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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.

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Structure of SeqAΔ(41–59)-A25R bound to a hemimethylated GATC repeat. (a) Ribbon diagram of the SeqAΔ(41–59)-A25R monomer with helices in purple and strands in pink. The junction between residues Gln40 and Lys60 is indicated with a black arrowhead. (b) SeqAΔ(41–59)-A25R dimer bound to DNA. Protomer A (encompassing residues 1–40/60–181) is shown in purple and protomer B (encompassing residues 1–35/60–181) is shown in green. The disordered linker in protomer B is shown as a green dotted line. DNA binding loops are shown in red with the side-chains of residues Asn150 and Asn152 as red sticks. Ile21 at the tip of the αA–αB loops are shown as sticks. The hemimethylated DNA is shown in orange (methylated strand) and light yellow (unmethylated strand) with the methylated adenine in brown. (c) Ribbon diagram depicting how the C-terminal domain breaks the two-fold symmetry of the N-terminal domain. The SeqAΔ(41–59)-A25R dimer is shown in purple (protomer A) and green (protomer B). Protomer A superimposed onto the N-terminus of protomer B is shown in light-grey. The first α-helix on the C-terminal domain (αC) is labeled for reference. The grey arrow indicates the rotation of the C-terminal domain around Lys34 (red asterisk and sticks). The cartoon illustrates the transformation from (b to c). (d) Sequence alignment of SeqA from Escherichia coli K-12, Salmonella enterica serovar Typhi str. CT18, Yersinia pestis CO92, Klebsiella pneumoniae MGH 78578 and Vibrio cholerae 623-39 (top to bottom). Secondary structure motifs from SeqAΔ(41–59)-A25R are shown. The deletion and point mutations in SeqAΔ(41–59)-A25R are marked with asterisks. Ala25 and hydrophobic residues within the linker region are highlighted in yellow. The variable linker between the two functional domains is shadowed in grey. DNA-binding residues are highlighted in red and those involved in reciprocal salt-bridges between neighbour C-terminal domains in green and blue. Residues (a–c) were prepared using PyMol (42).
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Figure 2: Structure of SeqAΔ(41–59)-A25R bound to a hemimethylated GATC repeat. (a) Ribbon diagram of the SeqAΔ(41–59)-A25R monomer with helices in purple and strands in pink. The junction between residues Gln40 and Lys60 is indicated with a black arrowhead. (b) SeqAΔ(41–59)-A25R dimer bound to DNA. Protomer A (encompassing residues 1–40/60–181) is shown in purple and protomer B (encompassing residues 1–35/60–181) is shown in green. The disordered linker in protomer B is shown as a green dotted line. DNA binding loops are shown in red with the side-chains of residues Asn150 and Asn152 as red sticks. Ile21 at the tip of the αA–αB loops are shown as sticks. The hemimethylated DNA is shown in orange (methylated strand) and light yellow (unmethylated strand) with the methylated adenine in brown. (c) Ribbon diagram depicting how the C-terminal domain breaks the two-fold symmetry of the N-terminal domain. The SeqAΔ(41–59)-A25R dimer is shown in purple (protomer A) and green (protomer B). Protomer A superimposed onto the N-terminus of protomer B is shown in light-grey. The first α-helix on the C-terminal domain (αC) is labeled for reference. The grey arrow indicates the rotation of the C-terminal domain around Lys34 (red asterisk and sticks). The cartoon illustrates the transformation from (b to c). (d) Sequence alignment of SeqA from Escherichia coli K-12, Salmonella enterica serovar Typhi str. CT18, Yersinia pestis CO92, Klebsiella pneumoniae MGH 78578 and Vibrio cholerae 623-39 (top to bottom). Secondary structure motifs from SeqAΔ(41–59)-A25R are shown. The deletion and point mutations in SeqAΔ(41–59)-A25R are marked with asterisks. Ala25 and hydrophobic residues within the linker region are highlighted in yellow. The variable linker between the two functional domains is shadowed in grey. DNA-binding residues are highlighted in red and those involved in reciprocal salt-bridges between neighbour C-terminal domains in green and blue. Residues (a–c) were prepared using PyMol (42).

Mentions: SeqAΔ(41–59)-A25R was crystallized in complex with a hemimethylated duplex containing two GATC sequences separated by 9 bp (27). The structure was solved by molecular replacement using the structures of the N- and C-terminal domains of SeqA (PDB codes 1XRX and 1LRR, respectively) and refined using standard protocols in REFMAC and PHENIX.REFINE (29,30). The asymmetric unit contains two identical protein–DNA complexes related by a 2-fold axis. The final model comprises two copies of protomer A (residues 1–40/60–181), two copies of protomer B (residues 1–35 and 60–181), two copies of the hemimethylated DNA duplex (with the exception of nucleotide Cyt2 from the unmethylated strands), 32 water molecules and four 2-methyl-2,4-pentanediol (MPD) molecules (Table 1 and Figure 2). Over 97% of the residues lie in the most favored regions of the Ramachandran plot, and none in disallowed regions.Figure 2.


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)

Structure of SeqAΔ(41–59)-A25R bound to a hemimethylated GATC repeat. (a) Ribbon diagram of the SeqAΔ(41–59)-A25R monomer with helices in purple and strands in pink. The junction between residues Gln40 and Lys60 is indicated with a black arrowhead. (b) SeqAΔ(41–59)-A25R dimer bound to DNA. Protomer A (encompassing residues 1–40/60–181) is shown in purple and protomer B (encompassing residues 1–35/60–181) is shown in green. The disordered linker in protomer B is shown as a green dotted line. DNA binding loops are shown in red with the side-chains of residues Asn150 and Asn152 as red sticks. Ile21 at the tip of the αA–αB loops are shown as sticks. The hemimethylated DNA is shown in orange (methylated strand) and light yellow (unmethylated strand) with the methylated adenine in brown. (c) Ribbon diagram depicting how the C-terminal domain breaks the two-fold symmetry of the N-terminal domain. The SeqAΔ(41–59)-A25R dimer is shown in purple (protomer A) and green (protomer B). Protomer A superimposed onto the N-terminus of protomer B is shown in light-grey. The first α-helix on the C-terminal domain (αC) is labeled for reference. The grey arrow indicates the rotation of the C-terminal domain around Lys34 (red asterisk and sticks). The cartoon illustrates the transformation from (b to c). (d) Sequence alignment of SeqA from Escherichia coli K-12, Salmonella enterica serovar Typhi str. CT18, Yersinia pestis CO92, Klebsiella pneumoniae MGH 78578 and Vibrio cholerae 623-39 (top to bottom). Secondary structure motifs from SeqAΔ(41–59)-A25R are shown. The deletion and point mutations in SeqAΔ(41–59)-A25R are marked with asterisks. Ala25 and hydrophobic residues within the linker region are highlighted in yellow. The variable linker between the two functional domains is shadowed in grey. DNA-binding residues are highlighted in red and those involved in reciprocal salt-bridges between neighbour C-terminal domains in green and blue. Residues (a–c) were prepared using PyMol (42).
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Figure 2: Structure of SeqAΔ(41–59)-A25R bound to a hemimethylated GATC repeat. (a) Ribbon diagram of the SeqAΔ(41–59)-A25R monomer with helices in purple and strands in pink. The junction between residues Gln40 and Lys60 is indicated with a black arrowhead. (b) SeqAΔ(41–59)-A25R dimer bound to DNA. Protomer A (encompassing residues 1–40/60–181) is shown in purple and protomer B (encompassing residues 1–35/60–181) is shown in green. The disordered linker in protomer B is shown as a green dotted line. DNA binding loops are shown in red with the side-chains of residues Asn150 and Asn152 as red sticks. Ile21 at the tip of the αA–αB loops are shown as sticks. The hemimethylated DNA is shown in orange (methylated strand) and light yellow (unmethylated strand) with the methylated adenine in brown. (c) Ribbon diagram depicting how the C-terminal domain breaks the two-fold symmetry of the N-terminal domain. The SeqAΔ(41–59)-A25R dimer is shown in purple (protomer A) and green (protomer B). Protomer A superimposed onto the N-terminus of protomer B is shown in light-grey. The first α-helix on the C-terminal domain (αC) is labeled for reference. The grey arrow indicates the rotation of the C-terminal domain around Lys34 (red asterisk and sticks). The cartoon illustrates the transformation from (b to c). (d) Sequence alignment of SeqA from Escherichia coli K-12, Salmonella enterica serovar Typhi str. CT18, Yersinia pestis CO92, Klebsiella pneumoniae MGH 78578 and Vibrio cholerae 623-39 (top to bottom). Secondary structure motifs from SeqAΔ(41–59)-A25R are shown. The deletion and point mutations in SeqAΔ(41–59)-A25R are marked with asterisks. Ala25 and hydrophobic residues within the linker region are highlighted in yellow. The variable linker between the two functional domains is shadowed in grey. DNA-binding residues are highlighted in red and those involved in reciprocal salt-bridges between neighbour C-terminal domains in green and blue. Residues (a–c) were prepared using PyMol (42).
Mentions: SeqAΔ(41–59)-A25R was crystallized in complex with a hemimethylated duplex containing two GATC sequences separated by 9 bp (27). The structure was solved by molecular replacement using the structures of the N- and C-terminal domains of SeqA (PDB codes 1XRX and 1LRR, respectively) and refined using standard protocols in REFMAC and PHENIX.REFINE (29,30). The asymmetric unit contains two identical protein–DNA complexes related by a 2-fold axis. The final model comprises two copies of protomer A (residues 1–40/60–181), two copies of protomer B (residues 1–35 and 60–181), two copies of the hemimethylated DNA duplex (with the exception of nucleotide Cyt2 from the unmethylated strands), 32 water molecules and four 2-methyl-2,4-pentanediol (MPD) molecules (Table 1 and Figure 2). Over 97% of the residues lie in the most favored regions of the Ramachandran plot, and none in disallowed regions.Figure 2.

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