User:Bianca Varney/Bacterial Replication Termination

From Proteopedia

(Difference between revisions)
Jump to: navigation, search
Line 1: Line 1:
-
Bacterial replication termination is mediated by replication terminator proteins that bind to polar inverted repeats approximately 180 degrees from the origin of replication (OriC).Replication terminator proteins bind bacterial DNA at termination (Ter) sites. When the replication forks meet with terminator proteins bound to Ter sites, replication is arrested, and DNA polymerase falls off the bacterial chromosome. However, protein-Ter interactions are orientation specific, and will only arrest the replication forks traveling in on one direction; either clockwise or anticlockwise. This means that one replication fork will be arrested at a Ter site, while the other fork, traveling in the opposite direction, will pass through the site unimpeded, allowing the entire bacterial chromosome to be copied. The area loaded with Ter sites in the chromosome is called a "replication fork trap".
+
Bacterial replication termination is mediated by replication terminator proteins that bind to polar inverted repeats approximately 180 degrees from the origin of replication (OriC). Replication terminator proteins bind bacterial DNA at termination (Ter) sites. When the replication forks meet with terminator proteins bound to Ter sites, replication is arrested, and DNA polymerase falls off the bacterial chromosome. However, protein-Ter interactions are orientation specific, and will only arrest the replication forks traveling in on one direction; either clockwise or anticlockwise. This means that one replication fork will be arrested at a Ter site, while the other fork, traveling in the opposite direction, will pass through the site unimpeded, allowing the entire bacterial chromosome to be copied. The area loaded with Ter sites in the chromosome is called a "replication fork trap".
==Mechanism==
==Mechanism==
Line 7: Line 7:
==Replication Terminator Protein (''Bacillus subtilis'')==
==Replication Terminator Protein (''Bacillus subtilis'')==
-
The replication terminator protein (RTP) is a homodimer composed of two 14.5 kDa subunits. Each RTP monomer consists of four α helical and three β strands, an unstructured N-terminal containing six amino acids, and an extended loop. The monomer comprises of a 4-turn α-helix (α1) connected to a β-strand (β1), from the N to C terminus. The β1 strand connects to α3, a 3-turn α-helix, via α2, a 3-turn helix. The α3 helix is attached to a β-pleated sheet. Antiparallel in structure it contains the β2 and β3 strands proximal to the β1 strand. The C-terminal contains the last α helix, the 8-turn α4 helix. The connections between the secondary structures are short, with the exception of the elements connecting α2-->α3 and β2-->β3. The latter is an important structure in dimer-dimer interactions, as it is predicted to contact the structure of an adjacent dimer. This was backed by evidence from site-directed mutagenesis as mutations in this region, specifically residue 34 and 82, results in no dimer-dimer interaction. The loop and strands create a wing, of the winged helix. The winged helix is involved in regions of DNA binding, and analysis has revealed that the RTP protein has structural homology to the H5 histone protein, due to this similar region. Although it is important to note that no significant similarity between sequences was determined between RTP and H5 histone protein. The RTP protein contains three major structural domains for its specific functionality, including DNA-binding, DnaB interaction and dimer-dimer interaction domains. Biochemical and mutational studies have identified particular residues that are vital for the functionality of the RT protein. Manna et al have identified that mutation within a hydrophobic region at residues Glu-30 and Tyr-33 causes the loss of contrahelicase ability. These mutations do not affect dimer-dimer interactions or DNA binding activity and indicate that simple DNA binding is not able to block the replication fork. This provided evidence that RTP and the replication fork machinery interact specifically.
+
The replication terminator protein (RTP) is a homodimer composed of two 14.5 kDa subunits. The RTP protein contains three major structural domains for its specific functionality, including DNA-binding, DnaB interaction and dimer-dimer interaction domains. Biochemical and mutational studies have identified particular residues that are vital for the functionality of the RT protein. Manna et al have identified that mutation within a hydrophobic region at residues Glu-30 and Tyr-33 causes the loss of contrahelicase ability. These mutations do not affect dimer-dimer interactions or DNA binding activity and indicate that simple DNA binding is not able to block the replication fork. This provided evidence that RTP and the replication fork machinery interact specifically.
The RTP is organized into a dimer by the association of their long α helices within the C-terminus. The ‘winged helix’ is believed to be involved as the major DNA-binding domain however two of the α helices, at the centre of the protein, and two β strands, in the outer regions, have been suggested to fit adjacently into the major and minor grooves respectively. The unstructured N terminal region is may also have a role in DNA-binding.
The RTP is organized into a dimer by the association of their long α helices within the C-terminus. The ‘winged helix’ is believed to be involved as the major DNA-binding domain however two of the α helices, at the centre of the protein, and two β strands, in the outer regions, have been suggested to fit adjacently into the major and minor grooves respectively. The unstructured N terminal region is may also have a role in DNA-binding.
 +
 +
<Structure load='try2' size='200' frame='true' align='right' caption='RTP complexed to the Ter site' scene='Insert optional scene name here' />
==Terminus Utilization Substance (''Escherichia coli'')==
==Terminus Utilization Substance (''Escherichia coli'')==
-
The Terminus Utilization Sequence (Tus) structure is organized into two discontinuous domains (N terminal and C terminal) that consist of α helical and β sheets that straddle the DNA helix (figure ??). Two antiparallel pairs of β strands that form an interdomain, are crossed bordered by three smaller strands connect the β sheet areas of the two domains providing a large central cleft that is positively charged into which the double helix (deformed locally) can fit, such that the two domains flank the DNA. The interdomain β strands, which makes up the DNA-binding domain) that sit across from the DNA, accesses a deepened major groove and contacts several of the bases within this groove, and is responsible for Ter sequence recognition and binding to DNA. The strength of Tus binding to the DNA is increased the by bordering β sheet regions that insert approxiametly perpendicular into the major groove and by electrostatic attractions between the majority of the phosphates along the DNA backbone in the 13bp binding region and the protein. The α helices I, II and III are amphipathic and bundle parallel to the DNA in antiparallel runs. The helicase-blocking face of Tus is completed by α helices IV and V and loops L1 and L2 that lie in the N-terminal domain and by α helices VI and VII that lie in the C-terminal domain. Of the four loops, three (L1, L2 and L3) are located at the fork blocking end of the protein, while L4 contacts bases in the minor groove of the DNA at the permissive end. The other major β sheet, that is not involved in DNA binding, but stabilizes this activity through hydrophobic interactions in the lower regions of the N terminal domain and makes up a significant amount of the N domains hydrophobic. The hydrophobic core is contributed majorily by the α helices including helices I, II and II in the N domain and helices VI and VII in the C domain, with smaller contributions from the β sheets.
+
The Terminus Utilization Sequence (Tus) structure is organized into two discontinuous domains (N terminal and C terminal) that consist of α helical and β sheets that straddle the DNA helix. Two antiparallel pairs of β strands that form an interdomain providing a large positively charged central cleft, which adopts the double helix. The interdomain β strands, which makes up the DNA-binding domain, accesses a deepened major groove, making base contact. This is responsible for Ter sequence recognition and DNA binding.
-
The Tus-Ter complexs effect on deforming the DNA structure can merely be speculated on. X-ray crystallography studies (figure ??) have shown that when bound to Tus the double stranded DNA is extensiblley deformed from its canonical B-form. The DNA is observed to be significantly unwound, creating a deeper major groove, bending the DNA to approximately 200 and decreasing the helical twist from 34.60 to 29.5,0 caused by the binding of this protein into the DNA. However the fragment of DNA using in the crystal experiments does not continue beyond the binding of the protein and consequently provides little structural information about how the DNA is deformed beyond its complex with protein and the structure of the nonblocking face of the complex (figure 7 must check this out).
+
X-ray crystallography studies (figure ??) have shown that when bound to Tus the double stranded DNA is extensiblley deformed. The DNA is observed to be significantly unwound, creating a deeper major groove, bending the DNA to approximately 20 degrees and decreasing the helical twist from 34.60 to 29.5,0 caused by the binding of this protein into the DNA.
Tus is unrelated structurally to the replication termination protein despite their similar functions.
Tus is unrelated structurally to the replication termination protein despite their similar functions.

Revision as of 06:20, 18 May 2011

Bacterial replication termination is mediated by replication terminator proteins that bind to polar inverted repeats approximately 180 degrees from the origin of replication (OriC). Replication terminator proteins bind bacterial DNA at termination (Ter) sites. When the replication forks meet with terminator proteins bound to Ter sites, replication is arrested, and DNA polymerase falls off the bacterial chromosome. However, protein-Ter interactions are orientation specific, and will only arrest the replication forks traveling in on one direction; either clockwise or anticlockwise. This means that one replication fork will be arrested at a Ter site, while the other fork, traveling in the opposite direction, will pass through the site unimpeded, allowing the entire bacterial chromosome to be copied. The area loaded with Ter sites in the chromosome is called a "replication fork trap".

Contents

Mechanism

Bacterial replication termination has been well studied in E. coli and B. subtilis. The proteins involved in this termination process differ structurally in these two bacterium, although each contains similar contrahelicase activity and performs similar functions in arresting replication. The replication termini sequences are located in two clusters approximately 180 degrees from the origin of replication, and are orientated such that each cluster has opposite polarity and are therefore inverted repeats. This means that counterclockwise replication forks can move beyond the first cluster of ter sites, the left terminus, but are arrested at the second cluster, the right terminus, containing correct polarity. Similarly, the clockwise fork will proceed through the left cluster but will be arrested at the right. The bipartite Ter nucleotide sequence is overlapping and each inverted repeat contains a core (IRIB) and an axillary (IRIA) sites. RTP binds to these sequences, resulting in the impediment the replication fork helicase.

Replication Terminator Protein (Bacillus subtilis)

The replication terminator protein (RTP) is a homodimer composed of two 14.5 kDa subunits. The RTP protein contains three major structural domains for its specific functionality, including DNA-binding, DnaB interaction and dimer-dimer interaction domains. Biochemical and mutational studies have identified particular residues that are vital for the functionality of the RT protein. Manna et al have identified that mutation within a hydrophobic region at residues Glu-30 and Tyr-33 causes the loss of contrahelicase ability. These mutations do not affect dimer-dimer interactions or DNA binding activity and indicate that simple DNA binding is not able to block the replication fork. This provided evidence that RTP and the replication fork machinery interact specifically.

The RTP is organized into a dimer by the association of their long α helices within the C-terminus. The ‘winged helix’ is believed to be involved as the major DNA-binding domain however two of the α helices, at the centre of the protein, and two β strands, in the outer regions, have been suggested to fit adjacently into the major and minor grooves respectively. The unstructured N terminal region is may also have a role in DNA-binding.

RTP complexed to the Ter site

Drag the structure with the mouse to rotate

Terminus Utilization Substance (Escherichia coli)

The Terminus Utilization Sequence (Tus) structure is organized into two discontinuous domains (N terminal and C terminal) that consist of α helical and β sheets that straddle the DNA helix. Two antiparallel pairs of β strands that form an interdomain providing a large positively charged central cleft, which adopts the double helix. The interdomain β strands, which makes up the DNA-binding domain, accesses a deepened major groove, making base contact. This is responsible for Ter sequence recognition and DNA binding.

X-ray crystallography studies (figure ??) have shown that when bound to Tus the double stranded DNA is extensiblley deformed. The DNA is observed to be significantly unwound, creating a deeper major groove, bending the DNA to approximately 20 degrees and decreasing the helical twist from 34.60 to 29.5,0 caused by the binding of this protein into the DNA.

Tus is unrelated structurally to the replication termination protein despite their similar functions.

Biological Significance

The role of the replication fork arrest was primarily believed to be of great importance for the faithful termination of replication, segregation of chromosomes and faithful inheritance of a stable genome. However recent studies where the rtp and tus genes of B.subtilis and E.coli, respectively, were knocked out, suggested that this role is dispensable. Indeed, bacterial systems that have mutations within these genes can survive in the environment and appear identical in both growth rate and cell morphology compared to wildtype bacteria, suggesting that replication termination is not a requirement for cytokinesis [4]. It has recently been suggested that this form of termination may have roles in aiding the co-ordination and optimization of recombination events preceding replication in bacteria, and preventing over-replication. It is also suggested that termination may occur by specific dif sites, conserved sites that are located near the terminus region that are involved in homologous recombination. In fact the dif-terminus hypothesis proposes that termination occurs at or near these sites, where after termination of the replication forks, the dif-sites would undergo site-specific recombination, and that this would resolve the dimer chromosomes and complete replication

References

Proteopedia Page Contributors and Editors (what is this?)

Bianca Varney

Retrieved from "http://52.214.119.220/wiki/index.php/User:Bianca_Varney/Bacterial_Replication_Termination"
Personal tools