Residues at the Carboxy Terminus of T<sub>4</sub> DNA Polymerase Are Important Determinants for Interaction with the Polymerase Accessory Proteins

Goodrich, Leo D.; Lin, Tsung‐I; Spicer, Eleanor K.; Jones, Christopher T.; Konigsberg, William H.

doi:10.1021/bi9708949

Cited by 13 publications

(6 citation statements)

References 36 publications

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“…This is commonly observed for the corresponding regions of other polymerases in the absence of substrate [9,[21][22][23][24]. In the DNA polymerases from bacteriophage T4 and RB69, the thumb subdomains also provide a C-terminal element that interacts with the processivity clamp [25,26]. In D. Tok Pol, the corresponding region (residues 757-773) is disordered.…”

Section: General Description Of the Structurementioning

confidence: 83%

Crystal structure of an archaebacterial DNA polymerase

et al. 1999

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The structure of D. Tok Pol confirms that the modes of binding of the template and extrusion of newly synthesized duplex DNA are likely to be similar in both Pol II and Pol I type DNA polymerases. However, the mechanism by which the newly synthesized product transits in and out of the proofreading exonuclease domain has to be quite different. The discovery of a domain that seems to be an RNA-binding module raises the possibility that Pol II family members interact with RNA.

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Section: General Description Of the Structurementioning

confidence: 83%

Crystal structure of an archaebacterial DNA polymerase

et al. 1999

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“…The importance of the COOH terminus of gp43 was also investigated by others (9). Additionally, the COOH termini of gp33 and gp55 have been shown to be essential for interaction with gp45 (10).…”

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confidence: 98%

The Carboxyl Terminus of the Bacteriophage T4 DNA Polymerase Contacts Its Sliding Clamp at the Subunit Interface

Alley

Jones

Soumillion³

et al. 1999

Journal of Biological Chemistry

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The location of the interaction of the COOH terminus of the bacteriophage T4 DNA polymerase with its trimeric, circular sliding clamp has been established. A peptide corresponding to the COOH terminus of the DNA polymerase was labeled with a fluorophore and fluorescence spectroscopy used to show that it forms a specific complex with the sliding clamp by virtue of its low K D value (7.1 ؎ 1.0 M). The same peptide was labeled with a photoaffinity probe and cross-linked to the sliding clamp. Mass spectrometry of tryptic digests determined the sole linkage point to be Ala-159 on the sliding clamp, an amino acid that lies on the subunit interface. These results demonstrate that the COOH terminus of the DNA polymerase is inserted into the subunit interface of its sliding clamp, thereby conferring processivity to the DNA polymerase.Multiprotein complexes lie at the heart of DNA replication. In bacteriophage T4, the DNA polymerase (gp43), its processivity factor or sliding clamp (gp45), and the clamp-loader (gp44/62) form the holoenzyme that is loaded onto DNA in a multistep process requiring hydrolysis of ATP (1-3). gp44/62 (a 4:1 complex of gp44 and gp62) is only a transient species in the holoenzyme assembly process, hydrolyzing ATP to load gp45 onto DNA and then chaperoning gp43 onto the gp45/DNA complex (4). Likewise, the intermediate states of assembly are distinct and transient (3,5). The x-ray crystal structures of gp45 from bacteriophage T4 1 and gp43 from bacteriophage RB69 (63% identity with gp43 from T4 (6)) are known (7), but the precise relative orientations of the proteins during and after the holoenzyme assembly process are unknown. X-ray crystallography should be able to eventually determine the structures of stable multiprotein complexes such as the final holoenzyme structure, but other methods will be required to determine the structures of transient or unstable structures during the holoenzyme assembly process. We have chosen to use fluorescence spectroscopy and mass spectrometry to map the connection point between the COOH terminus of gp43 and gp45, the interaction that is at the core of the holoenzyme and confers processivity to gp43 (8).We have shown previously that the COOH terminus of gp43 is essential for forming the holoenzyme, and a peptide corresponding to the COOH terminus of gp43 inhibits the interaction of gp45 and gp44/62, thereby inhibiting formation of the holoenzyme (8). The importance of the COOH terminus of gp43 was also investigated by others (9). Additionally, the COOH termini of gp33 and gp55 have been shown to be essential for interaction with gp45 (10). gp33 is a transcriptional co-activator (11), and gp55 is a -family protein (12), and their interaction with gp45 is required in late RNA transcription (13). The sequences of the COOH termini of gp33, gp55, and gp43, as well as the sequence of the COOH-terminal peptide of gp43 (peptide 1), are shown in Fig. 1. These three proteins have a common (S/T)LDFL motif, suggesting a shared mode of interaction with gp45. gp45 has shown to ...

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“…Relative to the analogous proteins of E. coli (β clamp) and yeast (PCNA), the 45 protein is unstable on DNA ( 20 ; Sexton and Benkovic, unpublished experiments). Recently, the interaction between the 45 protein and the 43 protein was targeted to the carboxyl-terminal sequence of the polymerase ( , ). This contact increases the resident time of the holoenzyme on DNA ( , ).…”

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confidence: 99%

Clamp Subunit Dissociation Dictates Bacteriophage T4 DNA Polymerase Holoenzyme Disassembly

1998

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Clamp proteins confer processivity to the DNA polymerase during DNA replication. These oligomeric proteins are loaded onto DNA by clamp loader protein complexes in an ATP-dependent manner. The mechanism by which the trimeric bacteriophage T4 clamp protein (the 45 protein) loads and dissociates from DNA was investigated as a function of its intersubunit protein-protein interactions. These interactions were continuously monitored using a fluorescence resonance energy transfer (FRET) based assay. A cysteine mutant of the 45 protein was constructed to facilitate site-specific incorporation of a fluorescent probe at the subunit interface. This site was chosen such that FRET was observed between the introduced fluorescent probe and a tryptophan residue located on the opposing subunit. By use of this fluorescently labeled 45 protein, it was possible to obtain an estimate of an apparent trimer dissociation constant from either a cooperative (0.08 +/- 0.04 microM2 at 25 degrees C) or a noncooperative (0.51 microM and 0.17 microM at 25 degrees C) model. Upon mixing the fluorescently labeled 45 protein with a 45 protein containing 4-fluorotryptophan, a nonfluorescent tryptophan analogue, subunit exchange between the two variants of the 45 protein was observed according to a reduction in intersubunit FRET. Subunit exchange rate constants measured in the presence or absence of the clamp loader (44/62 complex), the polymerase (43 protein), and/or a primer template DNA substrate demonstrate (a) that the 45 protein is not loaded onto DNA by subunit exchange and (b) that the disassembly dissociation of a stalled holoenzyme from DNA is dictated by 45 protein subunit dissociation.

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Residues at the Carboxy Terminus of T₄ DNA Polymerase Are Important Determinants for Interaction with the Polymerase Accessory Proteins

Cited by 13 publications

References 36 publications

Crystal structure of an archaebacterial DNA polymerase

Crystal structure of an archaebacterial DNA polymerase

The Carboxyl Terminus of the Bacteriophage T4 DNA Polymerase Contacts Its Sliding Clamp at the Subunit Interface

Clamp Subunit Dissociation Dictates Bacteriophage T4 DNA Polymerase Holoenzyme Disassembly

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Residues at the Carboxy Terminus of T4 DNA Polymerase Are Important Determinants for Interaction with the Polymerase Accessory Proteins

Cited by 13 publications

References 36 publications

Crystal structure of an archaebacterial DNA polymerase

Crystal structure of an archaebacterial DNA polymerase

The Carboxyl Terminus of the Bacteriophage T4 DNA Polymerase Contacts Its Sliding Clamp at the Subunit Interface

Clamp Subunit Dissociation Dictates Bacteriophage T4 DNA Polymerase Holoenzyme Disassembly

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Residues at the Carboxy Terminus of T₄ DNA Polymerase Are Important Determinants for Interaction with the Polymerase Accessory Proteins