A specific subdomain in φ29 DNA polymerase confers both processivity and strand-displacement capacity

Abstract

Recent crystallographic studies of 29 DNA polymerase have provided structural insights into its strand displacement and processivity. A specific insertion named terminal protein region 2 (TPR2), present only in protein-primed DNA polymerases, together with the exonuclease, thumb, and palm subdomains, forms two tori capable of interacting with DNA. To analyze the functional role of this insertion, we constructed a 29 DNA polymerase deletion mutant lacking TPR2 amino acid residues Asp-398 to Glu-420. Biochemical analysis of the mutant DNA polymerase indicates that its DNA-binding capacity is diminished, drastically decreasing its processivity. In addition, removal of the TPR2 insertion abolishes the intrinsic capacity of 29 DNA polymerase to perform strand displacement coupled to DNA synthesis. Therefore, the biochemical results described here directly demonstrate that TPR2 plays a critical role in strand displacement and processivity.protein-primed replication ͉ terminal protein region ͉ helicase-like activity ͉ DNA-binding stability D NA replication is a complex multistep process that involves a wide range of proteins and enzymatic activities (1, 2). DNA synthetic activity is provided by DNA polymerases that add nucleotides to the 3Ј-OH end of a primer strand guided by base pairing with the template strand. Polymerases involved in DNA replication are referred to as replicases to distinguish them from other DNA polymerases whose synthetic activities play a role in processes such as DNA repair or recombination. In most DNA replication systems, replication fork movement along the duplex DNA requires an unwinding activity to separate the strands as replication progresses (1, 2). Generally, such activity is not intrinsic to the replicase but is provided either by monomeric or multimeric enzymes called helicases, which melt the dsDNA in an ATP-dependent fashion. In addition, the intrinsic processivity (number of nucleotides incorporated per single DNA polymerase͞DNA-binding event) of most replicases is not high enough to account for the replication of an entire genome, and therefore processivity factors are also required to hold the DNA replicase on the template strand (1, 2).Bacteriophage 29 DNA polymerase is a protein-primed DNA-dependent replicase belonging to the eukaryotic-type family of DNA polymerases (family B). Other members of this family include polymerases with cellular, bacterial, and viral origins (3). 29 DNA polymerase, like many other replicative polymerases, contains both 5Ј-3Ј synthetic and 3Ј-5Ј degradative activities within a single polypeptide chain. Its intrinsic insertion discrimination of 10 4 to 10 6 (4) is further improved 100-fold (5) through proofreading by the exonuclease domain. An extensive mutational analysis of 29 DNA polymerase served to identify the catalytic residues required for these two activities, as well as those responsible for the stabilization of the primer terminus at the respective active sites; these residues are evolutionarily conserved in most DNA polymerases (reviewed ...

DNA polymerase from temperate phage Bam35 is endowed with processive polymerization and abasic sites translesion synthesis capacity

“…As mentioned in the introductory section, protein-primed DNA polymerases contain two insertions, TPR1 and TPR2, which in the case of Φ29DNAP are involved in interactions with the TP and in processivity and strand displacement, respectively (22,23). Recent work has suggested that TPR2 also may be required for TLS capacity in a transposon-derived protein-primed DNA polymerase (54).…”

“…This designation refers to their capacity, apparent for adenoviruses as well as different bacteriophage enzymes (20,21), to perform genome replication primed by a specific protein, termed terminal protein (TP), that becomes covalently linked to the 5′ DNA ends. Identification and annotation of these polymerases in databases is based on the presence of two specific amino acid insertions, TPR1 and TPR2, involved in the interaction with TP and in processivity and strand displacement capacity, respectively (22,23).…”

The φ29 DNA polymerase:protein-primer structure suggests a model for the initiation to elongation transition

“…By analogy to a right hand, polymerase domains are conventionally divided into palm, finger, and thumb subdomains, which are involved in binding the catalytic metal ions, the incoming dNTP, and the duplex product DNA, respectively (Steitz et al, 1994). The two subdomains that are unique to protein-primed polymerases, TPR1 and TPR2, are insertions in the palm subdomain involved in interaction with terminal protein (Dufour et al, 2000(Dufour et al, , 2003, and in processivity and strand displacement (Kamtekar et al, 2004;Rodríguez et al, 2005).…”

My scientific life

“…We made a deletion in the TPR2 subdomain and found that the resulting DNA polymerase had a reduced DNA binding and had lost its processivity and stranddisplacement capacity. 18 The crystal structure of the ø29 DNA polymerase/TP heterodimer was also obtained in collaboration with Tom Steitz's laboratory 11 showing three domains in the TP: the N-terminal domain that has sequence-independent DNA binding capacity and does not interact with the DNA polymerase; the intermediate domain that interacts with the TPR1 subdomain of the polymerase; and the priming domain, which contains Ser232 and occupies the same binding cleft in the polymerase as duplex DNA does during elongation. The N-terminal domain is responsible for the nucleoid association of the TP.…”