a GC-rich region, a DnaA-trio and an AT-rich region. We show that the DnaA-trio submodule is crucial for DNA unwinding, possibly because it enables proper DnaA oligomerization on ssDNA. However, we also observed the reverse effect: DNA unwinding, enabling subsequent DnaA–ssDNA oligomer formation—stabilized DnaA binding to box ts1. This suggests the interplay between DnaA binding to ssDNA and dsDNA upon DNA unwinding. Further investigation of the ts1 DnaA box revealed that this box, together with the newly identified c-ATP DnaA box in oriC1, constitute a new class of ATP–DnaA boxes. Indeed, in vitro ATP–DnaA unwinds H. pylori oriC more efficiently than ADP–DnaA. Our results expand the understanding of H. pylori orisome formation, indicating another regulatory pathway of H. pylori orisome assembly.

scholarly journals Transcription of alpha-specific genes in Saccharomyces cerevisiae: DNA sequence requirements for activity of the coregulator alpha 1.

1993 ◽  
Vol 13 (11) ◽  
pp. 6866-6875 ◽  
Author(s):  
D C Hagen ◽  
L Bruhn ◽  
C A Westby ◽  
G F Sprague
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Dna Sequences ◽  
Point Mutations ◽  
Transcription Activation ◽  
Specific Gene ◽  
Binding Assays ◽  
Weak Binding

Transcription activation of alpha-specific genes in Saccharomyces cerevisiae is regulated by two proteins, MCM1 and alpha 1, which bind to DNA sequences, called P'Q elements, found upstream of alpha-specific genes. Neither MCM1 nor alpha 1 alone binds efficiently to P'Q elements. Together, however, they bind cooperatively in a manner that requires both the P' sequence, which is a weak binding site for MCM1, and the Q sequence, which has been postulated to be the binding site for alpha 1. We analyzed a collection of point mutations in the P'Q element of the STE3 gene to determine the importance of individual base pairs for alpha-specific gene transcription. Within the 10-bp conserved Q sequence, mutations at only three positions strongly affected transcription activation in vivo. These same mutations did not affect the weak binding to P'Q displayed by MCM1 alone. In vitro DNA binding assays showed a direct correlation between the ability of the mutant sequences to form ternary P'Q-MCM1-alpha 1 complexes and the degree to which transcription was activated in vivo. Thus, the ability of alpha 1 and MCM1 to bind cooperatively to P'Q elements is critical for activation of alpha-specific genes. In all natural alpha-specific genes the Q sequence is adjacent to the degenerate side of P'. To test the significance of this geometry, we created several novel juxtapositions of P, P', and Q sequences. When the Q sequence was opposite the degenerate side, the composite QP' element was inactive as a promoter element in vivo and unable to form stable ternary QP'-MCM1-alpha 1 complexes in vitro. We also found that addition of a Q sequence to a strong MCM1 binding site allows the addition of alpha 1 to the complex. This finding, together with the observation that Q-element point mutations affected ternary complex formation but not the weak binding of MCM1 alone, supports the idea that the Q sequence serves as a binding site for alpha 1.

scholarly journals Role of Papillomavirus E1 Initiator Dimerization in DNA Replication

2005 ◽  
Vol 79 (13) ◽  
pp. 8661-8664 ◽  
Author(s):  
Stephen Schuck ◽  
Arne Stenlund
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Severe Effect ◽  
Origin Of Dna Replication ◽  
Initiation Of Dna Replication

ABSTRACT Viral initiator proteins are polypeptides that form oligomeric complexes on the origin of DNA replication (ori). These complexes carry out a multitude of functions related to initiation of DNA replication, and although many of these functions have been characterized biochemically, little is understood about how the complexes are assembled. Here we demonstrate that loss of one particular interaction, the dimerization between E1 DNA binding domains, has a severe effect on DNA replication in vivo but has surprisingly modest effects on most individual biochemical activities in vitro. We conclude that the dimer interaction is primarily required for initial recognition of ori.

Sign in / Sign up

Export Citation Format

Share Document