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

Abstract

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 ...

Creating a dynamic picture of the sliding clamp during T4 DNA polymerase holoenzyme assembly by using fluorescence resonance energy transfer

“…This mode of interaction is inconsistent with the experimental solution data, which would suggest the gp43 interaction to be in the subunit interface of gp45. The C-terminal tail of gp43 is required for holoenzyme assembly (22) and interacts with residues in the subunit interface of gp45 (21). This proposed protein-protein interaction, where gp45 ''bites'' down on the C-terminal tail of gp43, is an important contact point partially responsible for the highly processive DNA replisome.…”

“…This final state of gp45 K in holoenzyme assembly has an open interface distance of about 11 Å. It has been suggested previously that the C terminus of gp43 is inserted into the subunit interface of gp45 (21). Although the C-terminal tail of gp43 is largely unstructured, interaction with gp45 could allow the tail to become more structured when it interacts with gp45 in the subunit interface.…”

“…Photocrosslinking has determined that gp44͞62 and gp43 interact with gp45 on the same ''rough'' face of the clamp (18)(19)(20). Recently, we have shown that the C-terminal tail of gp43 crosslinks to amino acid residues within the subunit interface of gp45 (21) and is absolutely required for holoenzyme assembly (22).…”

The dynamic processivity of the T4 DNA polymerase during replication

“…The polymerase C-terminal region was shown to interact with the interdomain loop of one of the clamp subunits. However, solution studies revealed a clamp structure with one open and two closed interfaces (20) and strongly argued for an interaction between the polymerase C terminus and the open clamp subunit interface (21,22). The x-ray data and its associated holoenzyme model which used the weaker interdomain binding site of gp45 was predicted, however, to play some role in holoenzyme assembly, DNA replication, translesion bypass, or other replication associated processes (19,21).…”

Dissection of the Bacteriophage T4 Late Promoter Complex

“…The similar C-terminal motifs of gp33, gp55 and gp43 are necessary for binding to gp45 in solution; the C-termini of gp55 as well as gp43 are also sufficient for gp45 binding (Alley et al, 1999;Wong and Geiduschek, 1998). To test the functional equivalence of these motifs, they were exchanged in gp55 and gp33 in all combinations and tested for compatibility with transcriptional activation.…”