Primosomes:

 

DnaA- and PriA-dependent primosomes: two distinct replication complexes for replication of Escherichia coli chromosome. Masai H, Arai K. Department of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo, 108, Japan. hisao@ims.u-tokyo.ac.jp

 

Primosomes are complex of proteins that produce RNA primer at the time of DNA replication. Enzymatic analyses of primosome assembly at chromosomal and plasmid origins as well as that at single-stranded DNA replication origins revealed that the presence of two distinct primosomes in Escherichia coli for primer RNA synthesis and duplex unwinding.

 The DnaA-dependent primosome is assembled at oriC, the chromosomal origin of Escherichia coli, as well as at the A site, a single-stranded DNA hairpin containing a DnaA box sequence within its stem.

 In contrast, PriA protein recognizes a small stem-loop, called n'-pas (primosome assembly site), and initiates assembly of the phiX174-type PriA- primosome in conjunction with other prepriming proteins. Genetic analyses of the prepriming proteins required specifically for the latter primosome strongly suggested that it is responsible for RecA-dependent, DnaA/oriC-independent replication.  

Furthermore, primosome assembly in replication of various plasmids may also be classified into either DnaA-dependent or PriA-dependent types. Escherichia coli possess two distinct, mutually exclusive primosomes which are differentially utilized by the chromosome as well as by the plasmids. PriA protein appears to be conserved in a wide range of prokaryotic species, and the same will be discussed and its possible biological functions ex. PriA-dependent primosome in responses to DNA damages.

 

 

Kinds of Primosomes:

 

Dna-A Dependent Primosomes

Pri-A dependent Primosomes

 

 

Chromosomes: E.coli-DNA

Ori-C

Present;

operates

In damaged E.coli DNA

Ori-Ms (isdr)

Yes, operates

Plasmids:

 

 

 

 

 

F-

Ori

Yes

-

-

-

PSc 101

Ori

Yes

Col-E1

Ori Col E-1

Yes, operates

R6K

Ori

Yes

pBR322

Ori pBR

Yes

RK2

Ori

Yes

RSF 1030

Ori

Yes

Rts 1

Ori

Yes

Col e2 ori

Ori cool E 2

Yes

SS DNA:

Ss DNA Ori

Nil

Phi X 174

Ori (-), and Ori (+)

Yes

Requirements for primosome formation:

 

 

 

 

 

Dna binding proteins

Dna-A

 

Pri-A?

 

 

 

Hu

 

Pri-B

 

 

 

 

 

Pri-C

 

 

 

 

 

 

 

 

Helicase

Dna-B

 

Dna-B, Dna-T

 

 

 

Dna-C

Assists DnaB

Dna-C

 

 

Priming

Dna-G

 

Dna-G

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Pri-A dependent Primosome Component:

 

Pri-A Primosome component

Gene

Mol.wt (KD)

Subunit

 Function

Pri-A

 

76

Monomer

 

Pri-B

 

11.5

Dimer

 

Pri-C

 

33

Monomer

 

Dna-T

 

22

Trimer

 

Dna-B

 

50

Hexamer

 

Dna-C

 

29

Monomer

 

Dna-G

 

60

Monomer

 

RNA-pol

 

 

Pentamer (Holozyme)

Lays primer on leading strand

RNase-H

 

Dimer

 

By exonuclease activity produces a primer

 

 

 

 

 

 

Note: In Col E 1, on lagging strand, in addition to the above, requires Rom protein (7.5kd), a monomer.

 

 

Components of primosome in E. coli:

 

Protein

Gene

Activities and Functions

PriA

priA

helicase, 3' to 5' movement, site recognition

PriB

priB

 

PriC

priC

 

DnaT

dnaT

needed to add DnaB‑DnaC complex to preprimosome

DnaC

dnaC

forms complex with DnaB

DnaB

dnaB

helicase, 5' to 3' movement. DNA dependent ATPase.

DnaG

dnaG

synthesize primer

 

Five different proteins are found in a prepriming complex, PriA, PriB, PriC, DnaT, and DnaB (Table above), Dna A and  DnaC are also needed for the assembly of this complex. In the case of X174 viral DNA template coated with SSB, PriA recognizes a primer assembly site. The proteins PriB and PriC are then added to form a complex. The hexameric protein DnaB is in a complex with six molecules of DnaC when it is not on the DNA. In an ATP-dependent process, and with help from DnaT, DnaB is transferred to the template and DnaC is released.

 

In contrast to the other DNA polymerases discussed, terminal deoxynucleotidyl transferase does not require a template. It adds dNTPs (as dNMP) to the 3' end of DNA, using that 3' hydroxyl as a primer. It is found in differentiating lymphocytes, and appears to be used physiologically to introduce somatic mutations in immunoglobulin genes. In the laboratory, it is used to add "homopolymeric tails" to the ends of DNA molecules by incubating a linear DNA with one particular dNTP and terminal deoxynucleotidyl transferase.

 

The pre-priming complex and DnaG bind and make the active primosome. Although the role of each of the proteins in the primosome is not yet clear, information is available on some of the steps in primosome action. The preferred binding site on the template for primase is CTG (in E.coli), with the T being used as the template for the first nucleotide of the primers. A high affinity complex between DnaB and ATP forms or stabilizes a secondary structure in the single-stranded template DNA that is used by primase; this is thought to be how DnaB "activates" the primase to begin synthesis. After ATP hydrolysis by DnaB, the low affinity ADP-DnaB dissociates from the template. The primosome can now move to the next site for primer synthesis.

 

 

 

 

 

 

Another depiction PriA-primosomes involved PhiX174 DNA replication; multiple primers are produced. http://shomusbiology.weebly.com/

Figure: Assembly and migration of the primosome. After assembly of the pre-priming complex, DnaG joins the complex to complete the primosome. After synthesis of the primer (dark black line), the primosome moves to the next site for synthesis. This tracking along the SSB-coated single stranded DNA requires ATP hydrolysis and causes dissociation of some of the SSB. In the diagram, the primosome is shown moving in a 5' to 3' direction along the template strand (clockwise), which is the opposite of the direction of primer synthesis. This would be the direction of movement catalyzed by DnaB. The primosome also contains PriA, which catalyzes movement along single-stranded DNA in the opposite direction. Once primers have been synthesized, DNA polymerase III can synthesize Okazaki fragments from them, the primers are excised and gaps repaired by DNA polymerase I, and then the fragments are ligated together.

 

 

 

 

 

 

Components required for Dna-A dependent Primosomes:

 

Types

Proteins

Mol.wt (KD) & subunits

 

Functions

Dna binding proteins

Dna-A

50, monomers

 

Bind to Dna-A boxes induce opening at 13 mer regions

 

Hu

 

 

Binds to Ori-C, induces DNA bending

 

 

 

 

 

 

 

 

 

 

Helicase

Dna-B

50, hexamer

 

Binds to lagging strand at fork region and acts as the motor protein in unwinding DNA helix, in 5’. 3’ direction

 

Dna-C

, Monomer (1:1)

 

Assists Dna-B in 1:1 manner

Priming

Dna-G

60, monomer

 

Lays RNA primer of lagging strand

 

 

Primers:

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Models of primosomes and their activity during primer synthesis.

 

From the following article

Coupling DNA unwinding activity with primer synthesis in the bacteriophage T4 primosome

Maria Manosas, Michelle M Spiering, Zhihao Zhuang, Stephen J Benkovic & Vincent Croquette.

 

Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com

Schematic representation of three possible models for helicase and primase interaction during primer synthesis (left) and the real-time DNA extension traces expected for each model (right). (a) In the pausing model, the helicase and primase temporarily stop translocating during priming. (b) In the disassembly model, the primase dissociates from the helicase to synthesize a primer while the helicase continues unwinding DNA. (c) In the DNA looping model, the primosome remains intact, and DNA unwound during priming forms a loop.

Assembly of the bacteriophage T4 primosome: Single-molecule and ensemble studies

1.           Zhiquan Zhang * , , Michelle M. Spiering , , Michael A. Trakselis , §,

2.           Faoud T. Ishmael , , Jun Xi , Stephen J. Benkovic , and Gordon G. Hammes * ,

 

Fig. 4.

Assembly mechanism of the T4 lagging-strand primosome on fDNA. The gp32 protein binds to fDNA with either subsequent or concurrent binding of gp59. Subsequently, gp41 binds to gp59 and is loaded onto the fDNA in the presence of nucleotide. ATP hydrolysis is required for gp41 to displace gp32 and gp59, either directly or by translocation. The gp61 protein then binds and interacts closely with gp41 on fDNA. In the absence of gp32 and gp59, both gp41 and gp61 bind to DNA.

 

The coordination between the continuous leading strand DNA synthesis and the discontinuous lagging strand counterpart is regulated by a subassembly of the replisome called the primosome. In the T4 system the primosome is formed by a hexameric helicase (gp41) that unwinds dsDNA and an oligomeric primase (gp61) that synthesizes RNA primers to initiate repetitive Okazaki fragment synthesis. Three possible models have been suggested to explain how helicase is able to unwind dsDNA translocating 5′ to 3′ on the lagging strand while primase travels in the opposite direction (3′ to 5′) in order to synthesize an RNA primer. In the first model (pausing), the helicase temporarily pauses or stops translocating to allow for primer synthesis and then resumes unwinding the DNA; helicase pausing would necessitate the pausing of the entire replisome while a primer is being synthesized. In the second model (disassembly), one or more primase subunits dissociate from the helicase and remain behind to synthesize a primer while the helicase and any remaining primase subunits continue to translocate along the lagging strand. In this model, leading strand synthesis would continue uninterrupted; however, new primase subunits might need to be recruited for each cycle of Okazaki fragment synthesis. In the third model (DNA looping), the primosome remains intact and the DNA that is continuously unwound by the helicase during primer synthesis forms a loop which is released once the primer is transferred to the lagging strand polymerase.

 

 

Three priming mechanisms;

Interaction of the primase with the helicase is necessary for primer synthesis at a lagging-strand priming site. The primase makes primers in the opposite direction to helicase movement, leaving three ways by which the replisome might resolve this directional problem. a, The whole replisome can pause for primer synthesis; b, it can promptly release the primase; or c, as described for the first time by Pandey et al.1 and Manosas et al.2, the replisome can continue to move forward while the primase–helicase interaction persists. This produces a priming loop that eventually collapses into the lagging-strand trombone loop, probably on transfer of the primer to the lagging-strand polymerase.Three priming mechanisms; Nicholas E. Dixon; http://www.nature.com/

 

 

Figure: A model for the activities of helicases DnaB and PriA at replication fork: In this model, the replication machinery in a large Replisome (polymerase and primosome) is postulated to be stationary, with the DNA strands moving through it like fabric through a sewing machine. After unwinding the duplex DNA at the replication fork, the DnaB helicase can also track along single stranded DNA in a 5' to 3' direction. If the helicase is stationary, then the template strand for lagging strand synthesis moves in the direction of the upper gray arrow in the diagram, away from the replication fork. This template strand is also bound to the PriA helicase at the "back" end of the replisome. The 3' to 5' tracking activity of PriA will also pull the template strand, now in the direction of the bottom gray arrow. The result of the DnaB and PriA tracking activities is to pull the template for lagging strand synthesis into a loop, which is tethered to the replisome at both ends. Primase is also in the replisome, and can synthesize primers along this strand at appropriate intervals (thick blue lines), and one of the core polymerases of the DNA polymerase III holoenzyme can synthesize an Okazaki fragment. Newly synthesized DNA is a thick violet line, and parental DNA strands are thick black lines.

 

 

 

Nucleotide sequences and possible stem-loop structures of n'-pas isolated from various plasmid DNAs. Eight newly isolated n'-pas in addition to previously reported ones were classified into five groups on the basis of their primary structures. n'-pas of X174 phage, R100 plasmid and F•f2b belong to group I, n'-pas on the lagging strand of ColE1-type plasmids (ssiA) and n' sites of F•f5, F•f7 and ColE2 plasmid to group II, n'-pas of the F•f2a to group III, n'-pas on the leading strand of ColE1-type plasmids (ssiB) to group IV, and n'-pas from Rts1 plasmid to group V. Each group is represented by the sequences of the first listing described above. The sequences indicated by boxes show some sequence similarities, although none of them are conserved among all the groups. The group I and group II were described before. Hisao Masai and Ken-ichi Arai. http://www.bioscience.org/

 

A Specific Docking Site for DNA Polymerase α-Primase on the SV40 Helicase Is Required for Viral Primosome Activity, but Helicase Activity Is Dispensable*

1.           Hao HuangKun ZhaoDiana R. Arnett and  Ellen Fanning1

1.           From the Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37235-1634; 1 To whom correspondence should be addressed. Tel.: 615-343-5677; Fax: 615-343-6707; E-mail: Ellen.fanning@vanderbilt.edu.

Replication of simian virus 40 (SV40) DNA, a model for eukaryotic chromosomal replication, can be reconstituted in vitro using the viral helicase (large tumor antigen, or Tag) and purified human proteins.

DNA polymerase α-primase (pol-prim), constituting the viral primosome: an example for an eukaryote: http://www.jbc.org

Like the well characterized primosomes of phages T7 and T4, this trio of proteins coordinates parental DNA unwinding with primer synthesis to initiate the leading strand at the viral origin and each Okazaki fragment on the lagging strand template. We recently determined the structure of a previously unrecognized pol-prim domain (p68N) that docks on Tag, identified the p68N surface that contacts Tag, and demonstrated its vital role in primosome function. Here, we identify the p68N-docking site on Tag by using structure-guided mutagenesis of the Tag helicase surface. A charge reverse substitution in Tag disrupted both p68N-binding and primosome activity but did not affect docking with other pol-prim subunits. Unexpectedly, the substitution also disrupted Tag ATPase and helicase activity, suggesting a potential link between p68N docking and ATPase activity. To assess this possibility, we examined the primosome activity of Tag with a single residue substitution in the Walker B motif. Although this substitution abolished ATPase and helicase activity as expected, it did not reduce pol-prim docking on Tag or primosome activity on single-stranded DNA, indicating that Tag ATPase is dispensable for primosome activity in vitro.