Limits...
Structural insights into the cooperative binding of SeqA to a tandem GATC repeat.

Chung YS, Brendler T, Austin S, Guarné A - Nucleic Acids Res. (2009)

Bottom Line: The structure delineates how SeqA forms a high-affinity complex with DNA and it suggests why SeqA only recognizes GATC sites at certain spacings.The SeqA-DNA complex also unveils additional protein-protein interaction surfaces that mediate the formation of higher ordered complexes upon binding to newly replicated DNA.Based on this data, we propose a model describing how SeqA interacts with newly replicated DNA within the origin of replication and at the replication forks.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biomedical Sciences, Health Sciences Center, McMaster University, 1200 Main Street West, Hamilton, ON L8N 3Z5, Canada.

ABSTRACT
SeqA is a negative regulator of DNA replication in Escherichia coli and related bacteria that functions by sequestering the origin of replication and facilitating its resetting after every initiation event. Inactivation of the seqA gene leads to unsynchronized rounds of replication, abnormal localization of nucleoids and increased negative superhelicity. Excess SeqA also disrupts replication synchrony and affects cell division. SeqA exerts its functions by binding clusters of transiently hemimethylated GATC sequences generated during replication. However, the molecular mechanisms that trigger formation and disassembly of such complex are unclear. We present here the crystal structure of a dimeric mutant of SeqA [SeqADelta(41-59)-A25R] bound to tandem hemimethylated GATC sites. The structure delineates how SeqA forms a high-affinity complex with DNA and it suggests why SeqA only recognizes GATC sites at certain spacings. The SeqA-DNA complex also unveils additional protein-protein interaction surfaces that mediate the formation of higher ordered complexes upon binding to newly replicated DNA. Based on this data, we propose a model describing how SeqA interacts with newly replicated DNA within the origin of replication and at the replication forks.

Show MeSH

Related in: MedlinePlus

Recognition of hemimethylated DNA by SeqAΔ(41–59)-A25R. (a) Diagram of the protein–DNA interactions. The purple and green shadow boxes indicate the SeqAΔ(41–59)-A25R protomer that interacts with each GATC site with hydrogen bonds shown in blue and van der Waals interactions in red. The methylated adenines are labeled in red and the disordered Cyt2 is shadowed with a grey box. (b) Detail of the interaction between SeqAΔ(41–59)-A25R and the methylated and unmetylated A–T base pairs. The refined model is shown as sticks with protomer A shown in purple (left panel), protomer B in green (right panel), the methylated DNA strand in orange and the unmethylated DNA strand in yellow with the 2Fo−Fc electron density maps contoured at 1 σ. Hydrogen bonds are shown as black dashed lines with distances labeled. (c) Electrophoretic mobility shift assays of the oligonucleotide used for crystallization (80 nM) when incubated with increasing quantities of SeqAΔ(41–59)-A25R (nM). (d) Detail of helix αB in the two protomers of the dimer. Red arrows indicate the path of the main chain on each protomer. Panels (b and d) were prepared using PyMol (42).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2691817&req=5

Figure 3: Recognition of hemimethylated DNA by SeqAΔ(41–59)-A25R. (a) Diagram of the protein–DNA interactions. The purple and green shadow boxes indicate the SeqAΔ(41–59)-A25R protomer that interacts with each GATC site with hydrogen bonds shown in blue and van der Waals interactions in red. The methylated adenines are labeled in red and the disordered Cyt2 is shadowed with a grey box. (b) Detail of the interaction between SeqAΔ(41–59)-A25R and the methylated and unmetylated A–T base pairs. The refined model is shown as sticks with protomer A shown in purple (left panel), protomer B in green (right panel), the methylated DNA strand in orange and the unmethylated DNA strand in yellow with the 2Fo−Fc electron density maps contoured at 1 σ. Hydrogen bonds are shown as black dashed lines with distances labeled. (c) Electrophoretic mobility shift assays of the oligonucleotide used for crystallization (80 nM) when incubated with increasing quantities of SeqAΔ(41–59)-A25R (nM). (d) Detail of helix αB in the two protomers of the dimer. Red arrows indicate the path of the main chain on each protomer. Panels (b and d) were prepared using PyMol (42).

Mentions: In protomer A, the linker is completely ordered and mainly helical, with helix αB encompassing residues Arg25 to Ser39 (Figure 2A–C). However, the electron density of this linker in protomer B was very weak. Consequently, residues Ser36 to Gln40 were not included in the final model (Figure 2B). Lys34 is the pivotal point that re-orients the C-terminal domains towards the target DNA. While in protomer A Lys34 is part of helix αB, Lys34 exchanges the orientations of its main and side chains in protomer B, breaking the 2-fold symmetry (Figures 2C and 3D). Note that the two protomers of the dimer cannot adopt the conformation seen in protomer A concurrently as this would cause steric hindrance at residues Ala37-Gln40 (Figure 2C). Therefore, Lys34 may be an intrinsically flexible point in the SeqA protein even in the absence of DNA.Figure 3.


Structural insights into the cooperative binding of SeqA to a tandem GATC repeat.

Chung YS, Brendler T, Austin S, Guarné A - Nucleic Acids Res. (2009)

Recognition of hemimethylated DNA by SeqAΔ(41–59)-A25R. (a) Diagram of the protein–DNA interactions. The purple and green shadow boxes indicate the SeqAΔ(41–59)-A25R protomer that interacts with each GATC site with hydrogen bonds shown in blue and van der Waals interactions in red. The methylated adenines are labeled in red and the disordered Cyt2 is shadowed with a grey box. (b) Detail of the interaction between SeqAΔ(41–59)-A25R and the methylated and unmetylated A–T base pairs. The refined model is shown as sticks with protomer A shown in purple (left panel), protomer B in green (right panel), the methylated DNA strand in orange and the unmethylated DNA strand in yellow with the 2Fo−Fc electron density maps contoured at 1 σ. Hydrogen bonds are shown as black dashed lines with distances labeled. (c) Electrophoretic mobility shift assays of the oligonucleotide used for crystallization (80 nM) when incubated with increasing quantities of SeqAΔ(41–59)-A25R (nM). (d) Detail of helix αB in the two protomers of the dimer. Red arrows indicate the path of the main chain on each protomer. Panels (b and d) were prepared using PyMol (42).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2691817&req=5

Figure 3: Recognition of hemimethylated DNA by SeqAΔ(41–59)-A25R. (a) Diagram of the protein–DNA interactions. The purple and green shadow boxes indicate the SeqAΔ(41–59)-A25R protomer that interacts with each GATC site with hydrogen bonds shown in blue and van der Waals interactions in red. The methylated adenines are labeled in red and the disordered Cyt2 is shadowed with a grey box. (b) Detail of the interaction between SeqAΔ(41–59)-A25R and the methylated and unmetylated A–T base pairs. The refined model is shown as sticks with protomer A shown in purple (left panel), protomer B in green (right panel), the methylated DNA strand in orange and the unmethylated DNA strand in yellow with the 2Fo−Fc electron density maps contoured at 1 σ. Hydrogen bonds are shown as black dashed lines with distances labeled. (c) Electrophoretic mobility shift assays of the oligonucleotide used for crystallization (80 nM) when incubated with increasing quantities of SeqAΔ(41–59)-A25R (nM). (d) Detail of helix αB in the two protomers of the dimer. Red arrows indicate the path of the main chain on each protomer. Panels (b and d) were prepared using PyMol (42).
Mentions: In protomer A, the linker is completely ordered and mainly helical, with helix αB encompassing residues Arg25 to Ser39 (Figure 2A–C). However, the electron density of this linker in protomer B was very weak. Consequently, residues Ser36 to Gln40 were not included in the final model (Figure 2B). Lys34 is the pivotal point that re-orients the C-terminal domains towards the target DNA. While in protomer A Lys34 is part of helix αB, Lys34 exchanges the orientations of its main and side chains in protomer B, breaking the 2-fold symmetry (Figures 2C and 3D). Note that the two protomers of the dimer cannot adopt the conformation seen in protomer A concurrently as this would cause steric hindrance at residues Ala37-Gln40 (Figure 2C). Therefore, Lys34 may be an intrinsically flexible point in the SeqA protein even in the absence of DNA.Figure 3.

Bottom Line: The structure delineates how SeqA forms a high-affinity complex with DNA and it suggests why SeqA only recognizes GATC sites at certain spacings.The SeqA-DNA complex also unveils additional protein-protein interaction surfaces that mediate the formation of higher ordered complexes upon binding to newly replicated DNA.Based on this data, we propose a model describing how SeqA interacts with newly replicated DNA within the origin of replication and at the replication forks.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biomedical Sciences, Health Sciences Center, McMaster University, 1200 Main Street West, Hamilton, ON L8N 3Z5, Canada.

ABSTRACT
SeqA is a negative regulator of DNA replication in Escherichia coli and related bacteria that functions by sequestering the origin of replication and facilitating its resetting after every initiation event. Inactivation of the seqA gene leads to unsynchronized rounds of replication, abnormal localization of nucleoids and increased negative superhelicity. Excess SeqA also disrupts replication synchrony and affects cell division. SeqA exerts its functions by binding clusters of transiently hemimethylated GATC sequences generated during replication. However, the molecular mechanisms that trigger formation and disassembly of such complex are unclear. We present here the crystal structure of a dimeric mutant of SeqA [SeqADelta(41-59)-A25R] bound to tandem hemimethylated GATC sites. The structure delineates how SeqA forms a high-affinity complex with DNA and it suggests why SeqA only recognizes GATC sites at certain spacings. The SeqA-DNA complex also unveils additional protein-protein interaction surfaces that mediate the formation of higher ordered complexes upon binding to newly replicated DNA. Based on this data, we propose a model describing how SeqA interacts with newly replicated DNA within the origin of replication and at the replication forks.

Show MeSH
Related in: MedlinePlus